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A Practical Guide to Writing Quantitative and Qualitative Research Questions and Hypotheses in Scholarly Articles

Edward barroga.

1 Department of General Education, Graduate School of Nursing Science, St. Luke’s International University, Tokyo, Japan.

Glafera Janet Matanguihan

2 Department of Biological Sciences, Messiah University, Mechanicsburg, PA, USA.

The development of research questions and the subsequent hypotheses are prerequisites to defining the main research purpose and specific objectives of a study. Consequently, these objectives determine the study design and research outcome. The development of research questions is a process based on knowledge of current trends, cutting-edge studies, and technological advances in the research field. Excellent research questions are focused and require a comprehensive literature search and in-depth understanding of the problem being investigated. Initially, research questions may be written as descriptive questions which could be developed into inferential questions. These questions must be specific and concise to provide a clear foundation for developing hypotheses. Hypotheses are more formal predictions about the research outcomes. These specify the possible results that may or may not be expected regarding the relationship between groups. Thus, research questions and hypotheses clarify the main purpose and specific objectives of the study, which in turn dictate the design of the study, its direction, and outcome. Studies developed from good research questions and hypotheses will have trustworthy outcomes with wide-ranging social and health implications.

INTRODUCTION

Scientific research is usually initiated by posing evidenced-based research questions which are then explicitly restated as hypotheses. 1 , 2 The hypotheses provide directions to guide the study, solutions, explanations, and expected results. 3 , 4 Both research questions and hypotheses are essentially formulated based on conventional theories and real-world processes, which allow the inception of novel studies and the ethical testing of ideas. 5 , 6

It is crucial to have knowledge of both quantitative and qualitative research 2 as both types of research involve writing research questions and hypotheses. 7 However, these crucial elements of research are sometimes overlooked; if not overlooked, then framed without the forethought and meticulous attention it needs. Planning and careful consideration are needed when developing quantitative or qualitative research, particularly when conceptualizing research questions and hypotheses. 4

There is a continuing need to support researchers in the creation of innovative research questions and hypotheses, as well as for journal articles that carefully review these elements. 1 When research questions and hypotheses are not carefully thought of, unethical studies and poor outcomes usually ensue. Carefully formulated research questions and hypotheses define well-founded objectives, which in turn determine the appropriate design, course, and outcome of the study. This article then aims to discuss in detail the various aspects of crafting research questions and hypotheses, with the goal of guiding researchers as they develop their own. Examples from the authors and peer-reviewed scientific articles in the healthcare field are provided to illustrate key points.

DEFINITIONS AND RELATIONSHIP OF RESEARCH QUESTIONS AND HYPOTHESES

A research question is what a study aims to answer after data analysis and interpretation. The answer is written in length in the discussion section of the paper. Thus, the research question gives a preview of the different parts and variables of the study meant to address the problem posed in the research question. 1 An excellent research question clarifies the research writing while facilitating understanding of the research topic, objective, scope, and limitations of the study. 5

On the other hand, a research hypothesis is an educated statement of an expected outcome. This statement is based on background research and current knowledge. 8 , 9 The research hypothesis makes a specific prediction about a new phenomenon 10 or a formal statement on the expected relationship between an independent variable and a dependent variable. 3 , 11 It provides a tentative answer to the research question to be tested or explored. 4

Hypotheses employ reasoning to predict a theory-based outcome. 10 These can also be developed from theories by focusing on components of theories that have not yet been observed. 10 The validity of hypotheses is often based on the testability of the prediction made in a reproducible experiment. 8

Conversely, hypotheses can also be rephrased as research questions. Several hypotheses based on existing theories and knowledge may be needed to answer a research question. Developing ethical research questions and hypotheses creates a research design that has logical relationships among variables. These relationships serve as a solid foundation for the conduct of the study. 4 , 11 Haphazardly constructed research questions can result in poorly formulated hypotheses and improper study designs, leading to unreliable results. Thus, the formulations of relevant research questions and verifiable hypotheses are crucial when beginning research. 12

CHARACTERISTICS OF GOOD RESEARCH QUESTIONS AND HYPOTHESES

Excellent research questions are specific and focused. These integrate collective data and observations to confirm or refute the subsequent hypotheses. Well-constructed hypotheses are based on previous reports and verify the research context. These are realistic, in-depth, sufficiently complex, and reproducible. More importantly, these hypotheses can be addressed and tested. 13

There are several characteristics of well-developed hypotheses. Good hypotheses are 1) empirically testable 7 , 10 , 11 , 13 ; 2) backed by preliminary evidence 9 ; 3) testable by ethical research 7 , 9 ; 4) based on original ideas 9 ; 5) have evidenced-based logical reasoning 10 ; and 6) can be predicted. 11 Good hypotheses can infer ethical and positive implications, indicating the presence of a relationship or effect relevant to the research theme. 7 , 11 These are initially developed from a general theory and branch into specific hypotheses by deductive reasoning. In the absence of a theory to base the hypotheses, inductive reasoning based on specific observations or findings form more general hypotheses. 10

TYPES OF RESEARCH QUESTIONS AND HYPOTHESES

Research questions and hypotheses are developed according to the type of research, which can be broadly classified into quantitative and qualitative research. We provide a summary of the types of research questions and hypotheses under quantitative and qualitative research categories in Table 1 .

Research questions in quantitative research

In quantitative research, research questions inquire about the relationships among variables being investigated and are usually framed at the start of the study. These are precise and typically linked to the subject population, dependent and independent variables, and research design. 1 Research questions may also attempt to describe the behavior of a population in relation to one or more variables, or describe the characteristics of variables to be measured ( descriptive research questions ). 1 , 5 , 14 These questions may also aim to discover differences between groups within the context of an outcome variable ( comparative research questions ), 1 , 5 , 14 or elucidate trends and interactions among variables ( relationship research questions ). 1 , 5 We provide examples of descriptive, comparative, and relationship research questions in quantitative research in Table 2 .

Hypotheses in quantitative research

In quantitative research, hypotheses predict the expected relationships among variables. 15 Relationships among variables that can be predicted include 1) between a single dependent variable and a single independent variable ( simple hypothesis ) or 2) between two or more independent and dependent variables ( complex hypothesis ). 4 , 11 Hypotheses may also specify the expected direction to be followed and imply an intellectual commitment to a particular outcome ( directional hypothesis ) 4 . On the other hand, hypotheses may not predict the exact direction and are used in the absence of a theory, or when findings contradict previous studies ( non-directional hypothesis ). 4 In addition, hypotheses can 1) define interdependency between variables ( associative hypothesis ), 4 2) propose an effect on the dependent variable from manipulation of the independent variable ( causal hypothesis ), 4 3) state a negative relationship between two variables ( null hypothesis ), 4 , 11 , 15 4) replace the working hypothesis if rejected ( alternative hypothesis ), 15 explain the relationship of phenomena to possibly generate a theory ( working hypothesis ), 11 5) involve quantifiable variables that can be tested statistically ( statistical hypothesis ), 11 6) or express a relationship whose interlinks can be verified logically ( logical hypothesis ). 11 We provide examples of simple, complex, directional, non-directional, associative, causal, null, alternative, working, statistical, and logical hypotheses in quantitative research, as well as the definition of quantitative hypothesis-testing research in Table 3 .

Research questions in qualitative research

Unlike research questions in quantitative research, research questions in qualitative research are usually continuously reviewed and reformulated. The central question and associated subquestions are stated more than the hypotheses. 15 The central question broadly explores a complex set of factors surrounding the central phenomenon, aiming to present the varied perspectives of participants. 15

There are varied goals for which qualitative research questions are developed. These questions can function in several ways, such as to 1) identify and describe existing conditions ( contextual research question s); 2) describe a phenomenon ( descriptive research questions ); 3) assess the effectiveness of existing methods, protocols, theories, or procedures ( evaluation research questions ); 4) examine a phenomenon or analyze the reasons or relationships between subjects or phenomena ( explanatory research questions ); or 5) focus on unknown aspects of a particular topic ( exploratory research questions ). 5 In addition, some qualitative research questions provide new ideas for the development of theories and actions ( generative research questions ) or advance specific ideologies of a position ( ideological research questions ). 1 Other qualitative research questions may build on a body of existing literature and become working guidelines ( ethnographic research questions ). Research questions may also be broadly stated without specific reference to the existing literature or a typology of questions ( phenomenological research questions ), may be directed towards generating a theory of some process ( grounded theory questions ), or may address a description of the case and the emerging themes ( qualitative case study questions ). 15 We provide examples of contextual, descriptive, evaluation, explanatory, exploratory, generative, ideological, ethnographic, phenomenological, grounded theory, and qualitative case study research questions in qualitative research in Table 4 , and the definition of qualitative hypothesis-generating research in Table 5 .

Qualitative studies usually pose at least one central research question and several subquestions starting with How or What . These research questions use exploratory verbs such as explore or describe . These also focus on one central phenomenon of interest, and may mention the participants and research site. 15

Hypotheses in qualitative research

Hypotheses in qualitative research are stated in the form of a clear statement concerning the problem to be investigated. Unlike in quantitative research where hypotheses are usually developed to be tested, qualitative research can lead to both hypothesis-testing and hypothesis-generating outcomes. 2 When studies require both quantitative and qualitative research questions, this suggests an integrative process between both research methods wherein a single mixed-methods research question can be developed. 1

FRAMEWORKS FOR DEVELOPING RESEARCH QUESTIONS AND HYPOTHESES

Research questions followed by hypotheses should be developed before the start of the study. 1 , 12 , 14 It is crucial to develop feasible research questions on a topic that is interesting to both the researcher and the scientific community. This can be achieved by a meticulous review of previous and current studies to establish a novel topic. Specific areas are subsequently focused on to generate ethical research questions. The relevance of the research questions is evaluated in terms of clarity of the resulting data, specificity of the methodology, objectivity of the outcome, depth of the research, and impact of the study. 1 , 5 These aspects constitute the FINER criteria (i.e., Feasible, Interesting, Novel, Ethical, and Relevant). 1 Clarity and effectiveness are achieved if research questions meet the FINER criteria. In addition to the FINER criteria, Ratan et al. described focus, complexity, novelty, feasibility, and measurability for evaluating the effectiveness of research questions. 14

The PICOT and PEO frameworks are also used when developing research questions. 1 The following elements are addressed in these frameworks, PICOT: P-population/patients/problem, I-intervention or indicator being studied, C-comparison group, O-outcome of interest, and T-timeframe of the study; PEO: P-population being studied, E-exposure to preexisting conditions, and O-outcome of interest. 1 Research questions are also considered good if these meet the “FINERMAPS” framework: Feasible, Interesting, Novel, Ethical, Relevant, Manageable, Appropriate, Potential value/publishable, and Systematic. 14

As we indicated earlier, research questions and hypotheses that are not carefully formulated result in unethical studies or poor outcomes. To illustrate this, we provide some examples of ambiguous research question and hypotheses that result in unclear and weak research objectives in quantitative research ( Table 6 ) 16 and qualitative research ( Table 7 ) 17 , and how to transform these ambiguous research question(s) and hypothesis(es) into clear and good statements.

a These statements were composed for comparison and illustrative purposes only.

b These statements are direct quotes from Higashihara and Horiuchi. 16

a This statement is a direct quote from Shimoda et al. 17

The other statements were composed for comparison and illustrative purposes only.

CONSTRUCTING RESEARCH QUESTIONS AND HYPOTHESES

To construct effective research questions and hypotheses, it is very important to 1) clarify the background and 2) identify the research problem at the outset of the research, within a specific timeframe. 9 Then, 3) review or conduct preliminary research to collect all available knowledge about the possible research questions by studying theories and previous studies. 18 Afterwards, 4) construct research questions to investigate the research problem. Identify variables to be accessed from the research questions 4 and make operational definitions of constructs from the research problem and questions. Thereafter, 5) construct specific deductive or inductive predictions in the form of hypotheses. 4 Finally, 6) state the study aims . This general flow for constructing effective research questions and hypotheses prior to conducting research is shown in Fig. 1 .

Research questions are used more frequently in qualitative research than objectives or hypotheses. 3 These questions seek to discover, understand, explore or describe experiences by asking “What” or “How.” The questions are open-ended to elicit a description rather than to relate variables or compare groups. The questions are continually reviewed, reformulated, and changed during the qualitative study. 3 Research questions are also used more frequently in survey projects than hypotheses in experiments in quantitative research to compare variables and their relationships.

Hypotheses are constructed based on the variables identified and as an if-then statement, following the template, ‘If a specific action is taken, then a certain outcome is expected.’ At this stage, some ideas regarding expectations from the research to be conducted must be drawn. 18 Then, the variables to be manipulated (independent) and influenced (dependent) are defined. 4 Thereafter, the hypothesis is stated and refined, and reproducible data tailored to the hypothesis are identified, collected, and analyzed. 4 The hypotheses must be testable and specific, 18 and should describe the variables and their relationships, the specific group being studied, and the predicted research outcome. 18 Hypotheses construction involves a testable proposition to be deduced from theory, and independent and dependent variables to be separated and measured separately. 3 Therefore, good hypotheses must be based on good research questions constructed at the start of a study or trial. 12

In summary, research questions are constructed after establishing the background of the study. Hypotheses are then developed based on the research questions. Thus, it is crucial to have excellent research questions to generate superior hypotheses. In turn, these would determine the research objectives and the design of the study, and ultimately, the outcome of the research. 12 Algorithms for building research questions and hypotheses are shown in Fig. 2 for quantitative research and in Fig. 3 for qualitative research.

EXAMPLES OF RESEARCH QUESTIONS FROM PUBLISHED ARTICLES

• EXAMPLE 1. Descriptive research question (quantitative research)
• - Presents research variables to be assessed (distinct phenotypes and subphenotypes)
• “BACKGROUND: Since COVID-19 was identified, its clinical and biological heterogeneity has been recognized. Identifying COVID-19 phenotypes might help guide basic, clinical, and translational research efforts.
• RESEARCH QUESTION: Does the clinical spectrum of patients with COVID-19 contain distinct phenotypes and subphenotypes? ” 19
• EXAMPLE 2. Relationship research question (quantitative research)
• - Shows interactions between dependent variable (static postural control) and independent variable (peripheral visual field loss)
• “Background: Integration of visual, vestibular, and proprioceptive sensations contributes to postural control. People with peripheral visual field loss have serious postural instability. However, the directional specificity of postural stability and sensory reweighting caused by gradual peripheral visual field loss remain unclear.
• Research question: What are the effects of peripheral visual field loss on static postural control ?” 20
• EXAMPLE 3. Comparative research question (quantitative research)
• - Clarifies the difference among groups with an outcome variable (patients enrolled in COMPERA with moderate PH or severe PH in COPD) and another group without the outcome variable (patients with idiopathic pulmonary arterial hypertension (IPAH))
• “BACKGROUND: Pulmonary hypertension (PH) in COPD is a poorly investigated clinical condition.
• RESEARCH QUESTION: Which factors determine the outcome of PH in COPD?
• STUDY DESIGN AND METHODS: We analyzed the characteristics and outcome of patients enrolled in the Comparative, Prospective Registry of Newly Initiated Therapies for Pulmonary Hypertension (COMPERA) with moderate or severe PH in COPD as defined during the 6th PH World Symposium who received medical therapy for PH and compared them with patients with idiopathic pulmonary arterial hypertension (IPAH) .” 21
• EXAMPLE 4. Exploratory research question (qualitative research)
• - Explores areas that have not been fully investigated (perspectives of families and children who receive care in clinic-based child obesity treatment) to have a deeper understanding of the research problem
• “Problem: Interventions for children with obesity lead to only modest improvements in BMI and long-term outcomes, and data are limited on the perspectives of families of children with obesity in clinic-based treatment. This scoping review seeks to answer the question: What is known about the perspectives of families and children who receive care in clinic-based child obesity treatment? This review aims to explore the scope of perspectives reported by families of children with obesity who have received individualized outpatient clinic-based obesity treatment.” 22
• EXAMPLE 5. Relationship research question (quantitative research)
• - Defines interactions between dependent variable (use of ankle strategies) and independent variable (changes in muscle tone)
• “Background: To maintain an upright standing posture against external disturbances, the human body mainly employs two types of postural control strategies: “ankle strategy” and “hip strategy.” While it has been reported that the magnitude of the disturbance alters the use of postural control strategies, it has not been elucidated how the level of muscle tone, one of the crucial parameters of bodily function, determines the use of each strategy. We have previously confirmed using forward dynamics simulations of human musculoskeletal models that an increased muscle tone promotes the use of ankle strategies. The objective of the present study was to experimentally evaluate a hypothesis: an increased muscle tone promotes the use of ankle strategies. Research question: Do changes in the muscle tone affect the use of ankle strategies ?” 23

EXAMPLES OF HYPOTHESES IN PUBLISHED ARTICLES

• EXAMPLE 1. Working hypothesis (quantitative research)
• - A hypothesis that is initially accepted for further research to produce a feasible theory
• “As fever may have benefit in shortening the duration of viral illness, it is plausible to hypothesize that the antipyretic efficacy of ibuprofen may be hindering the benefits of a fever response when taken during the early stages of COVID-19 illness .” 24
• “In conclusion, it is plausible to hypothesize that the antipyretic efficacy of ibuprofen may be hindering the benefits of a fever response . The difference in perceived safety of these agents in COVID-19 illness could be related to the more potent efficacy to reduce fever with ibuprofen compared to acetaminophen. Compelling data on the benefit of fever warrant further research and review to determine when to treat or withhold ibuprofen for early stage fever for COVID-19 and other related viral illnesses .” 24
• EXAMPLE 2. Exploratory hypothesis (qualitative research)
• - Explores particular areas deeper to clarify subjective experience and develop a formal hypothesis potentially testable in a future quantitative approach
• “We hypothesized that when thinking about a past experience of help-seeking, a self distancing prompt would cause increased help-seeking intentions and more favorable help-seeking outcome expectations .” 25
• “Conclusion
• Although a priori hypotheses were not supported, further research is warranted as results indicate the potential for using self-distancing approaches to increasing help-seeking among some people with depressive symptomatology.” 25
• EXAMPLE 3. Hypothesis-generating research to establish a framework for hypothesis testing (qualitative research)
• “We hypothesize that compassionate care is beneficial for patients (better outcomes), healthcare systems and payers (lower costs), and healthcare providers (lower burnout). ” 26
• Compassionomics is the branch of knowledge and scientific study of the effects of compassionate healthcare. Our main hypotheses are that compassionate healthcare is beneficial for (1) patients, by improving clinical outcomes, (2) healthcare systems and payers, by supporting financial sustainability, and (3) HCPs, by lowering burnout and promoting resilience and well-being. The purpose of this paper is to establish a scientific framework for testing the hypotheses above . If these hypotheses are confirmed through rigorous research, compassionomics will belong in the science of evidence-based medicine, with major implications for all healthcare domains.” 26
• EXAMPLE 4. Statistical hypothesis (quantitative research)
• - An assumption is made about the relationship among several population characteristics ( gender differences in sociodemographic and clinical characteristics of adults with ADHD ). Validity is tested by statistical experiment or analysis ( chi-square test, Students t-test, and logistic regression analysis)
• “Our research investigated gender differences in sociodemographic and clinical characteristics of adults with ADHD in a Japanese clinical sample. Due to unique Japanese cultural ideals and expectations of women's behavior that are in opposition to ADHD symptoms, we hypothesized that women with ADHD experience more difficulties and present more dysfunctions than men . We tested the following hypotheses: first, women with ADHD have more comorbidities than men with ADHD; second, women with ADHD experience more social hardships than men, such as having less full-time employment and being more likely to be divorced.” 27
• “Statistical Analysis
• ( text omitted ) Between-gender comparisons were made using the chi-squared test for categorical variables and Students t-test for continuous variables…( text omitted ). A logistic regression analysis was performed for employment status, marital status, and comorbidity to evaluate the independent effects of gender on these dependent variables.” 27

EXAMPLES OF HYPOTHESIS AS WRITTEN IN PUBLISHED ARTICLES IN RELATION TO OTHER PARTS

• EXAMPLE 1. Background, hypotheses, and aims are provided
• “Pregnant women need skilled care during pregnancy and childbirth, but that skilled care is often delayed in some countries …( text omitted ). The focused antenatal care (FANC) model of WHO recommends that nurses provide information or counseling to all pregnant women …( text omitted ). Job aids are visual support materials that provide the right kind of information using graphics and words in a simple and yet effective manner. When nurses are not highly trained or have many work details to attend to, these job aids can serve as a content reminder for the nurses and can be used for educating their patients (Jennings, Yebadokpo, Affo, & Agbogbe, 2010) ( text omitted ). Importantly, additional evidence is needed to confirm how job aids can further improve the quality of ANC counseling by health workers in maternal care …( text omitted )” 28
• “ This has led us to hypothesize that the quality of ANC counseling would be better if supported by job aids. Consequently, a better quality of ANC counseling is expected to produce higher levels of awareness concerning the danger signs of pregnancy and a more favorable impression of the caring behavior of nurses .” 28
• “This study aimed to examine the differences in the responses of pregnant women to a job aid-supported intervention during ANC visit in terms of 1) their understanding of the danger signs of pregnancy and 2) their impression of the caring behaviors of nurses to pregnant women in rural Tanzania.” 28
• EXAMPLE 2. Background, hypotheses, and aims are provided
• “We conducted a two-arm randomized controlled trial (RCT) to evaluate and compare changes in salivary cortisol and oxytocin levels of first-time pregnant women between experimental and control groups. The women in the experimental group touched and held an infant for 30 min (experimental intervention protocol), whereas those in the control group watched a DVD movie of an infant (control intervention protocol). The primary outcome was salivary cortisol level and the secondary outcome was salivary oxytocin level.” 29
• “ We hypothesize that at 30 min after touching and holding an infant, the salivary cortisol level will significantly decrease and the salivary oxytocin level will increase in the experimental group compared with the control group .” 29
• EXAMPLE 3. Background, aim, and hypothesis are provided
• “In countries where the maternal mortality ratio remains high, antenatal education to increase Birth Preparedness and Complication Readiness (BPCR) is considered one of the top priorities [1]. BPCR includes birth plans during the antenatal period, such as the birthplace, birth attendant, transportation, health facility for complications, expenses, and birth materials, as well as family coordination to achieve such birth plans. In Tanzania, although increasing, only about half of all pregnant women attend an antenatal clinic more than four times [4]. Moreover, the information provided during antenatal care (ANC) is insufficient. In the resource-poor settings, antenatal group education is a potential approach because of the limited time for individual counseling at antenatal clinics.” 30
• “This study aimed to evaluate an antenatal group education program among pregnant women and their families with respect to birth-preparedness and maternal and infant outcomes in rural villages of Tanzania.” 30
• “ The study hypothesis was if Tanzanian pregnant women and their families received a family-oriented antenatal group education, they would (1) have a higher level of BPCR, (2) attend antenatal clinic four or more times, (3) give birth in a health facility, (4) have less complications of women at birth, and (5) have less complications and deaths of infants than those who did not receive the education .” 30

Research questions and hypotheses are crucial components to any type of research, whether quantitative or qualitative. These questions should be developed at the very beginning of the study. Excellent research questions lead to superior hypotheses, which, like a compass, set the direction of research, and can often determine the successful conduct of the study. Many research studies have floundered because the development of research questions and subsequent hypotheses was not given the thought and meticulous attention needed. The development of research questions and hypotheses is an iterative process based on extensive knowledge of the literature and insightful grasp of the knowledge gap. Focused, concise, and specific research questions provide a strong foundation for constructing hypotheses which serve as formal predictions about the research outcomes. Research questions and hypotheses are crucial elements of research that should not be overlooked. They should be carefully thought of and constructed when planning research. This avoids unethical studies and poor outcomes by defining well-founded objectives that determine the design, course, and outcome of the study.

Disclosure: The authors have no potential conflicts of interest to disclose.

Author Contributions:

• Conceptualization: Barroga E, Matanguihan GJ.
• Methodology: Barroga E, Matanguihan GJ.
• Writing - original draft: Barroga E, Matanguihan GJ.
• Writing - review & editing: Barroga E, Matanguihan GJ.

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Quantitative methods emphasize objective measurements and the statistical, mathematical, or numerical analysis of data collected through polls, questionnaires, and surveys, or by manipulating pre-existing statistical data using computational techniques . Quantitative research focuses on gathering numerical data and generalizing it across groups of people or to explain a particular phenomenon.

Babbie, Earl R. The Practice of Social Research . 12th ed. Belmont, CA: Wadsworth Cengage, 2010; Muijs, Daniel. Doing Quantitative Research in Education with SPSS . 2nd edition. London: SAGE Publications, 2010.

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Characteristics of Quantitative Research

Your goal in conducting quantitative research study is to determine the relationship between one thing [an independent variable] and another [a dependent or outcome variable] within a population. Quantitative research designs are either descriptive [subjects usually measured once] or experimental [subjects measured before and after a treatment]. A descriptive study establishes only associations between variables; an experimental study establishes causality.

Quantitative research deals in numbers, logic, and an objective stance. Quantitative research focuses on numeric and unchanging data and detailed, convergent reasoning rather than divergent reasoning [i.e., the generation of a variety of ideas about a research problem in a spontaneous, free-flowing manner].

Its main characteristics are :

• The data is usually gathered using structured research instruments.
• The results are based on larger sample sizes that are representative of the population.
• The research study can usually be replicated or repeated, given its high reliability.
• Researcher has a clearly defined research question to which objective answers are sought.
• All aspects of the study are carefully designed before data is collected.
• Data are in the form of numbers and statistics, often arranged in tables, charts, figures, or other non-textual forms.
• Project can be used to generalize concepts more widely, predict future results, or investigate causal relationships.
• Researcher uses tools, such as questionnaires or computer software, to collect numerical data.

The overarching aim of a quantitative research study is to classify features, count them, and construct statistical models in an attempt to explain what is observed.

  Things to keep in mind when reporting the results of a study using quantitative methods :

• Explain the data collected and their statistical treatment as well as all relevant results in relation to the research problem you are investigating. Interpretation of results is not appropriate in this section.
• Report unanticipated events that occurred during your data collection. Explain how the actual analysis differs from the planned analysis. Explain your handling of missing data and why any missing data does not undermine the validity of your analysis.
• Explain the techniques you used to "clean" your data set.
• Choose a minimally sufficient statistical procedure ; provide a rationale for its use and a reference for it. Specify any computer programs used.
• Describe the assumptions for each procedure and the steps you took to ensure that they were not violated.
• When using inferential statistics , provide the descriptive statistics, confidence intervals, and sample sizes for each variable as well as the value of the test statistic, its direction, the degrees of freedom, and the significance level [report the actual p value].
• Avoid inferring causality , particularly in nonrandomized designs or without further experimentation.
• Use tables to provide exact values ; use figures to convey global effects. Keep figures small in size; include graphic representations of confidence intervals whenever possible.
• Always tell the reader what to look for in tables and figures .

NOTE:   When using pre-existing statistical data gathered and made available by anyone other than yourself [e.g., government agency], you still must report on the methods that were used to gather the data and describe any missing data that exists and, if there is any, provide a clear explanation why the missing data does not undermine the validity of your final analysis.

Babbie, Earl R. The Practice of Social Research . 12th ed. Belmont, CA: Wadsworth Cengage, 2010; Brians, Craig Leonard et al. Empirical Political Analysis: Quantitative and Qualitative Research Methods . 8th ed. Boston, MA: Longman, 2011; McNabb, David E. Research Methods in Public Administration and Nonprofit Management: Quantitative and Qualitative Approaches . 2nd ed. Armonk, NY: M.E. Sharpe, 2008; Quantitative Research Methods. Writing@CSU. Colorado State University; Singh, Kultar. Quantitative Social Research Methods . Los Angeles, CA: Sage, 2007.

Basic Research Design for Quantitative Studies

Before designing a quantitative research study, you must decide whether it will be descriptive or experimental because this will dictate how you gather, analyze, and interpret the results. A descriptive study is governed by the following rules: subjects are generally measured once; the intention is to only establish associations between variables; and, the study may include a sample population of hundreds or thousands of subjects to ensure that a valid estimate of a generalized relationship between variables has been obtained. An experimental design includes subjects measured before and after a particular treatment, the sample population may be very small and purposefully chosen, and it is intended to establish causality between variables. Introduction The introduction to a quantitative study is usually written in the present tense and from the third person point of view. It covers the following information:

• Identifies the research problem -- as with any academic study, you must state clearly and concisely the research problem being investigated.
• Reviews the literature -- review scholarship on the topic, synthesizing key themes and, if necessary, noting studies that have used similar methods of inquiry and analysis. Note where key gaps exist and how your study helps to fill these gaps or clarifies existing knowledge.
• Describes the theoretical framework -- provide an outline of the theory or hypothesis underpinning your study. If necessary, define unfamiliar or complex terms, concepts, or ideas and provide the appropriate background information to place the research problem in proper context [e.g., historical, cultural, economic, etc.].

Methodology The methods section of a quantitative study should describe how each objective of your study will be achieved. Be sure to provide enough detail to enable the reader can make an informed assessment of the methods being used to obtain results associated with the research problem. The methods section should be presented in the past tense.

• Study population and sampling -- where did the data come from; how robust is it; note where gaps exist or what was excluded. Note the procedures used for their selection;
• Data collection – describe the tools and methods used to collect information and identify the variables being measured; describe the methods used to obtain the data; and, note if the data was pre-existing [i.e., government data] or you gathered it yourself. If you gathered it yourself, describe what type of instrument you used and why. Note that no data set is perfect--describe any limitations in methods of gathering data.
• Data analysis -- describe the procedures for processing and analyzing the data. If appropriate, describe the specific instruments of analysis used to study each research objective, including mathematical techniques and the type of computer software used to manipulate the data.

Results The finding of your study should be written objectively and in a succinct and precise format. In quantitative studies, it is common to use graphs, tables, charts, and other non-textual elements to help the reader understand the data. Make sure that non-textual elements do not stand in isolation from the text but are being used to supplement the overall description of the results and to help clarify key points being made. Further information about how to effectively present data using charts and graphs can be found here .

• Statistical analysis -- how did you analyze the data? What were the key findings from the data? The findings should be present in a logical, sequential order. Describe but do not interpret these trends or negative results; save that for the discussion section. The results should be presented in the past tense.

Discussion Discussions should be analytic, logical, and comprehensive. The discussion should meld together your findings in relation to those identified in the literature review, and placed within the context of the theoretical framework underpinning the study. The discussion should be presented in the present tense.

• Interpretation of results -- reiterate the research problem being investigated and compare and contrast the findings with the research questions underlying the study. Did they affirm predicted outcomes or did the data refute it?
• Description of trends, comparison of groups, or relationships among variables -- describe any trends that emerged from your analysis and explain all unanticipated and statistical insignificant findings.
• Discussion of implications – what is the meaning of your results? Highlight key findings based on the overall results and note findings that you believe are important. How have the results helped fill gaps in understanding the research problem?
• Limitations -- describe any limitations or unavoidable bias in your study and, if necessary, note why these limitations did not inhibit effective interpretation of the results.

Conclusion End your study by to summarizing the topic and provide a final comment and assessment of the study.

• Summary of findings – synthesize the answers to your research questions. Do not report any statistical data here; just provide a narrative summary of the key findings and describe what was learned that you did not know before conducting the study.
• Recommendations – if appropriate to the aim of the assignment, tie key findings with policy recommendations or actions to be taken in practice.
• Future research – note the need for future research linked to your study’s limitations or to any remaining gaps in the literature that were not addressed in your study.

Black, Thomas R. Doing Quantitative Research in the Social Sciences: An Integrated Approach to Research Design, Measurement and Statistics . London: Sage, 1999; Gay,L. R. and Peter Airasain. Educational Research: Competencies for Analysis and Applications . 7th edition. Upper Saddle River, NJ: Merril Prentice Hall, 2003; Hector, Anestine. An Overview of Quantitative Research in Composition and TESOL . Department of English, Indiana University of Pennsylvania; Hopkins, Will G. “Quantitative Research Design.” Sportscience 4, 1 (2000); "A Strategy for Writing Up Research Results. The Structure, Format, Content, and Style of a Journal-Style Scientific Paper." Department of Biology. Bates College; Nenty, H. Johnson. "Writing a Quantitative Research Thesis." International Journal of Educational Science 1 (2009): 19-32; Ouyang, Ronghua (John). Basic Inquiry of Quantitative Research . Kennesaw State University.

Strengths of Using Quantitative Methods

Quantitative researchers try to recognize and isolate specific variables contained within the study framework, seek correlation, relationships and causality, and attempt to control the environment in which the data is collected to avoid the risk of variables, other than the one being studied, accounting for the relationships identified.

Among the specific strengths of using quantitative methods to study social science research problems:

• Allows for a broader study, involving a greater number of subjects, and enhancing the generalization of the results;
• Allows for greater objectivity and accuracy of results. Generally, quantitative methods are designed to provide summaries of data that support generalizations about the phenomenon under study. In order to accomplish this, quantitative research usually involves few variables and many cases, and employs prescribed procedures to ensure validity and reliability;
• Applying well established standards means that the research can be replicated, and then analyzed and compared with similar studies;
• You can summarize vast sources of information and make comparisons across categories and over time; and,
• Personal bias can be avoided by keeping a 'distance' from participating subjects and using accepted computational techniques .

Babbie, Earl R. The Practice of Social Research . 12th ed. Belmont, CA: Wadsworth Cengage, 2010; Brians, Craig Leonard et al. Empirical Political Analysis: Quantitative and Qualitative Research Methods . 8th ed. Boston, MA: Longman, 2011; McNabb, David E. Research Methods in Public Administration and Nonprofit Management: Quantitative and Qualitative Approaches . 2nd ed. Armonk, NY: M.E. Sharpe, 2008; Singh, Kultar. Quantitative Social Research Methods . Los Angeles, CA: Sage, 2007.

Limitations of Using Quantitative Methods

Quantitative methods presume to have an objective approach to studying research problems, where data is controlled and measured, to address the accumulation of facts, and to determine the causes of behavior. As a consequence, the results of quantitative research may be statistically significant but are often humanly insignificant.

Some specific limitations associated with using quantitative methods to study research problems in the social sciences include:

• Quantitative data is more efficient and able to test hypotheses, but may miss contextual detail;
• Uses a static and rigid approach and so employs an inflexible process of discovery;
• The development of standard questions by researchers can lead to "structural bias" and false representation, where the data actually reflects the view of the researcher instead of the participating subject;
• Results provide less detail on behavior, attitudes, and motivation;
• Researcher may collect a much narrower and sometimes superficial dataset;
• Results are limited as they provide numerical descriptions rather than detailed narrative and generally provide less elaborate accounts of human perception;
• The research is often carried out in an unnatural, artificial environment so that a level of control can be applied to the exercise. This level of control might not normally be in place in the real world thus yielding "laboratory results" as opposed to "real world results"; and,
• Preset answers will not necessarily reflect how people really feel about a subject and, in some cases, might just be the closest match to the preconceived hypothesis.

Research Tip

Finding Examples of How to Apply Different Types of Research Methods

SAGE publications is a major publisher of studies about how to design and conduct research in the social and behavioral sciences. Their SAGE Research Methods Online and Cases database includes contents from books, articles, encyclopedias, handbooks, and videos covering social science research design and methods including the complete Little Green Book Series of Quantitative Applications in the Social Sciences and the Little Blue Book Series of Qualitative Research techniques. The database also includes case studies outlining the research methods used in real research projects. This is an excellent source for finding definitions of key terms and descriptions of research design and practice, techniques of data gathering, analysis, and reporting, and information about theories of research [e.g., grounded theory]. The database covers both qualitative and quantitative research methods as well as mixed methods approaches to conducting research.

SAGE Research Methods Online and Cases

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Writing Quantitative Research Studies

• Reference work entry
• First Online: 13 January 2019
• Cite this reference work entry

• Ankur Singh 2 ,
• Adyya Gupta 3 &
• Karen G. Peres 4  

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Summarizing quantitative data and its effective presentation and discussion can be challenging for students and researchers. This chapter provides a framework for adequately reporting findings from quantitative analysis in a research study for those contemplating to write a research paper. The rationale underpinning the reporting methods to maintain the credibility and integrity of quantitative studies is outlined. Commonly used terminologies in empirical studies are defined and discussed with suitable examples. Key elements that build consistency between different sections (background, methods, results, and the discussion) of a research study using quantitative methods in a journal article are explicated. Specifically, recommended standard guidelines for randomized controlled trials and observational studies for reporting and discussion of findings from quantitative studies are elaborated. Key aspects of methodology that include describing the study population, sampling strategy, data collection methods, measurements/variables, and statistical analysis which informs the quality of a study from the reviewer’s perspective are described. Effective use of references in the methods section to strengthen the rationale behind specific statistical techniques and choice of measures has been highlighted with examples. Identifying ways in which data can be most succinctly and effectively summarized in tables and graphs according to their suitability and purpose of information is also detailed in this chapter. Strategies to present and discuss the quantitative findings in a structured discussion section are also provided. Overall, the chapter provides the readers with a comprehensive set of tools to identify key strategies to be considered when reporting quantitative research.

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Quantitative Research

Case Study 3: Application of Quantitative Methodology

Bhaumik S, Arora M, Singh A, Sargent JD. Impact of entertainment media smoking on adolescent smoking behaviours. Cochrane Database Syst Rev. 2015;6:1–12. https://doi.org/10.1002/14651858.CD011720 .

Article   Google Scholar  

Dickersin K, Manheimer E, Wieland S, Robinson KA, Lefebvre C, McDonald S. Development of the Cochrane Collaboration’s CENTRAL register of controlled clinical trials. Eval Health Prof. 2002;25(1):38–64.

Google Scholar  

Docherty M, Smith R. The case for structuring the discussion of scientific papers: much the same as that for structuring abstracts. Br Med J. 1999;318(7193):1224–5.

Greenland S, Pearl J, Robins JM. Causal diagrams for epidemiologic research. Epidemiology. 1999;10(1):37–48.

Horton R. The rhetoric of research. Br Med J. 1995;310(6985):985–7.

Kool B, Ziersch A, Robinson P, Wolfenden L, Lowe JB. The ‘Seven deadly sins’ of rejected papers. Aust N Z J Public Health. 2016;40(1):3–4.

Mannocci A, Saulle R, Colamesta V, D’Aguanno S, Giraldi G, Maffongelli E, et al. What is the impact of reporting guidelines on public health journals in Europe? The case of STROBE, CONSORT and PRISMA. J Public Health. 2015;37(4):737–40.

Rothwell PM. External validity of randomised controlled trials: “to whom do the results of this trial apply?”. Lancet. 2005;365(9453):82–93.

Schulz KF, Altman DG, Moher D. CONSORT 2010 statement: updated guidelines for reporting parallel group randomised trials. PLoS Med. 2010;7(3):e1000251.

Szklo M. Quality of scientific articles. Rev Saude Publica. 2006;40 Spec no:30–5.

Vandenbroucke JP, von Elm E, Altman DG, Gotzsche PC, Mulrow CD, Pocock SJ, et al. Strengthening the reporting of observational studies in epidemiology (STROBE): explanation and elaboration. PLoS Med. 2007;4(10):e297.

Weiss NS, Koepsell TD, Psaty BM. Generalizability of the results of randomized trials. Arch Intern Med. 2008;168(2):133–5.

Singh A, Gupta A, Peres MA, Watt RG, Tsakos G, Mathur MR. Association between tooth loss and hypertension among a primarily rural middle aged and older Indian adult population. J Public Health Dent. 2016;76:198–205.

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Ankur Singh

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Adyya Gupta

Australian Research Centre for Population Oral Health (ARCPOH), Adelaide Dental School, The University of Adelaide, Adelaide, SA, Australia

Karen G. Peres

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Singh, A., Gupta, A., Peres, K.G. (2019). Writing Quantitative Research Studies. In: Liamputtong, P. (eds) Handbook of Research Methods in Health Social Sciences. Springer, Singapore. https://doi.org/10.1007/978-981-10-5251-4_117

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Example of a Quantitative Research Paper

Posted by Rene Tetzner | Sep 4, 2021 | How To Get Published | 0 |

Example of a Quantitative Research Paper for Students & Researchers This example of a quantitative research paper is designed to help students and other researchers who are learning how to write about their work. The reported research observes the behaviour of restaurant customers, and example paragraphs are combined with instructions for logical argumentation. Authors are encouraged to observe a traditional structure for organising quantitative research papers, to formulate research questions, working hypotheses and investigative tools, to report results accurately and thoroughly, and to present thoughtful interpretation and logical discussion of evidence.

The structure of the example and the nature of its contents follow the recommendations of the   Publication Manual of the American Psychological Association . This APA style calls for parenthetical author–date citations in the paper’s main text (with page numbers when material is quoted) and a final list of complete references for all sources cited, so I have given a few sample references here. Content has been kept as simple as possible to focus attention on the way in which the paper presents the research process and its results. As is the case in many research projects, the more the author learns and thinks about the topic, the more complex the issues become, and here the researcher discusses a hypothesis that proved incorrect. An APA research paper would normally include additional elements such as an abstract, keywords and perhaps tables, figures and appendices similar to those referred to in the example. These elements have been eliminated for brevity here, so do be sure to check the APA   Manual   (or any other guidelines you are following) for the necessary instructions.

Surprises at a Local “Family” Restaurant: Example Quantitative Research Paper

A quantitative research paper with that title might start with a paragraph like this:

Quaintville, located just off the main highway only five miles from the university campus, may normally be a sleepy community, but recent plans to close the only fast-food restaurant ever to grace its main street have been met with something of a public outcry. Regular clients argue that Pudgy’s Burgers fills a vital function and will be sorely missed. As the editor of the  Quaintville Times  would have it, “good old Pudgy’s is the only restaurant in Quaintville where a working family can still get a decent meal for a fair buck, and a comfortable place to eat it too, out of the winter wind where the kids can run about and play a bit” (Chapton, 2017, p. A3). On the other hand, the most outspoken of Quaintville residents in favour of the planned closure look forward to the eradication of a local eyesore and tend to consider the restaurant more of “a hazard than a benefit to the health of some of our poorest families” (“Local dive,” 2017, p. 1).

Following this opening a brief introduction to published scholarship and other issues associated with the problem would be appropriate, so here the researcher might add a paragraph or two discussing:

• A selection of recently published studies that investigate the effect of inexpensive fast-food restaurants on the health of low-income families, especially their children (Shunts, 2013; Whinner, 2015). • Fast-food restaurants that have responded to criticism about the quality of their food by offering healthy menu items. This could be enhanced with evidence that when such choices are available, they are rare purchases for many families (Parkson, 2016), particularly in small towns and rural areas (Shemble, 2017). • The interesting trend in several independent studies suggesting that families form a much smaller portion of the clientele of fast-food restaurants than anticipated.

Explaining how the current research is related to the published scholarship as well as the specific problem is vital. Here, for instance, the author might be thinking that Pudgy’s, which has healthy menu items as well as the support of so many long-term residents, will prove an exception to the trends revealed by other studies. Research questions and hypotheses should be constructed to articulate and explore that idea. Research questions, for instance, could be developed from that claim in the   Quaintville Times   as well as from the published scholarship:

• Do families constitute the majority of Pudgy’s regular clientele? • Does the restaurant offer a decent family meal for a fair price? • Do families linger in the restaurant’s comfort and warmth?> • Do children use the indoor play area provided by the restaurant?

Working hypotheses can be constructed by anticipating answers to these questions. The example paper assumes a simple hypothesis something along the lines of “Families do indeed constitute the majority of Pudgy’s clientele.” The exact opposite supposition would work as well – “Families do not constitute the majority of Pudgy’s clientele” – and so would hypotheses exploring and combining other aspects of the situation, such as “Pudgy’s healthy menu options and indoor play area are positive and appealing considerations for families” or “The comfortable atmosphere of Pudgy’s with its play area makes it much more than a restaurant for local families.”

The exact wording of your questions and hypotheses will ultimately depend on your focus and aims, but certain terms, concepts and categories may require definition to ensure precision in communicating your ideas to readers. Here, for instance, exactly what is meant by ‘a family,’ ‘a decent meal,’ ‘a fair price’ and even ‘comfortable’ could be briefly but carefully defined. A general statement about your understanding of how the current research will explore the problem, answer your questions and test your hypotheses is usually required as well, setting the stage for the more detailed Method section that follows. This statement might be something as simple as “I intend to observe the restaurant’s customers over a two-month period with the objective of learning about Pudgy’s clientele and measuring the use and value of the establishment for local families.” On the other hand, outlining your research might require a paragraph or two of introductory discussion.

Method Whether a brief general statement or a longer explanation of how the research will proceed appears among your introductory material, it is in the Method section that you should report exactly what you did to conduct your investigation, explain the conditions and controls you applied to increase the reliability and value of your research, and reveal any difficulties you encountered. For example:

My observations took place at Pudgy’s Burgers in January and February of 2018. Each session was approximately four hours long, and I aimed to obtain an equivalent number of observations for all opening hours of the week (the restaurant’s hours are listed in Table 1), but course requirements made this difficult. Tuesday and Thursday afternoons are therefore underrepresented, and observations from 1:00 pm to 5:00 pm on two consecutive Tuesdays (6 and 13 February) are the work of my classmate, Jake Jenkins. Without his assistance, I could not have met my objective of gathering observations for every opening hour of the week at least twice (Table 2 outlines the overall pattern of observation sessions). Serving staff at the restaurant assure me that I have now “seen ‘em all,” so I believe my observations have resulted in a representative sampling of local customers over two months when that “winter wind” has been especially busy about its work.

To avoid detection by the customers I was observing and the possibility of altering their behaviour, I obtained permission from Pudgy’s manager, Mr Jobson, to sit at the staff table in a dark and quiet corner of the restaurant where clients never go. This table is labelled in the plan of Pudgy’s Burgers and its grounds that I have included as Figure 1. From there I could see the customers both at the service counter and at their tables, but they could not see me, at least not clearly, and if they did, they paid me no more attention than they did the restaurant employees. From the staff table I could also see the row of indoor park-style children’s toys running down the north wall of windows, as well as the take out lane and the people waiting in their cars.

A Method section often features subheadings to separate and present particularly important aspects of the research methodology, such as the Customer Fact Sheet developed and used by the author of this study.

The Customer Fact Sheet Recording thorough and equivalent information about every Pudgy’s customer I observed was crucial for quantifying and analysing the results of my study. I therefore prepared a Customer Fact Sheet (included as Appendix I at the end of this paper) for gathering key pieces of information and recording observations about each individual, couple or group who purchased food or beverages. This sheet ensured that vital details such as date, weather conditions, time of arrival, eat in or take out order, number in party, approximate age of individuals, food purchased, food consumed, healthy choices, amount spent, who paid, dessert or extra beverage, children playing, interaction with other children and families, time of departure and other important details were recorded in every case. The Customer Fact Sheet proved particularly helpful when my classmate performed observations for me and was invaluable for evaluating the data I collected. I initially hoped to complete at least 500 of these Customer Fact Sheets and was pleased to increase that number by 100 for a total of 600 or an average of just over 10 per day over the 59 days of the study.

Notice in the three example paragraphs for the Method section that clear references to Tables 1 & 2, Figure 1 and Appendix I are provided to let readers know when and why these extra elements are relevant and helpful. Be sure also to include in your description of methods any additional approaches or sources of information that should be considered part of your research procedures, such as:

• Receipt information about customer purchases provided by the restaurant manager. • Conversations with restaurant servers who might confirm family relationships and estimated ages or tell you what was eaten and what was not by particular customer groups. • The analysis you performed to make sense of your results, such as counting customers, meals and behaviours and working out percentages and averages overall as well as for certain categories in order to answer the research questions.

Results The Results section is where you report what you discovered during your research, including the findings that do not support your hypothesis (or hypotheses) as well as those that do. Returning to your research questions to indicate exactly how the data you gathered answers them is an excellent way to stay focused and enable the selectivity that may be necessary to meet length requirements or maintain a clear line of argumentation. A Results section for the Pudgy’s research project might start like this:

The results of my investigation were both surprising and more complex than I had anticipated. I asked whether families constituted the majority of Pudgy’s clientele and assumed they did, but my research shows that they do not (see Figure 2 for information on customer categories). Even when the loosest definition of family as explained in my introduction is applied, only slightly over 25% (152) of the 600 Customer Fact Sheets record family visits to the restaurant. Among them fathers alone with their children are the most frequent patrons (68 Customer Fact Sheets or nearly 45% of the family category). The only day of the week on which families approach 50% of the restaurant’s customers is Sunday, particularly in the afternoon, when family groups account for 48% of the total customers averaged over the eight Sundays of observation. On all other days of the week, individual customers are the most frequent patrons, with their numbers hovering around 50% on most days. Single men visit the restaurant more often than any other customers and constitute as much as 61% of the clientele on a few weekday evenings.

The report of results might then continue by providing information about other categories of customer, what different types of customers ate and did, and any additional results that help answer the other research questions posed in the introductory paragraphs. Major trends revealed by the data should be reported, and both content and writing style should be clear and factual. Interpretation and discussion are best saved for the Discussion section except in those rare instances when guidelines indicate that research results and discussion should be combined in a single section.  Although you will need to inform readers about any mathematical or statistical analysis of your raw data if you have not already done so in the Method section, the raw data itself is usually not appropriate for a short research paper. Selecting the most convincing and relevant evidence as the focus is, however, and the raw data can usually be made available via a university’s website or a journal’s online archives for expert readers and future researchers.

Discussion The Discussion section of a quantitative paper is where you interpret your research results and discuss their implications. Here the hypotheses as well as the research questions established in the introductory material are important. Were your primary suppositions confirmed by your results or not? Be precise and concise as you discuss your findings, but keep in mind that matters need not be quite as black and white or as strictly factual as they were in the Results section. Your ideas and argument should be soundly based on the data you collected, of course, but the Discussion is the place for describing complexities and expressing uncertainties as well as offering interpretations and explanations. The following opening briefly restates primary findings, picks up other important threads from the Results section and sets the stage for discussing the complexities involved in assessing the true value of Pudgy’s to the Quaintville community:

Although I had anticipated that families constitute the majority of Pudgy’s clientele, the evidence gathered over two months of observation does not support this supposition. In fact, individuals are the most frequent customers, with groups of teenagers running a close second. These teenagers are often in the restaurant when families are and they sometimes sit on the indoor toys instead of at the plastic tables and chairs, which I can confirm as extremely uncomfortable. On a few occasions the presence of teenagers appeared to intimidate the children and prevent them from playing on the facilities intended for them. In accordance with Parkson (2016) and Shemble (2017), my research also showed that most families who eat at Pudgy’s do not choose the healthier low-fat menu items, with the limited number and extremely high prices of these items offering little incentive. The few parents who make healthy choices for themselves and their children often do not insist upon the children eating those items, adding waste (of both food and money) to the problem. Furthermore, although Pudgy’s prices for their more traditional fast-food items are the lowest in town, at least two of the restaurants in Quaintville offer equivalent meals for similar prices and far healthier ones for just a little more.

The claim, then, in the  Quaintville Times  that “good old Pudgy’s is the only restaurant in Quaintville where a working family can still get a decent meal for a fair buck, and a comfortable place to eat it too, out of the winter wind where the kids can run about and play a bit” (Chapton, 2017, p.A3) is revealed as more sentiment than fact. It would be equally erroneous, however, to insist that Pudgy’s Burgers has no value for the local community or to call it more of “a hazard…to the health of some of our poorest families” (“Local dive,” 2017, p.1) than any other restaurants serving burgers and chips in Quaintville. Indeed, I suspect those “poorest families” very rarely visit local restaurants at all, but my observations have revealed a great deal about who does eat at Pudgy’s, what they do when they are there and what kind of value the establishment actually has for Quaintville residents.

The discussion could then continue with information about the customers, behaviours and other issues that render the findings more complex and the restaurant more valuable to the community than the primary results noted above may indicate:

• Perhaps the restaurant serves a vital function as a social gathering place for all those single customers. Do they usually remain alone or do they meet up with others to linger and talk over coffee or lunch? • Do the teenagers who gather at Pudgy’s have an alternative place to meet out of the cold? In towns without recreation centres or other facilities for teens, restaurants with informal, open-door policies can be vital. Where might those teenagers go or what might they be doing were Pudgy’s not there? • Even though the evidence showed that families are not the most frequent customers, you may want to consider the value the restaurant has for the families who do use it. Those single fathers are certainly worthy of some attention, for instance, and perhaps family groups occasionally met up with other families, ate together and then lingered for dessert and talk as their children enjoyed the toys. This would be worth discussing too. • Less measurable considerations viewed through a qualitative research lens may be helpful as well, but the data collected through observations should support such discussions. Remember as you analyse your data, reflect on your findings, determine their meaning and develop your argument that it is important to keep the limitations of your methodology and thus of your results and their implications clearly in mind.

Offering recommendations is also standard in the Discussion section of a quantitative research paper, and here recommendations might be particularly useful if the franchise had not yet finalised its decision about closing Pudgy’s and was actively seeking community feedback. The researcher might suggest that Pudgy’s could better serve families by increasing the number of healthy food items on the menu, offering these for more affordable prices and making an effort to keep the teenagers off the children’s toys. Finally, the last part of a Discussion usually provides concluding comments, so summarising your key points and clearly articulating the main messages you want your readers to take away with them are essential. In some organisational templates, Conclusions are offered in a separate final section of the paper instead of at the end of the Discussion, so always check the guidelines.

References These references follow APA style, but since special fonts may not display properly in all online situations, please note that the titles of books and the names and volume numbers of journals are (and should be) in italic font. The list represents a sample only; a paper the length of the one posited in this example would almost certainly mention, discuss and list more than half a dozen studies and sources.

Chapton, D. (2017, September 29). Will Quaintville lose its favourite family restaurant?  Quaintville Times , pp. A1, A3. Local dive sees last days. (2017, Autumn).  Quaintville Community Newsletter , pp. 1–2. Shemble, M. (2017). Is anyone really eating healthy fast food in rural towns?  Country Food & Families ,  14 , 12–23. Shunts, P. (2013). The true cost of high-fat fast food for low-income families.  Journal of Family Health & Diet ,  37 , 3–19. Parkson, L. (2016). Family diets, fast foods and unhealthy choices. In S. Smith & J. Jones (eds.),  Modern diets and family health  (pp. 277–294). Philadelphia, PA: The Family Press. Whinner, N. (2015). Healthy families take time: The impact of fatty fast foods on child health.  Journal of Family Health & Diet ,  39 , 31–43.

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Example of a Quantitative Research Paper This example is organised into introductory material, method, results & discussion.

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Example of a Quantitative Research Paper for Students & Researchers

This example of a quantitative research paper is designed to help students and other r esearchers who are learning how to write about their work. The reported research obs erves the behaviour of restaurant customers, and example paragraphs are combined with instructions for logical argumentation. Authors are encouraged to observe a traditional structure for organising quantitative research papers, to formulate research que stions, working hypotheses and investigative tools, to report results accurately and thor oughly, and to present thoughtful interpretation and logical discussion of evidence.

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Food industry, the world over, is witnessing unprecedented increase in the number of multinational enterprises. These multinational enterprises, when deciding to expand their operations to a new country, have to make a choice between following uniform business strategies as in their home country or modify their strategies to suit the host country socioeconomic and political environment. Given the economic cost of modification of business strategies, the choice has widespread implications for the sustainability of multinational enterprises. The present paper argues that this decision-making is particularly critical in the case of multinational food enterprises because of large scale variability in food habits across countries and even within a country. Drawing from case studies of three multinational food enterprises in India, the paper points out that, in order to operate successfully in their host countries, the multinational food enterprises must adopt Glocalized strategies in marketing, product development, advertisement etc.

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Food is an important aspect of social culture and has a close relationship with economic development. The Chinese food culture has the characteristics of inheritability and development, and throughout the history of Chinese food culture, it has maintained its momentum of development since its primitive society. Neither the change of dynasty nor the change of social system has had a profound influence on it, and the philosophy of supplying enough food to people and food being the top priority was very popular. Eating was a top priority for people in China. Long ago, Confucius said that the desire for food and sex is part of human nature. As such, in the Chinese culture food became the priority. Because of the attention to diet, Chinese people would, when they had leisure time or abundant raw materials, work out a variety of food. Chinese cooking is flexible, which is characterized by saying that there is no fixed taste and what is delicious is valued. The beauty of food is one of the important roots of Chinese aesthetics, which inspires people with the stimulation of eating. Triggering art inspiration is the inevitable result of Chinese food culture pursuing complete and beautiful color, fragrance, taste, shape, and utensils. It makes food culture a comprehensive art containing multiple cultural connotations of diet, diet mentality, beautiful utensils and etiquette, food enjoyment and eating. Chinese foods have not only exquisite craftsmanship and rich nutrition, but also elegant and graceful names, which are literary and romantic, poetic and fancy. Food functions to not only satiate people’s hunger; it has also become an integral aspect of life enjoyment, which represents an essential component of food anthropology. Food anthropologists stress that changes in people’s eating habits not only depend on the local food culture, which may be specific to a given region, but also varies with economic development in different regions. Food anthropology, as a sub branch of applied anthropology, adapts anthropological theories and methods to study food industry, food culture, food consumption and food commerce. Seminal work in this regard has been provided by scholars and consultants in the field of food anthropology. This book describes the anthropological studies on Chinese foodways, outlines the Chinese food anthropology basic theories and methods. Anthropology in China is still at its development stage in China, while food anthropology is just at its initial stages of development. Nevertheless, China’s economic and social development, especially in ethnic minority regions in Western China, needs the theoretical guidance of some disciplines, including food anthropology, economic anthropology and business anthropology. At the same time, it has provided opportunities to develop food anthropology with the Chinese characteristics. Therefore, when Chinese scholars are learning and adopting Western food anthropology theories and methodologies, they must innovate and develop the related theories and methodologies with Chinese characteristics, so that they can better serve the well-off of the entire society.

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Methodology

• What Is Quantitative Research? | Definition, Uses & Methods

What Is Quantitative Research? | Definition, Uses & Methods

Published on June 12, 2020 by Pritha Bhandari . Revised on June 22, 2023.

Quantitative research is the process of collecting and analyzing numerical data. It can be used to find patterns and averages, make predictions, test causal relationships, and generalize results to wider populations.

Quantitative research is the opposite of qualitative research , which involves collecting and analyzing non-numerical data (e.g., text, video, or audio).

Quantitative research is widely used in the natural and social sciences: biology, chemistry, psychology, economics, sociology, marketing, etc.

• What is the demographic makeup of Singapore in 2020?
• How has the average temperature changed globally over the last century?
• Does environmental pollution affect the prevalence of honey bees?
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Table of contents

Quantitative research methods, quantitative data analysis, advantages of quantitative research, disadvantages of quantitative research, other interesting articles, frequently asked questions about quantitative research.

You can use quantitative research methods for descriptive, correlational or experimental research.

• In descriptive research , you simply seek an overall summary of your study variables.
• In correlational research , you investigate relationships between your study variables.
• In experimental research , you systematically examine whether there is a cause-and-effect relationship between variables.

Correlational and experimental research can both be used to formally test hypotheses , or predictions, using statistics. The results may be generalized to broader populations based on the sampling method used.

To collect quantitative data, you will often need to use operational definitions that translate abstract concepts (e.g., mood) into observable and quantifiable measures (e.g., self-ratings of feelings and energy levels).

Note that quantitative research is at risk for certain research biases , including information bias , omitted variable bias , sampling bias , or selection bias . Be sure that you’re aware of potential biases as you collect and analyze your data to prevent them from impacting your work too much.

Prevent plagiarism. Run a free check.

Once data is collected, you may need to process it before it can be analyzed. For example, survey and test data may need to be transformed from words to numbers. Then, you can use statistical analysis to answer your research questions .

Descriptive statistics will give you a summary of your data and include measures of averages and variability. You can also use graphs, scatter plots and frequency tables to visualize your data and check for any trends or outliers.

Using inferential statistics , you can make predictions or generalizations based on your data. You can test your hypothesis or use your sample data to estimate the population parameter .

First, you use descriptive statistics to get a summary of the data. You find the mean (average) and the mode (most frequent rating) of procrastination of the two groups, and plot the data to see if there are any outliers.

You can also assess the reliability and validity of your data collection methods to indicate how consistently and accurately your methods actually measured what you wanted them to.

Quantitative research is often used to standardize data collection and generalize findings . Strengths of this approach include:

• Replication

Repeating the study is possible because of standardized data collection protocols and tangible definitions of abstract concepts.

• Direct comparisons of results

The study can be reproduced in other cultural settings, times or with different groups of participants. Results can be compared statistically.

• Large samples

Data from large samples can be processed and analyzed using reliable and consistent procedures through quantitative data analysis.

• Hypothesis testing

Using formalized and established hypothesis testing procedures means that you have to carefully consider and report your research variables, predictions, data collection and testing methods before coming to a conclusion.

Despite the benefits of quantitative research, it is sometimes inadequate in explaining complex research topics. Its limitations include:

• Superficiality

Using precise and restrictive operational definitions may inadequately represent complex concepts. For example, the concept of mood may be represented with just a number in quantitative research, but explained with elaboration in qualitative research.

• Narrow focus

Predetermined variables and measurement procedures can mean that you ignore other relevant observations.

• Structural bias

Despite standardized procedures, structural biases can still affect quantitative research. Missing data , imprecise measurements or inappropriate sampling methods are biases that can lead to the wrong conclusions.

• Lack of context

Quantitative research often uses unnatural settings like laboratories or fails to consider historical and cultural contexts that may affect data collection and results.

If you want to know more about statistics , methodology , or research bias , make sure to check out some of our other articles with explanations and examples.

• Chi square goodness of fit test
• Degrees of freedom
• Null hypothesis
• Discourse analysis
• Control groups
• Mixed methods research
• Non-probability sampling
• Inclusion and exclusion criteria

Research bias

• Rosenthal effect
• Implicit bias
• Cognitive bias
• Selection bias
• Negativity bias
• Status quo bias

Quantitative research deals with numbers and statistics, while qualitative research deals with words and meanings.

Quantitative methods allow you to systematically measure variables and test hypotheses . Qualitative methods allow you to explore concepts and experiences in more detail.

In mixed methods research , you use both qualitative and quantitative data collection and analysis methods to answer your research question .

Data collection is the systematic process by which observations or measurements are gathered in research. It is used in many different contexts by academics, governments, businesses, and other organizations.

Operationalization means turning abstract conceptual ideas into measurable observations.

For example, the concept of social anxiety isn’t directly observable, but it can be operationally defined in terms of self-rating scores, behavioral avoidance of crowded places, or physical anxiety symptoms in social situations.

Before collecting data , it’s important to consider how you will operationalize the variables that you want to measure.

Reliability and validity are both about how well a method measures something:

• Reliability refers to the  consistency of a measure (whether the results can be reproduced under the same conditions).
• Validity   refers to the  accuracy of a measure (whether the results really do represent what they are supposed to measure).

If you are doing experimental research, you also have to consider the internal and external validity of your experiment.

Hypothesis testing is a formal procedure for investigating our ideas about the world using statistics. It is used by scientists to test specific predictions, called hypotheses , by calculating how likely it is that a pattern or relationship between variables could have arisen by chance.

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Academic Referencing: How to Cite a Research Paper

Learning how to conduct accurate, discipline-specific academic research can feel daunting at first. But, with a solid understanding of the reasoning behind why we use academic citations coupled with knowledge of the basics, you’ll learn how to cite sources with accuracy and confidence.

When it comes to academic research, citing sources correctly is arguably as important as the research itself. "Your instructors are expecting your work to adhere to these professional standards," said Amanda Girard , research support manager of Shapiro Library at Southern New Hampshire University (SNHU).

With Shapiro Library for the past three years, Girard manages the library’s research support services, which includes SNHU’s 24/7 library chat and email support. She holds an undergraduate degree in professional writing and a graduate degree in library and information science. She said that accurate citations show that you have done your research on a topic and are knowledgeable about current ideas from those actively working in the field.

In other words, when you cite sources according to the academic style of your discipline, you’re giving credit where credit is due.

Why Cite Sources?

Citing sources properly ensures you’re following high academic and professional standards for integrity and ethics.

“When you cite a source, you can ethically use others’ research. If you are not adequately citing the information you claim in your work, it would be considered plagiarism ,” said Shannon Geary '16 , peer tutor at SNHU.

Geary has an undergraduate degree in communication  from SNHU and has served on the academic support team for close to 2 years. Her job includes helping students learn how to conduct research  and write academically.

“In academic writing, it is crucial to state where you are receiving your information from,” she said. “Citing your sources ensures that you are following academic integrity standards.”

According to Geary and Girard, several key reasons for citing sources are:

• Access. Citing sources points readers to original sources. If anyone wants to read more on your topic, they can use your citations as a roadmap to access the original sources.
• Attribution. Crediting the original authors, researchers and experts  shows that you’re knowledgeable about current ideas from those actively working in the field and adhering to high ethical standards, said Girard.
• Clarity. “By citing your sources correctly, your reader can follow along with your research,” Girard said.
• Consistency. Adhering to a citation style provides a framework for presenting ideas within similar academic fields. “Consistent formatting makes accessing, understanding and evaluating an author's findings easier for others in related fields of study,” Geary said.
• Credibility. Proper citation not only builds a writer's authority but also ensures the reliability of the work, according to Geary.

Ultimately, citing sources is a formalized way for you to share ideas as part of a bigger conversation among others in your field. It’s a way to build off of and reference one another’s ideas, Girard said.

How Do You Cite an Academic Research Paper?

Any time you use an original quote or paraphrase someone else’s ideas, you need to cite that material, according to Geary.

“The only time we do not need to cite is when presenting an original thought or general knowledge,” she said.

While the specific format for citing sources can vary based on the style used, several key elements are always included, according to Girard. Those are:

• Title of source
• Type of source, such as a journal, book, website or periodical

By giving credit to the authors, researchers and experts you cite, you’re building credibility. You’re showing that your argument is built on solid research.

“Proper citation not only builds a writer's authority but also ensures the reliability of the work,” Geary said. “Properly formatted citations are a roadmap for instructors and other readers to verify the information we present in our work.”

Common Citation Styles in Academic Research

Certain disciplines adhere to specific citation standards because different disciplines prioritize certain information and research styles . The most common citation styles used in academic research, according to Geary, are:

• American Psychological Association, known as APA . This style is standard in the social sciences such as psychology, education and communication. “In these fields, research happens rapidly, which makes it exceptionally important to use current research,” Geary said.
• Modern Language Association, known as MLA . This style is typically used in literature and humanities because of the emphasis on literature analysis. “When citing in MLA, there is an emphasis on the author and page number, allowing the audience to locate the original text that is being analyzed easily,” Geary said.
• Chicago Manual of Style, known as Chicago . This style is typically used in history, business and sometimes humanities. “(Chicago) offers flexibility because of the use of footnotes, which can be seen as less distracting than an in-text citation,” Geary said.

The benefit of using the same format as other researchers within a discipline is that the framework of presenting ideas allows you to “speak the same language,” according to Girard.

How to Ensure Proper Citations

Keeping track of your research as you go is one of the best ways to ensure you’re citing appropriately and correctly based on the style that your academic discipline uses.

“Through careful citation, authors ensure their audience can distinguish between borrowed material and original thoughts, safeguarding their academic reputation and following academic honesty policies,” Geary said.

Some tips that she and Girard shared to ensure you’re citing sources correctly include:

• Keep track of sources as you work. Writers should keep track of their sources every time an idea is not theirs, according to Geary. “You don’t want to find the perfect research study and misplace its source information, meaning you’d have to omit it from your paper,” she said.
• Practice. Even experienced writers need to check their citations before submitting their work. “Citing requires us to pay close attention to detail, so always start your citation process early and go slow to ensure you don’t make mistakes,” said Geary. In time, citing sources properly becomes faster and easier.
• Use an Online Tool . Geary recommends the Shapiro Library citation guide . You can find sample papers, examples of how to cite in the different academic styles and up-to-date citation requirements, along with information and examples for APA, MLA and Chicago style citations.
• Work with a Tutor. A tutor can offer support along with tips to help you learn the process of academic research. Students at SNHU can connect with free peer tutoring through the Academic Support tab in their online courses, though many colleges and universities offer peer tutoring.

Find Your Program

How to cite a reference in academic writing.

A citation consists of two pieces: an in-text citation that is typically short and a longer list of references or works cited (depending on the style used) at the end of the paper.

“In-text citations immediately acknowledge the use of external source information and its exact location,” Geary said. While each style uses a slightly different format for in-text citations that reference the research, you may expect to need the page number, author’s name and possibly date of publication in parentheses at the end of a sentence or passage, according to Geary.

A longer entry listing the complete details of the resource you referenced should also be included on the references or works cited page at the end of the paper. The full citation is provided with complete details of the source, such as author, title, publication date and more, Geary said.

The two-part aspect of citations is because of readability. “You can imagine how putting the full citation would break up the flow of a paper,” Girard said. “So, a shortened version is used (in the text).”

“For example, if an in-text citation reads (Jones, 2024), the reader immediately knows that the ideas presented are coming from Jones’s work, and they can explore the comprehensive citation on the final page,” she said.

The in-text citation and full citation together provide a transparent trail of the author's process of engaging with research.

“Their combined use also facilitates further research by following a standardized style (APA, MLA, Chicago), guaranteeing that other scholars can easily connect and build upon their work in the future,” Geary said.

Developing and demonstrating your research skills, enhancing your work’s credibility and engaging ethically with the intellectual contributions of others are at the core of the citation process no matter which style you use.

A degree can change your life. Choose your program  from 200+ SNHU degrees that can take you where you want to go.

A former higher education administrator, Dr. Marie Morganelli is a career educator and writer. She has taught and tutored composition, literature, and writing at all levels from middle school through graduate school. With two graduate degrees in English language and literature, her focus — whether teaching or writing — is in helping to raise the voices of others through the power of storytelling. Connect with her on LinkedIn .

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Founded in 1932, and online since 1995, we’ve helped countless students reach their goals with flexible, career-focused programs . Our 300-acre campus in Manchester, NH is home to over 3,000 students, and we serve over 135,000 students online. Visit our about SNHU  page to learn more about our mission, accreditations, leadership team, national recognitions and awards.

A Quantitative Study of the Effect of English Civics Elements on Students’ Critical Thinking Skills in EFL Classrooms in a Big Data Environment

• September 2024
• Applied Mathematics and Nonlinear Sciences 9(1)
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• Published: 26 August 2024

Solid health care waste management practice in Ethiopia, a convergent mixed method study

• Yeshanew Ayele Tiruneh 1 ,
• L. M. Modiba 2 &
• S. M. Zuma 2  

BMC Health Services Research volume  24 , Article number:  985 ( 2024 ) Cite this article

31 Accesses

Metrics details

Introduction

Healthcare waste is any waste generated by healthcare facilities that is considered potentially hazardous to health. Solid healthcare waste is categorized into infectious and non-infectious wastes. Infectious waste is material suspected of containing pathogens and potentially causing disease. Non-infectious waste includes wastes that have not been in contact with infectious agents, hazardous chemicals, or radioactive substances, similar to household waste, i.e. plastic, papers and leftover foods.

This study aimed to investigate solid healthcare waste management practices and develop guidelines to improve solid healthcare waste management practices in Ethiopia. The setting was all health facilities found in Hossaena town.

A mixed-method study design was used. For the qualitative phase of this study, eight FGDs were conducted from 4 government health facilities, one FGD from each private health facility (which is 37 in number), and forty-five FGDs were conducted. Four FGDs were executed with cleaners; another four were only health care providers because using homogeneous groups promotes discussion. The remaining 37 FGDs in private health facilities were mixed from health professionals and cleaners because of the number of workers in the private facilities. For the quantitative phase, all health facilities and health facility workers who have direct contact with healthcare waste management practice participated in this study. Both qualitative and quantitative study participants were taken from the health facilities found in Hossaena town.

Seventeen (3.1%) health facility workers have hand washing facilities. Three hundred ninety-two (72.6%) of the participants agree on the availability of one or more personal protective equipment (PPE) in the facility ‘‘ the reason for the absence of some of the PPEs, like boots and goggles, and the shortage of disposable gloves owes to cost inflation from time to time and sometimes absent from the market’’ . The observational finding shows that colour-coded waste bins are available in 23 (9.6%) rooms. 90% of the sharp containers were reusable, and 100% of the waste storage bins were plastic buckets that were easily cleanable. In 40 (97.56%) health facilities, infectious wastes were collected daily from the waste generation areas to the final disposal points. Two hundred seventy-one (50.2%) of the respondents were satisfied or agreed that satisfactory procedures are available in case of an accident. Only 220 (40.8%) respondents were vaccinated for the Hepatitis B virus.

Hand washing facilities, personal protective equipment and preventive vaccinations are not readily available for health workers. Solid waste segregation practices are poor and showed that solid waste management practices (SWMP) are below the acceptable level.

Peer Review reports

Healthcare waste (HCW) encompasses all types of waste generated while providing health-related services, spanning activities such as diagnosis, immunization, treatment, and research. It constitutes a diverse array of materials, each presenting potential hazards to health and the environment. Within the realm of HCW, one finds secretions and excretions from humans, cultures, and waste containing a stock of infectious agents. Discarded plastic materials contaminated with blood or other bodily fluids, pathological wastes, and discarded medical equipment are classified as healthcare waste. Sharps, including needles, scalpels, and other waste materials generated during any healthcare service provision, are also considered potentially hazardous to health [ 1 ].

Healthcare waste in solid form (HCW) is commonly divided into two primary groups: infectious and non-infectious. The existence of pathogens in concentrations identifies infectious waste or amounts significant enough to induce diseases in vulnerable hosts [ 1 ] If healthcare facility waste is free from any combination with infectious agents, nearly 85% is categorized as non-hazardous waste, exhibiting characteristics similar to conventional solid waste found in households [ 2 ]. World Health Organization (WHO) recommends that appropriate colour-coded waste receptacles be available in all medical and other waste-producing areas [ 3 ].

Solid waste produced in the course of healthcare activities carries a higher potential for infection and injury than any other type of waste. Improper disposal of sharps waste increases the risk of disease transmission among health facility workers and general populations [ 1 ]. Inadequate and inappropriate handling of healthcare waste may have serious public health consequences and a significant environmental impact. The World Health Organization (2014) guidelines also include the following guidance for hand washing and the use of alcohol-based hand rubs: Wash hands before starting work, before entering an operating theatre, before eating, after touching contaminated objects, after using a toilet, and in all cases where hands are visibly soiled [ 4 ].

Among the infectious waste category, sharps waste is the most hazardous waste because of its ability to puncture the skin and cause infection [ 3 ]. Accidents or occurrences, such as near misses, spills, container damage, improper waste segregation, and incidents involving sharps, must be reported promptly to the waste management officer or an assigned representative [ 5 ].

Africa is facing a growing waste management crisis. While the volumes of waste generated in Africa are relatively small compared to developed regions, the mismanagement of waste in Africa already impacts human and environmental health. Infectious waste management has always remained a neglected public health problem in developing countries, resulting in a high burden of environmental pollution affecting the general masses. In Ethiopia, there is no updated separate regulation specific to healthcare waste management in the country to enforce the proper management of solid HCW [ 6 ].

In Ethiopia, like other developing countries, healthcare waste segregation practice was not given attention and did not meet the minimum HCWM standards, and it is still not jumped from paper. Previous study reveals that healthcare waste generation rates are significantly higher than the World Health Organization threshold, which ranges from 29.5–53.12% [ 7 , 8 ]. In Meneilk II Hospital, the proportion of infectious waste was 53.73%, and in the southern and northern parts of Ethiopia, it was 34.3 and 53%, respectively. Generally, this figure shows a value 3 to 4 times greater than the threshold value recommended by the World Health Organization [ 7 ].

Except for sharp wastes, segregation practice was poor, and all solid wastes were collected without respecting the colour-coded waste disposal system [ 9 ]. The median waste generation rate was found to vary from 0.361- 0.669 kg/patient/day, comprising 58.69% non-hazardous and 41.31% hazardous wastes. The amount of waste generated increased as the number of patients flow increased. Public hospitals generated a high proportion of total healthcare waste (59.22%) in comparison with private hospitals (40.48) [ 10 ]. The primary SHCW treatment and disposal mechanism was incineration, open burning, burring into unprotected pits and open dumping on municipal dumping sites as well as in the hospital backyard. Carelessness, negligence of the health workers, patients and cleaners, and poor commitment of the facility leaders were among the major causes of poor HCWM practice in Ethiopia [ 9 ]. This study aimed to investigate solid healthcare waste management practices and develop guidelines to improve solid healthcare waste management practices in Ethiopia.

The setting for this study was all health facilities found in Hossaena town, which is situated 232 kms from the capital city of Ethiopia, Addis Ababa, and 165 kms from the regional municipality of Hawasa. The health facilities found in the town were one university hospital, one private surgical centre, three government health centres, 17 medium clinics, and 19 small clinics were available in the city and; health facility workers who have direct contact with generating and disposal of HCW and those who are responsible as a manager of health facilities found in Hossaena town are the study settings. All health facilities except drug stores and health facility workers who have direct contact with healthcare waste generation participated in this study.

A mixed-method study design was used. For the quantitative part of this study, all healthcare workers who have direct contact with healthcare waste management practice participated in this study, and one focus group discussion from each health facility was used. Both of the study participants were taken from the same population. All health facility workers who have a role in healthcare waste management practice were included in the quantitative part of this study. The qualitative data collection phase used open-ended interviews, focus group discussions, and visual material analysis like posters and written materials. All FGDs were conducted by the principal investigator, one moderator, and one note-taker, and it took 50 to 75 min. 4–6 participants participated in each FGD.

According to Elizabeth (2018: 5), cited by Creswell and Plano (2007: 147), the mixed method is one of the research designs with philosophical assumptions as well as methods of inquiry. As a method, it focuses on collecting, analyzing, and mixing both quantitative and qualitative data in a single study. As a methodology, it involves philosophical assumptions guiding the direction of the collection and analysis and combining qualitative and quantitative approaches in many phases of the research project. The central premise is that using qualitative and quantitative approaches together provides a better understanding of the research problems than either approach alone.

The critical assumption of the concurrent mixed methods approach in this study is that quantitative and qualitative data provide different types of information, often detailed views of participants’ solid waste management practice qualitatively and scores on instruments quantitatively, and together, they yield results that should be the same. In this approach, the researcher collected quantitative and qualitative data almost simultaneously and analyzed them separately to cross-validate or compare whether the findings were similar or different between the qualitative and quantitative information. Concurrent approaches to the data collection process are less time-consuming than other types of mixed methods studies because both data collection processes are conducted on time and at the same visit to the field [ 11 ].

Data collection

The data collection involves collecting both quantitative and qualitative data simultaneously. The quantitative phase of this study assessed three components. Health care waste segregation practice, the availability of waste segregation equipment for HCW segregation, temporary storage facilities, transportation for final disposal, and disposal facilities data were collected using a structured questionnaire and observation of HCW generation. Recycling or re-using practice, waste treatment, the availability of the HCWM committee, and training data were collected.

Qualitative data collection

The qualitative phase of the data collection for this study was employed by using focus group discussions and semi-structured interviews about SHCWMP. Two focus group discussions (FGD) from each health facility were conducted in the government health facilities, one at the administrative level and one at the technical worker level, and one FGD was conducted for all private health facilities because of the number of available health facility workers. Each focus group has 4–6 individuals.

In this study, the qualitative and the quantitative data provide different information, and it is suitable for this study to compare and contrast the findings of the two results to obtain the best understanding of this research problem.

Quantitative data collection

The quantitative data were entered into Epi data version 3.1 to minimize the data entry mistakes and exported to the statistical package for social science SPSS window version 27.0 for analysis. A numeric value was assigned to each response in a database, cleaning the data, recoding, establishing a codebook, and visually inspecting the trends to check whether the data were typically distributed.

Data analysis

Data were analyzed quantitatively by using relevant statistical tools, such as SPSS. Descriptive statistics and the Pearson correlation test were used for the bivariate associations and analysis of variance (ANOVA) to compare the HCW generation rate between private and government health facilities and between clinics, health centres and hospitals in the town. Normality tests were performed to determine whether the sample data were drawn from a normally distributed population.

The Shapiro–Wilk normality tests were used to calculate a test statistic based on the sample data and compare it to critical values. The Shapiro–Wilk test is a statistical test used to assess whether a given sample comes from a normally distributed population. The P value greater than the significance level of 0.05 fails to reject the null hypothesis. It concludes that there is not enough evidence to suggest that the data does not follow the normal distribution. Visual inspection of a histogram, Q-Q plot, and P-P plot (probability-probability plot) was assessed.

Bivariate (correlation) analysis assessed the relationships between independent and dependent variables. Then, multiple linear regression analysis was used to establish the simple correlation matrices between different variables for investigating the strength relationships of the study variables in the analysis. In most variables, percentages and means were used to report the findings with a 95% confidence interval. Open-ended responses and focused group findings were undertaken by quantifying and coding the data to provide a thematic narrative explanation.

Appropriate and scientific care was taken to maintain the data quality before, during, and after data collection by preparing the proper data collection tools, pretesting the data collection tools, providing training for data collectors, and proper data entry practice. Data were cleaned on a daily basis during data collection practice, during data entry, and before analysis of its completeness and consistency.

Data analysis in a concurrent design consists of three phases. First, analyze the quantitative database in terms of statistical results. Second, analyze the qualitative database by coding the data and collapsing the codes into broad themes. Third comes the mixed-method data analysis. This is the analysis that consists of integrating the two databases. This integration consists of merging the results from both the qualitative and the quantitative findings.

Descriptive analysis was conducted to describe and summarise the data obtained from the samples used for this study. Reliability statistics for constructs, means and modes of each item, frequencies and percentage distributions, chi-square test of association, and correlations (Spearman rho) were used to portray the respondents’ responses.

All patient care-providing health facilities were included in this study, and the generation rate of healthcare waste and composition assessed the practice of segregation, collection, transportation, and disposal system was observed quantitatively using adopted and adapted structured questionnaires. To ensure representativeness, various levels of health facilities like hospitals, health centres, medium clinics, small clinics and surgical centres were considered from the town. All levels of health facilities are diagnosing, providing first aid services and treating patients accordingly.

The hospital and surgical centre found in the town provide advanced surgical service, inpatient service and food for the patients that other health facilities do not. The HCW generation rate was proportional to the number of patients who visited the health facilities and the type of service provided. The highest number of patients who visited the health facilities was in NEMMCSH; the service provided was diverse, and the waste generation rate was higher than that of other health facilities. About 272, 18, 15, 17, and 20 average patients visited the health facilities daily in NEMMCSH: government health centres, medium clinics, small clinics, and surgical centres. Paper and cardboard (141.65 kg), leftover food (81.71 kg), and contaminated gloves (42.96 kg) are the leading HCWs generated per day.

A total of 556 individual respondents from sampled health facilities were interviewed to complete the questionnaire. The total number of filled questionnaires was 540 (97.1) from individuals representing these 41 health facilities.

The principal investigator observed the availability of handwashing facilities near SHCW generation sites. 17(3.1%) of health facility workers had hand washing facilities near the health care waste generation and disposal site. Furthermore,10 (3.87%), 2 (2.1%), 2 (2.53%), 2 (2.1%), 1 (6.6%) of health facility workers had the facility of hand washing near the health care waste generation site in Nigist Eleni Mohamed Memorial Comprehensive Specialized Hospital (NEMMCSH), government health centres, medium clinics, small clinics, and surgical centre respectively. This finding was nearly the same as the study findings conducted in Myanmar; the availability of hand washing facilities near the solid health care waste generation was absent in all service areas [ 12 ]. The observational result was convergent with the response of facility workers’ response regarding the availabilities of hand washing facilities near to the solid health care waste generation sites.

The observational result was concurrent with the response of facility workers regarding the availability of hand-washing facilities near the solid health care waste generation sites.

The availability of personal protective equipment (PPE) was checked in this study. Three hundred ninety-two (72.6%) of the respondents agree on the facility’s availability of one or more personal protective equipment (PPE). The availability of PPEs in different levels of health facilities shows 392 (72.6%), 212 (82.2%), 56 (58.9%), 52 (65.8%), 60 (65.2%), 12 (75%) health facility workers in NEMMCSH, government health centres, medium clinics, small clinics, and surgical centres respectively agree to the presence of personal protective equipment in their department. The analysis further shows that the availability of masks for healthcare workers was above the mean in NEMMCSH and surgical centres.

Focus group participants indicated that health facilities did not volunteer to supply Personal protective equipment (PPEs) for the cleaning staff.

“We cannot purchase PPE by ourselves because of the salary paid for the cleaning staff.”

Cost inflation and the high cost of purchasing PPEs like gloves and boots are complained about by all (41) health facility owners.

“the reason for the absence of some of the PPEs like boots, goggles, and shortage of disposable gloves are owing to cost inflation from time to time and sometimes absent from the market is the reason why we do not supply PPE to our workers.”

Using essential personal protective equipment (PPEs) based on the risk (if the risk is a splash of blood or body fluid, use a mask and goggles; if the risk is on foot, use appropriate shoes) is recommended by the World Health Organization [ 13 ]. The mean availability of gloves in health facilities was 343 (63.5% (95% CI: 59.3–67.4). Private health institutions are better at providing gloves for their workers, 67.1%, 72.8%, and 62.5% in medium clinics, small clinics, and surgical centres, respectively, which is above the mean.

Research participants agree that.

‘‘ there is a shortage of gloves to give service in Nigist Eleni Mohamed Memorial Comprehensive Specialized Hospital (NEMMCSH) and government health centres .’’

Masks are the most available personal protective equipment for health facility workers compared to others. 65.4%, 55.6%, and 38% of the staff are available with gloves, plastic aprons and boots, respectively.

The mean availability of masks, heavy-duty gloves, boots, and aprons was 71.1%, 65.4%, 38%, and 44.4% in the study health facilities. Health facility workers were asked about the availability of different personal protective equipment, and 38% of the respondents agreed with the presence of boots in the facility. Still, the qualitative observational findings of this study show that all health facility workers have no shoes or footwear during solid health care waste management practice.

SHCW segregation practice was checked by observing the availability of SHCW collection bins in each patient care room. Only 4 (1.7%) of the room’s SHCW bins are collected segregated (non-infectious wastes segregated in black bins and infectious wastes segregated in yellow bins) based on the World Health Organization standard. Colour-coded waste bins, black for non-infectious and yellow for infectious wastes, were available in 23 (9.6%) rooms. 90% of the sharp containers were reusable, and 100% of the waste storage bins were plastic buckets that were easily cleanable. Only 6.7% of the waste bins were pedal operated and adequately covered, and the rest were fully opened, or a tiny hole was prepared on the container’s cover. All of the healthcare waste disposal bins in each health facility and at all service areas were away from the arm’s reach distance of the waste generation places, and this is contrary to World Health Organization SHCWM guidelines [ 13 ]. The observation result reveals that the reason for the above result was that medication trolleys were not used during medication or while healthcare providers provided any health services to patients.

Most medical wastes are incinerated. Burning solid and regulated medical waste generated by health care creates many problems. Medical waste incinerators emit toxic air pollutants and ash residues that are the primary source of environmental dioxins. Public concerns about incinerator emissions and the creation of federal regulations for medical waste incinerators are causing many healthcare facilities to rethink their choices in medical waste treatment. Health Care Without Harm [ 14 ], states that non-incineration treatment technologies are a growing and developing field. The U.S. National Academy of Science 2000 argued that the emission of pollutants during incineration is a potential risk to human health, and living or working near an incineration facility can have social, economic, and psychological effects [ 15 ].

The incineration of solid healthcare waste technology has been accepted and adopted as an effective method in Ethiopia. Incineration of healthcare waste can produce secondary waste and pollutants if the treatment facilities are not appropriately constructed, designed, and operated. It can be one of the significant sources of toxic substances, such as polychlorinated dibenzo-dioxins/dibenzofurans (PCDD/ PCDF), polyvinyl chloride (PVC), hexachlorobenzenes and polychlorinated biphenyls, and dioxins and furans that are known as hazardous pollutants. These pollutants may have undesirable environmental impacts on human and animal health, such as liver failure and cancer [ 15 , 16 ].

All government health facilities (4 in number) used incineration to dispose of solid waste. 88.4% and 100% of the wastes are incinerated in WUNEMMCSH and government health centres. This finding contradicts the study findings in the United States of America and Malaysia, in which 49–60% and 59–60 were incinerated, respectively, and the rest were treated using other technologies [ 15 , 16 ].

World Health Organization (2014:45) highlighted those critical elements of the appropriate operation of incinerators include effective waste reduction and waste segregation, placing incinerators away from populated areas, satisfactory engineered design, construction following appropriate dimensional plans, proper operation, periodic maintenance, and staff training and management are mandatory.

Solid waste collection times should be fixed and appropriate to the quantity of waste produced in each area of the health care facility. General waste should not be collected simultaneously or in the same trolley as infectious or hazardous wastes. The collection should be done daily for most wastes, with collection timed to match the pattern of waste generation during the day [ 13 ].

SHCW segregation practices were observed for 240 rooms in 41 health facilities that provide health services in the town. In government health centres, medium clinics, small clinics, and surgical centres, SHCW segregation practice was not based on the World Health Organization standard. All types of solid waste were collected in a single container near the generation area, and there were no colour-coded SHCW storage dust bins. Still, in NEMMCSH, in most of the service areas, colour-coded waste bins are available, and the segregation practice was not based on the standard. Only 3 (10%) of the dust bins collected the appropriate wastes according to the World Health Organization standard, and the rest were mixed with infectious and non-infectious SHCW.

Table 1 below shows health facility managers were asked about healthcare waste segregation practices, and 9 (22%) of the facility leaders responded that there is an appropriate solid healthcare waste segregation practice in their health facilities. Still, during observation, only 4 (1.7%) of the rooms in two (4.87%) of the facilities, SHCW bins collected the segregated wastes (non-infectious wastes segregated at the black bin and infectious wastes segregated at yellow bin) based on the world health organization standard. The findings of this study show there is a poor segregation practice, and all kinds of solid wastes are collected together.

In 40 (97.56%) health facilities, infectious wastes were collected daily from the waste generation areas to the final disposal points. During observation in one of the study health facilities, infectious wastes were not collected daily and left for days. Utility gloves, boots, and aprons are not available for cleaning staff to collect and transport solid healthcare wastes in all study health facilities. 29.26% of the facilities’ cleaning staff have a face mask, and 36.5% of the facilities remove waste bins from the service area when 3/4 full, and the rest were not removed or replaced with new ones. There is a separate container only in 2 health facilities for infectious and non-infectious waste segregation practice, and the rest were segregated and collected using single and non-colour coded containers.

At all of the facilities in the study area, SHCW was transported from the service areas to the disposal site were transported manually by carrying the collection container and there is no trolley for transportation. This finding was contrary to the study findings conducted in India, which show segregated waste from the generation site was being transported through the chute to the carts placed at various points on the hospital premises by skilled sanitary workers [ 17 ].

Only 2 out of 41 health facilities have temporary solid waste storage points at the facility. One of the temporary storage places was clean, and the other needed to be properly cleaned and unsightly. Two (100%) of the temporary storage areas are not fenced and have no restriction to an authorized person. Temporary storage areas are available only in two health facilities that are away from the service provision areas.

Observational findings revealed that pre-treatment of SHCW before disposal was not practised at all study health facilities. 95% of the facilities have no water supply for hand washing during and after solid healthcare waste generation, collection, and disposal.

The United States Agency estimated sharp injuries from medical wastes to health professionals and sanitary service personnel for toxic substances and disease registry. Most of the injuries are caused during the recapping of hypodermic needles before disposal into sharps containers [ 13 ]. Nearly half of the respondents, 245 (51.5%), are recapping needles after providing an injection to the patient. Recapping was more practised in NEMMCSH and surgical centres, which is 57.5% and 57.5%, respectively. In government health centres, medium clinics, and surgical centres, the recapping of used needles was practised below the mean, which is 47.9%, 48, and 43.8%, respectively. This finding was reasonable compared to the study findings of Doylo et al. [ 18 ] in western Ethiopia, where 91% of the health workers are recapping needles after injection [ 18 ]. The research finding shows that there is no significant association P-value of 0.82 between the training and recapping of needles after injection.

Focus group participants ’ response for appropriate SHCWMP regarding patients ’ and visitors ’ lack of knowledge on SHCW segregation practice

“The personal responsibilities of patients and visitors on solid HCW disposal should be explained to help appropriate safe waste management practice and maintain good hygiene .” “Providing waste management training and creating awareness are the two aspects of improving SHCW segregation practice.” “Training upgrades and creates awareness on hygiene for all workers.”

Sharp waste collection practices were observed in 240 rooms in the study health facilities, and 9.2% of the rooms used disposable sharp containers.

Sixty per cent (60%), 13.3%, 8.24%, and 15.71% of the sharps containers in NEMMCSH, government health centres, medium clinics, and small clinics, respectively, were using disposable sharps containers; sharps were disposed together with the sharps container, and surgical centre was using reusable sharp collection container. All disposable sharps containers in medium and small clinics used non-puncture-resistant or simple packaging carton boxes. 60% and 13.3% of the disposable sharps containers in NEMMCSH and the government health centre use purposefully manufactured disposable safety boxes.

Needle sticks injury reporting and occurrence

A total of 70 injuries were reported to the health facility manager in the last one year, and 44 of the injuries were reported by health professionals. The rest of the injuries were reported by supportive staff. These injuries were reported from 35 health facilities, and the remaining six health facilities did not report any cases of injury related to work; see Tables 2 and 3 below.

Accidents or incidents, including near misses, spillages, damaged containers, inappropriate segregation, and any incidents involving sharps, should be reported to the waste-management officer. Accidental contamination must be notified using a standard-format document. The cause of the accident or incident should be investigated by the waste-management officer (in case of waste) or another responsible officer, who should also take action to prevent a recurrence [ 13 ]. Two hundred seventy-one (50.2% (CI: 45.7–54.6) of the respondents agree that satisfactory procedures are available in case of an accident, while the remaining 269 (49.8%( CI: 45.4–54.3) of respondents do not agree on the availability of satisfactory procedures in case of an accident, see Table  4 below. The availability of satisfactory procedures in case of an accident is above the mean in medium clinics, which is 60.8%. 132(24.4%) of the staff are pricked by needle stick injury while providing health services. Nearly half of the respondents, 269 (49.8%), who have been exposed to needle stick injury do not get satisfactory procedures after being pricked by a needle, and those who have not been stung by a needle stick injury for the last year. 204 (37.8%) disagree with the presence of satisfactory procedures in the case of a needle stick injury. In NEMMCSH, 30.2% of the research participants were pricked by needle stick injury within one year of period, and 48.8% of those who were stung by needle stick injuries did not agree upon the presence of satisfactory procedures in case of needle stick injuries in the study hospital. 17.9% and 49.5%, 24.1% and 60.8%, 7.6% and 50% of the respondents are pricked by needle sticks, and they disagree on the availability of satisfactory procedures in case of accidents, respectively, in government health centres, medium clinics, small clinics, and surgical centre respectively.

One hundred seventy-seven (32.7% (CI:29.1–37) respondents were exposed to needle stick injury while working in the current health facilities. One hundred three (58.1%) and 26 (32.9%) needle stick injuries were reported from WUNEMMCSH and medium clinics, which is above the mean. One hundred thirty-two(24.7% (95%CI:20.7–28.1) of the respondents are exposed to needle stick injury within one year of the period. Seventy-eight(30.2%), 17 (17.9%), 19 (24.1%), 15 (16.3%), 3 (18.8%) of the staff are injured by needle sticks from NEMMCSH, government health centres, medium clinics, small clinics, and surgical centre staffs respectively within one year of service.

The mean availabilities of satisfactory procedures in case of accidents were 321 (59.4% (CI:55.4–63.7). Out of this, 13.7% of the staff is injured by needle sticks within one year before the survey. Except in NEMMCSH, the mean availabilities of satisfactory procedures were above the mean, which is 50%, 60%, 77.2%, 66.3%, and 81.3% in NEMMCSH, government health centres, medium clinics, small clinics, and surgical centres respectively.

Table 5 below shows that Hepatitis B, COVID-19, and tetanus toxoid vaccinations are the responses of the research participants to an open-ended question on which vaccine they took. The finding shows that 220 (40.8%) of the respondents were vaccinated to prevent themselves from health facility-acquired infection. One hundred fifty-six (70.9%) of the respondents are vaccinated to avoid themselves from Hep B infection. Fifty-nine (26%0.8) of the respondents were vaccinated to protect themselves from two diseases that are Hep B and COVID-19.

Appropriate health care waste management practice was assessed by using 12 questions: availability of colour-coded waste bins, foot-operated dust bins, elbow or foot-operated hand washing basin, personal protective equipment, training, role and responsibility of the worker, the presence of satisfactory procedures in case of an accident, incinerator, vaccination, guideline, onsite treatment, and the availability of poster. The mean of appropriate healthcare waste management practice was 55.58%. The mean of solid health care waste management practice based on the level of health facilities was summed and divided into 12 variables to get each health facility’s level of waste management practice. 64.9%, 45.58%, 49%, 46.9%, and 51.8% are the mean appropriate health care waste management practices in NEMMCSH, government health centres, medium clinics, small clinics, and surgical centres, respectively. In NEMMCSH, the practice of solid healthcare waste management shows above the mean, and the rest was below the mean of solid healthcare waste management practice.

Healthcare waste treatment and disposal practice

Solid waste treatment before disposal was not practised at all study health facilities. There is an incineration practice at all of the study health facilities, and the World Health Organization 2014 recommended three types of incineration practice for solid health care waste management: dual-chamber starved-air incinerators, multiple chamber incinerators, and rotary kilns incinerators. Single-chamber, drum, and brick incinerators do not meet the best available technique requirements of the Stockholm Convention guidelines [ 13 ]. The findings of this study show that none of the incinerators found in the study health facilities meet the minimum standards of solid healthcare waste incineration practice, and they need an air inlet to facilitate combustion. Eleven (26.82%) of the health facilities have an ash pit to dispose of burned SHCW; the majority, 30 (73.17%), dispose of the incinerated ash and burned needles in the municipal waste disposal site. In one out of 11 health facilities with an ash pit, one of the incinerators was built on the ash pit, and the incinerated ashes were disposed of in the ash pit directly. Pre-treatment of SHCW before disposal was not practised at all health facilities; see Table  6 below.

All government health facilities use incineration to dispose of solid waste. 88.4% and 100% of the solid wastes are incinerated in WUNEMMCS Hospital and government health centres, respectively. This finding was not similar to the other studies because other technologies like autoclave microwave and incineration were used for 59–60% of the waste [ 15 ]. Forty-one (100%) of the study facilities were using incinerators, and only 5 (12.19%) of the incinerators were constructed by using brick and more or less promising than others for incinerating the generated solid wastes without considering the emitting gases into the atmosphere and the residue chemicals and minerals in the ashes.

Research participants’ understanding of the environmental friendliness of health care waste management practice was assessed, and the result shows that more than half, 312(57%) of the research participants do not agree on the environmental friendliness of the waste disposal practices in the health facilities. The most disagreement regarding environmental friendliness was observed in NEMMCSH; 100 (38.8%) of the participants only agreed the practice was environmentally friendly of the service. Forty-four (46.3%), 37 (46.8%), 40 (43.5%), and 7 (43.8%) of the participants agree on the environmental friendliness of healthcare waste management practice in government health centres, medium clinics, small clinics, and surgical centres, respectively.

One hundred twenty-five (48.4%) and 39(42.4%) staff are trained in solid health care waste management practice in NEMMCSH and small clinic staff, respectively; this result shows above the mean. Twenty-seven (28.4%), 30 (38%), and 4 (25%) of the staff are trained in health care waste management practice in Government health centres, medium clinics, and surgical centres, respectively. The training has been significantly associated with needle stick injury, and the more trained staff are, the less exposed to needle stick injury. One hundred ninety-six (36.4%) of the participants answered yes to the question about the availability of trainers in the institution. 43.8% of the NEMMCSH staff agreed on the availability of trainers on solid health care waste management, which is above the mean, and 26.3%, 31.6%, 31.5%, and 25% for the government health centres, medium clinics, small clinics, and surgical centre respectively, which is below the mean.

Trained health professionals are more compliant with SHCWM standards, and the self-reported study findings of this study show that 41.7% (95%CI:37.7–46) of the research participants are trained in health care waste management practice. This finding was higher compared to the study findings of Sahiledengle in 2019 in the southeast of Ethiopia, shows 13.0% of healthcare workers received training related to HCWM in the past one year preceding the study period and significantly lower when compared to the study findings in Egypt which is 71% of the study participants were trained on SHCWM [ 8 , 19 , 20 ].

Three out of four government health facility leaders, 17 (45.94%) of private health facility leaders/owners of the clinic and 141 FGD participants complain about the absence of some PPEs like boots and aprons to protect themselves from infectious agents.

‘ ‘Masks, disposable gloves, and changing gowns are a critical shortage at all health facilities.’’

Cleaners in private health facilities are more exposed to infectious agents because of the absence of personal protective equipment. Except for the cleaning staff working in the private surgical centre, all cleaning staff 40 (97.56) of the health facilities complain about the absence of changing gowns and the fact that there are no boots in the facilities.

Cost inflation and the high cost of purchasing PPEs like gloves and boots are complained by all of (41) the health facility owners and the reason for the absence of some of the PPEs like boots, goggles, and shortage of disposable gloves. Sometimes, absence from the market is the reason why we do not supply PPE to our workers.

Thirty-four (82.92%) of the facility leaders are forwarded, and there is a high expense and even unavailability of some of the PPEs, which are the reasons for not providing PPEs for the workers.

‘‘Medical equipment and consumables importers and whole sellers are selective for importing health supplies, and because of a small number of importers in the country and specifically, in the locality, we can’t get materials used for health care waste management practice even disposable gloves. ’’

One of the facility leaders from a private clinic forwarded that before the advent of COVID-19 -19) personal protective equipment was more or less chip-and-get without difficulty. Still, after the advent of the first Japanese COVID-19 patient in Ethiopia, people outside the health facilities collect PPEs like gloves and masks and storing privately in their homes.

‘‘PPEs were getting expensive and unavailable in the market. Incinerator construction materials cost inflation, and the ownership of the facility building are other problems for private health facilities to construct standard incinerators.’’

For all of the focus group discussion participants except in NEMMCSH and two private health facilities, covered and foot-operated dust bins were absent or in a critical shortage compared to the needed ones.

‘‘ Waste bins are open and not colour-coded. The practice attracts flies and other insects. Empty waste bins are replaced without cleaning and disinfecting by using chlorine solution.’’ “HCW containers are not colour-coded, but we are trying to label infectious and non-infectious in Amharic languages.”

Another issue raised during focus group discussions is incineration is not the final disposal method. It needs additional disposal sites, lacks technology, is costly to construct a brick incinerator, lacks knowledge for health facility workers, shortage of man powers /cleaners, absence of environmental health professionals in health centres and all private clinics, and continues exposure to the staff for needle stick injury, foully smell, human scavengers, unsightly, fire hazard, and lack of water supply in the town are the major teams that FGD participants raise and forwarded the above issue as a problem to improve SHCWMP.

Focus group participants, during the discussion, raised issues that could be more comfortable managing SHCWs properly in their institution. Two of the 37 private health facilities are working in their own compound, and the remaining 35 are rented; because of this, they have difficulty constructing incinerators and ash removal pits and are not confident about investing in SHCWM systems. Staff negligence and involuntary abiding by the rules of the facilities were raised by four of the government health facilities, and it was difficult to punish those who violated the healthcare waste management rules because the health facility leaders were not giving appropriate attention to the problem.

Focus group participants forwarded recommendations on which interventions can improve the management of SHCW, and recommendations are summarised as follows:

“PPE should be available in quality and quantity for all health facility workers who have direct contact with SHCW.” “Scientific-based waste management technologies should be availed for health facilities.” “Continuous induction HCW management training should be provided to the workers. Law enforcement should be strengthened.” “Communal HCW management sites should be availed, especially for private health facilities.” “HCWM committee should be strengthened.” “Non-infectious wastes should be collected communally and transported to the municipal SHCW disposal places.” “Leaders should be knowledgeable on the SHCWM system and supervise the practice continuously.” “Patient and client should be oriented daily about HCW segregation practice.” “Regulatory bodies should supervise the health facilities before commencing and periodically between services .”

The above are the themes that FGD participants discussed and forwarded for the future improvements of SHAWMP in the study areas.

Lack of water supply in the town

Other issues raised during FGDs were health facilities’ lack of water supply. World Health Organization (2014: 89) highlights that water supply for the appropriate waste management system should be mandatory at any time in all health service delivery points.

Thirty-nine (95.12%) of the health facilities complain about the absence of water supply to improve HCW management practices and infection prevention and control practices in the facilities.

“We get water once per week, and most of the time, the water is available at night, and if we are not fetching as scheduled, we can’t get water the whole week”.

In this research, only those who have direct contact have participated in this study, and 434 (80.4%) of the respondents agree they have roles and responsibilities for appropriate solid health care waste management practice. The rest, 19.6%, do not agree with their commitment to manage health care wastes properly, even though they are responsible. Health facility workers in NEMMCSH and medium clinics know their responsibilities better than others, and their results show above the mean. 84.5%, 74.5%, 81%, 73.9% and 75% in NEMMCSH, Government health centres, medium clinics, small clinics, and surgical centres, respectively.

Establishing a policy and a legal framework, training personnel, and raising public awareness are essential elements of successful healthcare waste management. A policy can be viewed as a blueprint that drives decision-making at a political level and should mobilize government effort and resources to create the conditions to make changes in healthcare facilities. Three hundred and seventy-four (69.3%) of the respondents agree with the presence of any solid healthcare waste management policy in Ethiopia. The more knowledge above the mean (72.9%) on the presence of the policy is reported from NEMMCSH.

Self-reported level of knowledge on what to do in case of an accident revealed that 438 (81.1% CI: 77.6–84.3%) of the respondents knew what to do in case of an accident. Government health centre staff and medium clinic staff’s knowledge about what to do in case of an accident was above the mean (88.4% and 82.3%), respectively, and the rest were below the mean. The action performed after an occupational accident revealed that 56 (35.7%) of the respondents did nothing after any exposure to an accident. Out of 56 respondents who have done nothing after exposure, 47 (83.92%) of the respondents answered yes to their knowledge about what to do in case of an accident. Out of 157 respondents who have been exposed to occupational accidents, only 59 (37.6%) of the respondents performed the appropriate measures, 18 (11.5%), 9 (5.7%), 26 (16.6%), 6 (3.8%) of the respondents are taking prophylaxis, linked to the incident officer, consult the available doctors near to the department, and test the status of the patient (source of infection) respectively and the rest were not performing the scientific measures, that is only practising one of the following practices washing the affected part, squeezing the affected part to remove blood, cleaning the affected part with alcohol.

Health facility workers’ understanding of solid health care waste management practices was assessed by asking whether the current SHCWM practice needs improvement. Four hundred forty-nine (83.1%) health facility workers are unsatisfied with the current solid waste management practice at the different health facility levels, and they recommend changing it to a scientific one. 82.6%, 87.4%, 89.9%, 75%, and 81.3% of the respondents are uncomfortable or need to improve solid health care waste management practices in NEMMCSH, government health centres, medium clinics, small clinics, and surgical centres, respectively.

Lack of safety box, lack of colour-coded waste bins, lack of training, and no problems are the responses to the question problems encountered in managing SHCWMP. Two Hundred and Fifty (46.92%) and 232 (42.96%) of the respondents recommend the availability of safety boxes and training, respectively.

Four or 9.8% of the facilities have infection prevention and control (IPC) teams in the study health facilities. This finding differed from the study in Pakistan, where thirty per cent (30%) of the study hospitals had HCWM or infection control teams [ 21 ]. This study’s findings were similar to those conducted in Pakistan by Khan et al. [ 21 ], which confirmed that the teams were almost absent at the secondary and primary healthcare levels [ 20 ].

The availability of health care waste management policy report reveals that 69.3% (95% CI: 65.4–73) of the staff are aware of the presence of solid health care waste management policy in the institution. Availability of health care waste management policy was 188 (72.9%), 66 (69.5%), 53 (677.1%), 57 (62%), 10 (62.5%) in NEMMCSH, Government health centres, medium clinics, small clinics, and surgical centre respectively. Healthcare waste management policy availability was above the mean in NEMMCSH and government health centres; see Table  6 below.

Open-ended responses on the SHCWM practice of health facility workers were collected using the prepared interview guide, and the responses were analyzed using thematic analysis. All the answered questions were tallied on the paper and exported to Excel software for thematic analysis.

The study participants recommend.

“appropriate segregation practice at the point of generation” "health facility must avail all the necessary supplies that used for SHCWMP, punishment for those violating the rule of SHCWMP",

“waste management technologies should be included in solid waste management guidelines, and enforcement should be strengthened.”

The availability of written national or adopted/adapted SHCWM policies was observed at all study health facilities. Twenty eight (11.66%) of the rooms have either a poster or a written document of the national policy document. However, all staff working in the observed rooms have yet to see the inside content of the policy. The presence of the policy alone cannot bring change to SHCWMP. This finding shows that the presence of policy in the institution was reasonable compared to the study findings in Menelik II hospital in Addis Ababa, showing that HCWM regulations and any applicable facility-based policy and strategy were not found [ 22 ]. The findings of this study were less compared to the study findings in Pakistan; 41% of the health facilities had the policy document or internal rules for the HCWM [ 21 ].

Focus group participants have forwarded recommendations on which interventions can improve the management of SHCW, and recommendations are summarised as follows.

‘‘Supplies should be available in quality and quantity for all health facility workers with direct contact with SHCW. Scientific-based waste management technologies should be available for health facilities. Continues and induction health care waste management training should be provided to the workers. Law enforcement should be strengthened. Community healthcare waste management sites should be available, especially for private health facilities. HCWM committee should be strengthened. Non-infectious wastes should be collected communally and transported to the municipal SHCW disposal places. Leaders should be knowledgeable about the SHCWM system and supervise the practice continuously. Patients and clients should be oriented daily about health care waste segregation practices. Regulatory bodies should supervise the health facilities before commencing and periodically in between the service are the themes those FGD participants discussed and forward for the future improvements of SHCWMP in the study areas.’’

The availability of PPEs in different levels of health facilities shows 392 (72.6%), 212 (82.2%), 56 (58.9%), 52 (65.8%), 60 (65.2%), 12 (75%) health facility workers in NEMMCSH, government health centres, medium clinics, small clinics, and surgical centres respectively agree to the presence of personal protective equipment in their department. The availability of PPEs in this study was nearly two-fold when compared to the study findings in Myanmar, where 37.6% of the staff have PPEs [ 12 ].

The mean availability of masks, heavy-duty gloves, boots, and aprons was 71.1%, 65.4%, 38%, and 44.4% in the study health facilities. This finding shows masks are less available in the study health facilities compared to other studies. The availability of utility gloves, boots, and plastic aprons is good in this study compared to the study conducted by Banstola, D in Pokhara Sub-Metropolitan City [ 23 ].

The findings of this study show there is a poor segregation practice, and all kinds of solid wastes were collected together. This finding was similar to the study findings conducted in Addis Ababa, Ethiopia, by Debere et al. [ 24 ] and contrary to the study findings conducted in Nepal and India, which shows 50% and 65–75% of the surveyed health facilities were practising proper waste segregation systems at the point of generation without mixing general wastes with hazardous wastes respectively [ 9 , 17 ].

Ninety percent of private health facilities collect and transport SHCW generated in every service area and transport it to the disposal place by the collection container (no separate container to collect and transport the waste to the final disposal site). This finding was similar to the study findings of Debre Markos’s town [ 25 ]. At all of the facilities in the study area, SHCW was transported from the service areas to the disposal site manually by carrying the collection container, and there was no trolley for transportation. This finding was contrary to the study findings conducted in India, which show segregated waste from the generation site was being transported through the chute to the carts placed at various points on the hospital premises by skilled sanitary workers [ 17 ].

Observational findings revealed that pre-treatment of SHCW before disposal was not practised at all study health facilities. This study was contrary to the findings of Pullishery et al. [ 26 ], conducted in Mangalore, India, which depicted pre-treatment of the waste in 46% of the hospitals [ 26 ]. 95% of the facilities have no water supply for handwashing during and after solid healthcare waste generation, collection, and disposal. This finding was contrary to the study findings in Pakistan hospitals, which show all health facilities have an adequate water supply near the health care waste management sites [ 27 ].

Questionnaire data collection tools show that 129 (23.8%) of the staff needle stick injuries have occurred on health facility workers within one year of the period before the data collection. This finding was slightly smaller than the study findings of Deress et al. [ 25 ] in Debre Markos town, North East Ethiopia, where 30.9% of the workers had been exposed to needle stick injury one year prior to the study [ 25 ]. Reported and registered needle stick injuries in health facilities are less reported, and only 70 (54.2%) of the injuries are reported to the health facilities. This finding shows an underestimation of the risk and the problem, which was supported by the study conducted in Menilik II hospitals in Addis Ababa [ 22 ]. 50%, 33.4%, 48%, 52%, and 62.5% of needle stick injuries were not reported in NEMMCSH, Government health centres, medium clinics, small clinics, and surgical centres, respectively, to the health facility manager.

Nearly 1/3 (177 or 32.7%) of the staff are exposed to needle stick injuries. Needle stick injuries in health facilities are less reported, and only 73 (41.24%) of the injuries are reported to the health facilities within 12 months of the data collection. This finding is slightly higher than the study finding of Deress et al. [ 25 ] in Debere Markos, Ethiopia, in which 23.3% of the study participants had encountered needle stick/sharps injuries preceding 12 months of the data collection period [ 25 ].

Seventy-three injuries were reported to the health facility manager in the last one year, 44 of the injuries were reported by health professionals, and the rest were reported by supportive staff. These injuries were reported from 35(85.3%) health facilities; the remaining six have no report. These study findings were better than the findings of Khan et al. [ 21 ], in which one-third of the facilities had a reporting system for an incident, and almost the same percentage of the facilities had post-exposure procedures in both public and private sectors [ 21 ].

Within one year of the study period, 129 (23.88%) needle stick injuries occurred. However, needle stick injuries in health facilities are less reported, and only 70 (39.5%) of the injuries are reported to the health facilities. These findings were reasonable compared to the study findings of the southwest region of Cameroon, in which 50.9% (110/216) of all participants had at least one occupational exposure [ 28 , 29 ]. This result report shows a very high exposure to needle stick injury compared to the study findings in Brazil, which shows 6.1% of the research participants were injured [ 27 ].

The finding shows that 220 (40.8%) of the respondents were vaccinated to prevent themselves from health facility-acquired infection. One Hundred Fifty-six (70.9%) of the respondents are vaccinated in order to avoid themselves from Hep B infection. Fifty-nine (26%0.8) of the respondents were vaccinated to protect themselves from two diseases that are Hep B and COVID-19. This finding was nearly the same as the study findings of Deress et al. [ 7 ],in Ethiopia, 30.7% were vaccinated, and very low compared to the study findings of Qadir et al. [ 30 ] in Pakistan and Saha & Bhattacharjya India which is 66.67% and 66.17% respectively [ 25 , 30 , 31 ].

The incineration of solid healthcare waste technology has been accepted and adopted as an effective method in Ethiopia. These pollutants may have undesirable environmental impacts on human and animal health, such as liver failure and cancer [ 15 , 16 ]. All government health facilities use incineration to dispose of solid waste. 88.4% and 100% of the wastes are incinerated in WUNEMMCSH and government health centres, respectively. This finding contradicts the study findings in the United States of America and Malaysia, which are 49–60% and 59–60 are incinerated, respectively, and the rest are treated using other technologies [ 15 , 16 ].

All study health facilities used a brick or barrel type of incinerator. The incinerators found in the study health facilities need to meet the minimum standards of solid health care waste incineration practice. These findings were similar to the study findings of Nepal and Pakistan [ 32 ]. The health care waste treatment system in health facilities was found to be very unsystematic and unscientific, which cannot guarantee that there is no risk to the environment and public health, as well as safety for personnel involved in health care waste treatment. Most incinerators are not properly operated and maintained, resulting in poor performance.

All government health facilities use incineration to dispose of solid waste. All the generated sharp wastes are incinerated using brick or barrel incinerators, as shown in Fig.  1 above. This finding was consistent with the findings of Veilla and Samwel [ 33 ], who depicted that sharp waste generation is the same as sharps waste incinerated [ 33 ]. All brick incinerators were constructed without appropriate air inlets to facilitate combustion except in NEMMCSH, which is built at a 4-m height. These findings were similar to the findings of Tadese and Kumie at Addis Ababa [ 34 ].

Barrel and brick incinerators used in private clinic

Strengths and limitations

This is a mixed-method study; both qualitative and quantitative study design, data collection and analysis techniques were used to understand the problem better. The setting for this study was one town, which is found in the southern part of the country. It only represents some of the country’s health facilities, and it is difficult to generalize the findings to other hospitals and health centres. Another limitation of this study was that private drug stores and private pharmacies were not incorporated.

Conclusions

In the study, health facilities’ foot-operated solid waste dust bins are not available for healthcare workers and patients to dispose of the generated wastes. Health facility managers in government and private health institutions should pay more attention to the availability of colour-coded dust bins. Most containers are opened, and insects and rodents can access them anytime. Some of them are even closed (not foot-operated), leading to contamination of hands when trying to open them.

Healthcare waste management training is mandatory for appropriate healthcare waste disposal. Healthcare-associated exposure should be appropriately managed, and infection prevention and control training should be provided to all staff working in the health facilities.

Availability of data and materials

The authors declare that data for this work are available upon request to the first author.

Chartier, Y et al. Safe management of wastes from health-care activities. 2nd ed. WHO; 2014.

Tesfahun E, et al. Developing models for the prediction of hospital healthcare waste generation rate. Waste Manag Res. 2014;34(1):75–80.

Manzoor J, Sharma M. Impact of Biomedical Waste on Environment and Human Health. Environmental Claims Journal. 2019;31(4):311–34.

Article   Google Scholar  

Yves C, Jorge E, Ute P, Annette P, et al. Safe management of wastes from health-care activities. WHO 2nd ed. 2014.

OSHA. Occupational Safety and Health Administration, Guidelines for Healthcare Waste Management. 2023.

Godfrey L, Ahmed M, et al. Solid waste management in Africa: governance failure or development opportunity?. Intech open. 2019.

Deress T, Jemal M, Girma M, Adane K. Knowledge, attitude, and practice of waste handlers about medical waste management in Debre Markos town healthcare facilities, northwest Ethiopia. BMC Res Notes. 2019;12(1):146.

Article   PubMed   PubMed Central   Google Scholar  

Sahiledengle B. Self-reported healthcare waste segregation practice and its correlate among healthcare workers in hospitals of Southeast Ethiopia. BMC Health Serv Res. 2019;19(1):591.

Debalkie D, Kume A. Healthcare Waste Management: The Current Issue in Menellik II Referral Hospital, Ethiopia. Curr World Environ. 2017;12(1):42–52.

Debere MK, Gelaye KA, Alamdo AG, Trifa ZM. Assessment of the health care waste generation rates and its management system in hospitals of Addis Ababa, Ethiopia, 2011. BMC Public Health. 2013;13(28).

Creswell JW. Research design qualitative, quantitative, & mixed method approach. 4th ed. SAGE Publications, Inc.; 2014.

Win EM, Saw YM, Oo KL, Than TM, Cho SM, Kariya T, et al. Healthcare waste management at primary health centres in Mon State, Myanmar: the comparisons between hospital and non-hospital type primary health centres. Nagoya J Med Sci. 2019;81(1):81–91.

PubMed   PubMed Central   Google Scholar  

WHO. Safe management of wastes from health-care activities. 2nd ed. editor Chartier, Y et al. 2014. 

Richard B, Ben A, Kristian S. Health care without harm climate-smart health care series green paper number one. 2019.

Khadem Ghasemi M, Mohd YR. Advantages and Disadvantages of Healthcare Waste Treatment and Disposal Alternatives: Malaysian Scenario. Pol J Environ Stud. 2016;25(1):17–25.

Mohseni-Bandpei A, Majlesi M, Rafiee M, Nojavan S, Nowrouz P, Zolfagharpour H. Polycyclic aromatic hydrocarbons (PAHs) formation during the fast pyrolysis of hazardous health-care waste. Chemosphere. 2019;227:277–88.

Article   PubMed   CAS   Google Scholar  

Pandey A, Ahuja S, Madan M, Asthana AK. Bio-Medical Waste Managment in a Tertiary Care Hospital: An Overview. J Clin Diagn Res. 2016;10(11):DC01-DC3.

Doylo T, Alemayehu T, Baraki N. Knowledge and Practice of Health Workers about Healthcare Waste Management in Public Health Facilities in Eastern Ethiopia. J Community Health. 2019;44(2):284–91.

Article   PubMed   Google Scholar  

Hosny G, Samir S, Sharkawy R. An intervention significantly improve medical waste handling and management: A consequence of raising knowledge and practical skills of health care workers. Int J Health Sci.2018;12(4).

Khan EA, Sabeeh SM, Chaudhry MA, Yaqoob A, Kumar R. et al. Health care waste management in Pakistan: A situational analisis and way forward. Pak J Public Health. 2016;6(3).

Khan BA, Cheng L, Khan AA, Ahmed H. Healthcare waste management in Asian developing countries: A mini review. Waste Manag Res. 2019;37(9):863–75.

Debalkie D, Kumie A. Healthcare Waste Management: The Current Issue in Menellik II Referral Hospital. Ethiopia Current World Environment. 2017;12(1):42–52.

Banstola D, Banstola R, Nepal D, Baral P. Management of hospital solid wastes: A study in Pokhara sub metropolitan city. J Institute Med. 2017;31(1):68–74.

Debere MK, Gelaye KA, Alamdo AG, Trifa, ZM. Assessment of the HCW generation rates and its management system in hospitals of Addis Ababa, Ethiopia. BMC Public Health. 2014;13(28):1–9.

Deress T, Hassen F, Adane K, Tsegaye A. Assessment of Knowledge, Attitude, and Practice about Biomedical Waste Management and Associated Factors among the Healthcare Professionals at Debre Markos Town Healthcare Facilities. Northwest Ethiopia J Environ Public Health. 2018;2018:7672981.

PubMed   Google Scholar  

Pullishery F, Panchmal GS, Siddique S, Abraham A. Awareness, knowledge, and practices on bio-medical waste management among health care professionals in Mangalore- A cross sectional study. Integr Med. 2016;3(1):29–35.

Google Scholar  

Ream PS, Tipple AF, Salgado TA, Souza AC, Souza SM, Galdino-Junior H, et al. Hospital housekeepers: Victims of ineffective hospital waste management. Arch Environ Occup Health. 2016;71(5):273–80.

Ngwa CH, Ngoh EA, Cumber SN. Assessment of the knowledge, attitude and practice of health care workers in Fako division on post exposure prophylaxis to blood borne viruses: a hospital based cross-sectional study. Pan Afr Med J. 2018;31.

Health care waste managemnt in pakistan. a situation analysis and way forward. Pakistan Journal of Public Health. 2016;6(3):35–45.

Qadir DM, Murad DR, Faraz DN. Hospital Waste Management; Tertiary Care Hospitals. The Professional Medical Journal. 2016;23(07):802–6.

Saha A, Bhattacharjya H. Health-Care Waste Management in Public Sector of Tripura, North-East India: An Observational Study. Indian J Community Med. 2019;44(4):368–72.

Pullishery F, Panchmal G, Siddique S, Abraham A. Awareness, knowledge, and practices on bio-medical waste management among health care professionals in Mangalore- A cross sectional study. Integr Med. 2016;3(1):29–35.

Veilla EM, Samwel VM. Assessment of sharps waste management practices in a referral hospital. Afr J Environ Sci Technol. 2016;10(3):86–95.

Tadesse ML, Kumie A. Healthcare waste generation and management practice in government health centers of Addis Ababa. Ethiopia BMC Public Health. 2014;14:1221.

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Acknowledgements

The authors are grateful to the health facility leaders and ethical committees of the hospitals for their permission. The authors acknowledge the cooperation of the health facility workers who participated in this study.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Dr. Yeshanew Ayele Tiruneh is a researcher of this study; the principal investigator does all the proposal preparation, methodology, data collection, result and discussion, and manuscript writing. Professor LM Modiba and Dr. SM Zuma are supervisors for this study. They participated in the topic selection and modification to the final manuscript preparation by commenting on and correcting the study. Finally, the three authors read and approved the final version of the manuscript and agreed to submit the manuscript for publication.

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Tiruneh, Y.A., Modiba, L.M. & Zuma, S.M. Solid health care waste management practice in Ethiopia, a convergent mixed method study. BMC Health Serv Res 24 , 985 (2024). https://doi.org/10.1186/s12913-024-11444-8

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Inter-laboratory comparison of eleven quantitative or digital PCR assays for detection of proviral bovine leukemia virus in blood samples

• Aneta Pluta 1 , 13 ,
• Juan Pablo Jaworski 2 ,
• Casey Droscha 3 ,
• Sophie VanderWeele 3 ,
• Tasia M. Taxis 4 ,
• Stephen Valas 5 ,
• Dragan Brnić 6 ,
• Andreja Jungić 6 ,
• María José Ruano 7 ,
• Azucena Sánchez 7 ,
• Kenji Murakami 8 ,
• Kurumi Nakamura 8 ,
• Rodrigo Puentes 9 ,
• MLaureana De Brun 9 ,
• Vanesa Ruiz 2 ,
• Marla Eliana Ladera Gómez 10 ,
• Pamela Lendez 10 ,
• Guillermina Dolcini 10 ,
• Marcelo Fernandes Camargos 11 ,
• Antônio Fonseca 11 ,
• Subarna Barua 12 ,
• Chengming Wang 12 ,
• Aleksandra Giza 13 &
• Jacek Kuźmak 1  

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Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis and causes a persistent infection that can leave cattle with no symptoms. Many countries have been able to successfully eradicate BLV through improved detection and management methods. However, with the increasing novel molecular detection methods there have been few efforts to standardize these results at global scale. This study aimed to determine the interlaboratory accuracy and agreement of 11 molecular tests in detecting BLV. Each qPCR/ddPCR method varied by target gene, primer design, DNA input and chemistries. DNA samples were extracted from blood of BLV-seropositive cattle and lyophilized to grant a better preservation during shipping to all participants around the globe. Twenty nine out of 44 samples were correctly identified by the 11 labs and all methods exhibited a diagnostic sensitivity between 74 and 100%. Agreement amongst different assays was linked to BLV copy numbers present in samples and the characteristics of each assay (i.e., BLV target sequence). Finally, the mean correlation value for all assays was within the range of strong correlation. This study highlights the importance of continuous need for standardization and harmonization amongst assays and the different participants. The results underscore the need of an international calibrator to estimate the efficiency (standard curve) of the different assays and improve quantitation accuracy. Additionally, this will inform future participants about the variability associated with emerging chemistries, methods, and technologies used to study BLV. Altogether, by improving tests performance worldwide it will positively aid in the eradication efforts.

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Introduction

Bovine leukemia virus (BLV) is a deltaretrovirus from the Orthoretrovirinae subfamily of the Retroviridae family. An essential step in the BLV replication cycle is the integration of DNA copy of its RNA genome into the DNA of a host cell [ 1 ]. Once integrated, the proviral DNA is replicated along with the host’s DNA during cellular divisions, as for any cellular gene. The BLV is the etiologic agent of enzootic bovine leukosis (EBL). BLV causes a persistent infection in cattle, and in most cases this infection is asymptomatic [ 2 ]. In one-third of infected animals the infection progresses to a state of persistent lymphocytosis, and in 1 to 10% of infected cattle it develops into lymphosarcoma [ 2 ]. BLV induces high economic losses due to trade restrictions, replacement cost, reduced milk production, immunosuppression, and increased susceptibility to pneumonia, diarrhea, mastitis, and so on [ 3 , 4 , 5 , 6 ]. BLV is globally distributed with a high prevalence, except for Western Europe and Oceania, where the virus has been successfully eradicated through detection and elimination of BLV-infected animals [ 7 , 8 ]. The agar gel immunodiffusion and ELISA for the detection of BLV-specific antibodies in sera and milk are the World Organization for Animal Health (WOAH, founded as OIE) prescribed tests for serological diagnosis but ELISA, due to its high sensitivity and ability to test many samples at a very low cost, is highly recommended [ 9 ]. Despite the advantages of serologic testing, there are some scenarios in which direct detection of the BLV genomic fragment was important to improve BLV detection. The most frequent cases is the screening of calves with maternal antibodies, acute infection, animals without persistent antibody response and animal subproducts (i.e., semen). In this regard, nucleic acid amplification tests such as real-time quantitative PCR (qPCR) allows for a rapid and highly sensitive detection of BLV proviral DNA (BLV DNA) that can be used to test infected and asymptomatic animals, before the elicitation of anti-BLV specific antibodies and when proviral load (PVL) are still low [ 10 ]. Furthermore, qPCR assays can serve as confirmatory tests for the clarification of inconclusive and discordant serological test results usually associated with these cases [ 11 ]. For these reasons, the inclusion of qPCR in combination with other screening tests might increase control programs efficiency. Additionally, qPCR allows the estimation of BLV PVL which is important for studying the dynamics of BLV infection (i.e., basic research). Further, considering that BLV PVL correlates with the risk of BLV transmission, this feature of qPCR can be exploited for developing rational segregation programs [ 12 , 13 ]. The results of Kobayashi et al. suggest that high PVL is also a significant risk factor for progression to EBL and should therefore be used as a parameter to identify cattle for culling from the herd well before EBL progression [ 14 ]. Several qPCRs have been developed globally for the quantitation of BLV DNA. Although most assays have been properly validated by each developer, a proper standardization and harmonization of such tests is currently lacking. Considering that standardization and harmonization of qPCR methods and results are essential for comparisons of data from BLV laboratories around the world, this could directly impact international surveillance programs and collaborative research. We built a global collaborative network of BLV reference laboratories to evaluate the interlaboratory variability of different qPCRs and sponsored a harmonization of assays to hopefully impact international surveillance programs and research going forward.

In 2018 we conducted the first global trial of this kind to assess the interlaboratory variability of six qPCRs for the detection of BLV DNA [ 15 ]. Since this complex process is a continuous rather than a one-time effort, we now started a second study of this type. In this follow up study, we built a more comprehensive sample panel, accounting for a broader geographical diversification. Additionally, we increased the number of participants to ten collaborating laboratories plus one WOAH reference lab and tested novel methodologies including digital PCR (ddPCR) and FRET-qPCR. Finally, we established the next steps towards the international standardization of molecular assays for the detection of BLV DNA.

Materials and methods

Participants.

The eleven laboratories that took part in the study were:(i) the Auburn University College of Veterinary Medicine (Auburn, Alabama, United States): (ii) AntelBio, a division of CentralStar Cooperative (Michigan, United States); (iii) Laboratórios Federais de Defesa Agropecuária de Minas Gerais (LFDA-MG, Pedro Leopoldo, Brasil); (iv) Centro de Investigación Veterinaria de Tandil (CIVETAN, Buenos Aires, Argentina); (v) the Faculty of Agriculture Iwate University (Iwate, Japan); (vi) Universidad de la República de Uruguay (UdelaR, Montevideo, Uruguay); (vii) the Croatian Veterinary Institute (Zagreb, Croatia); (viii) Instituto Nacional de Tecnología Agropecuaria (INTA, Buenos Aires, Argentina); (ix) Laboratorio Central de Veterinaria (LCV, Madrid, Spain); (x) the National Veterinary Research Institute (NVRI, Puławy, Poland) and (xi) the French Agency for Food, Environmental and Occupational Health and Safety (Anses, Niort, France). All European laboratories participating in this study are acting as national reference laboratories for EBL, NVRI acts as WOAH reference laboratory for EBL, while the remaining laboratories are nationally renowned entities for BLV diagnostics. The eleven participating methods are referred to below as qPCR1 – qPCR5, ddPCR6, qPCR7 – qPCR11, respectively.

Sample collection and DNA extraction

A total of 42 DNA samples obtained from blood of naturally BLV-infected dairy cattle from Poland, Moldova, Pakistan, Ukraine, Canada and United States were used for this study. Thirty-six of them were archival DNA samples obtained between 2012–2018 as described in our previous studies on samples from Poland ( n  = 21) [ 16 , 17 ], Moldova ( n  = 4) [ 18 ], Pakistan ( n  = 5) [ 19 ] and Ukraine ( n  = 6) [ 15 , 20 ]. Between 2020–2021 6 peripheral blood and serum samples from naturally BLV-infected cattle were obtained from three dairy farms of Alberta, Canada and two dairy farms of Michigan, US. Serological testing and sample processing were conducted by the laboratories from which the samples originated. The genomic DNA from Canadian and US samples was extracted from whole blood using a Quick DNA Miniprep Plus kit (Zymo Research) and a DNeasy Blood & Tissue Kit (Qiagen), respectively in University of Calgary and Michigan State University and sent to the NVRI in the form of DNA solutions. Additionally, one plasmid DNA sample (pBLV344) was kindly supplied by Luc Willems (University of Liège, Belgium) and DNA extracted from FLK-BLV cells were included as positive controls. Finally, DNA extracted from PBL of a serologically negative cattle was included as negative control. At the NVRI, the DNA concentration in all samples was estimated by spectrophotometry using a NanoPhotometer (Implen). Each sample was divided into eleven identical aliquots containing between 800 and 4,000 ng of lyophilised genomic DNA. Eleven identical sets of these samples were lyophilized (Alpha 1–4 LSC basic, Martin Christ Gefriertrocknungsanlagen GmbH) and distributed to participating laboratories. At the NVRI, all samples were coded (identification [ 21 ] run numbers 1 to 44) to perform a blinded testing. The samples, together with instructions for their preparation (Additional file 1), were shipped by air at room temperature (RT).

Examination of DNA quality/stability

Since different extraction methods and lyophilization process were employed for the preparation of the DNA samples, it was necessary to test the quality of the DNA at the NVRI laboratory. For that purpose, one complete set of samples ( n  = 44) was tested by Fragment Analyzer (Agilent Technologies), before and after freeze-drying, to assess DNA quality by calculating a Genomic Quality Number (GQN) for every sample. Low GQN value (< 2.5) represents sheared or degraded DNA. A high GQN (> 9) represents undegraded DNA. In addition, quality of DNA was assessed by determination of copy number of the histone H3 family 3A ( H3F3A ) housekeeping gene using quantitative real-time PCR (qPCR) [ 22 ]. The qPCR results were expressed as the number of H3F3A gene copies per 300 ng of DNA in each sample. Grubbs´ test was performed to determine outliers. To test the stability of DNA, samples were stored for 20 days at RT (10 days) and at + 4 °C (10 days) and were retested by Fragment Analyzer and qPCR 21 days later. A Mann–Whitney U-test was used to compare the median values between fresh and stored samples (time 0 and time 1), respectively.

Description of BLV qPCR protocols used by participating laboratories

All participating laboratories performed their qPCR or ddPCR using a variety of different equipment, reagents, and reaction conditions, which had been set up, validated, and evaluated previously and are currently used as working protocols. The specific features of each of these protocols are described below and summarized in Table  1 .

All laboratories applied standard procedures for avoiding false-positive results indicative of DNA contamination, such as the use of separate rooms for preparing reaction mixtures, adding the samples, and performing the amplification reaction. One of the ten BLV qPCRs used LTR region and the remaining nine qPCRs used the pol gene as the target sequence for amplification, while the ddPCR amplified the env gene.

Method qPCR1

The BLV qPCR amplifying a 187-bp pol gene was performed according to a previously published methods [ 23 , 24 ]. A real-time fluorescence resonance energy transfer (FRET) PCR was carried out in a 20-μl PCR mixture containing 10 μl handmade reaction master mix and 10 μl genomic DNA. The PCR buffer was 4.5 mM MgCl2, 50 mM KCl, 20 mM Tris–HCl, pH 8.4, supplemented with 0.05% each Tween20 and Non-idet P-40, and 0.03% acetylated BSA (Roche Applied Science). For each 20 μl total reaction volume, the nucleotides were used at 0.2 mM each and 1.5 U Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA) was used. Primers were used at 1 μM, LCRed640 probe was used at 0.2 μM, and 6-FAM probe was used at 0.1 μM. Amplification was performed in the Roche Light Cycler 480 II (Roche Molecular Biochemicals) using 10 min denaturation step at 95 °C, followed by 18 high-stringency step-down thermal cycles and 30 low-stringency fluorescence acquisition cycles.

A plasmid containing the BLV-PCR amplicon region was diluted ten-fold from 1 × 10 5 copies to 10 copies per 10 µl and was used as a standard to measure the BLV copy numbers.

Method qPCR2

A BLV proviral load qPCR assay developed by AntelBio, a division of CentralStar Cooperative Inc. on Applied Biosystems 7500 Real-Time PCR system [ 25 , 33 ]. This multiplex assay amplifies the BLV pol gene along with the bovine β-actin gene and an internal amplification control, “Spike”. A quantitative TaqMan PCR was carried out in a 25-μl PCR mixture containing 12.5 µl of 2X InhibiTaq Multiplex HotStart qPCR MasterMix (Empirical Bioscience), 16 nM each BLV primer, 16 nM each β-actin primer, 8 nM each spike primer, 8 nM BLV FAM-probe, 8 nM β-actin Cy5-probe, 4 nM spike JOE-probe, 1 µl of an internal spike-in control (10,000 copies per µl), 7.25 µl of nuclease-free water and 4 µl of DNA sample for each qPCR reaction. The thermal PCR protocol was as follows: 95 °C for 10 min, 40 × (95 °C for 15 s, 60 °C for 1 min). Copy numbers of both the BLV pol gene and bovine β-Actin were derived using a plasmid containing target sequences, quantified by ddPCR, diluted 1 × 10 6 copies per µl to 10 copies per µl in tenfold dilutions. DNA concentrations of each sample were measured using a Qubit 4 Fluorometer and used in combination with the qPCR copy numbers to calculate BLV copies per 100 ng.

Method qPCR3

The qPCR assays for the BLV LTR gene were performed according to a previously published methods [ 26 ]. Genomic DNA was amplified by TaqMan PCR with 10 μl of GoTaq Probe qPCR Master Mix × 2 (Promega), 0.6 pmol/μl each primer, 0.3 pmol/µl double-quenched probe and 100 ng genomic DNA. Amplification was performed in the CFX96 cycler (BioRad) according to the protocol: 5 min denaturation at 95°C followed by 45 cycles (60 s at 94°C and 60 s at 60°C). The efficiency of each reaction was calculated from the serial dilution of DNA extracted from BLV persistently infected fetal lamb kidney (FLK) cells, starting at a concentration of 100 ng/µl [ 21 ]. The detection limit was tested using a plasmid containing the target of the qPCRs, starting at 10 3 ng/µl.

Method qPCR4

The quantitative real-time PCR was done with the primers for the BLV pol gene as previously described [ 34 ]. The qPCR reaction mix contained 1 × PCR Master Mix with SYBR Green (FastStart Universal SYBR Green Master Rox, Roche), 0.3 μM each primer and 30 ng of extracted genomic DNA. Amplification was performed in QuantStudio 5 Real-Time PCR System (Applied Biosystems) under the following conditions: 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C and 60 s at 60 °C. A standard curve of six tenfold serial dilutions of pBLV, containing 1 × 10 6 to 10 BLV copies, was built and run 3 times for validation of the method. The number of provirus copies per reaction (100 ng) was calculated.

Method qPCR5

BLV PVLs were determined by using qPCR kit, RC202 (Takara Bio, Shiga, Japan) [ 28 , 35 ]. This qPCR assay amplifies the BLV pol gene along with the bovine RPPH1 gene as an internal control. Briefly, 100 ng genomic DNA was amplified by TaqMan PCR with four primers for pol gene and RPPH1 gene according to the manufacturer’s instructions: 30 s denaturation at 95 °C followed by 45 cycles (5 s at 95 °C and 30 s at 60 °C). The qPCR was performed on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific K.K., Tokyo, Japan). Standard curve was generated by creating tenfold serial dilutions of the standard plasmid included in the kit. The standards for calibration ranged from 1 to 10 6 copies/reaction and were run in duplicate. The number of provirus copies per 100 ng was calculated.

Method ddPCR6

The digital droplet PCR (ddPCR) assay for the env gene of the BLV was performed using the protocol previously described by [ 28 , 29 ]. An absolute quantification by TaqMan ddPCR was performed in a typical 20-μl assay, 1 μl of DNA sample was mixed with 1 μl of each primer (10 μM), 0.5 μl of probe (10 μM), and 2 × Supermix emulsified with oil (Bio-Rad). The droplets were transferred to a 96-well plate (Eppendorf). The PCR assay was performed in a thermocycler (C1000 touch cycler; Bio-Rad) with the following parameters: initial denaturation of 10 min at 95 °C, then 40 cycles of 30 s at 94 °C, and 1 min at 58 °C, with final deactivation of the enzyme for 10 min at 98 °C. The presence of fluorescent droplets determined the number of resulting positive events that were analyzed in the software (QuantaSoft v.1.7.4; Bio-Rad), using dot charts. The number of provirus copies per 100 ng were calculated. Each sample was run in duplicate, and results were averaged.

Method qPCR7

This qPCR method for the BLV pol gene is a modified option of widely available quantitative TaqMan qPCR described by Rola-Łuszczak et al. [ 11 ], using the same primers and standards. A quantitative TaqMan PCR was performed in a 20 μl PCR mix containing 10 μl of 2 × ORA qPCR Probe ROX L Mix (highQu, Kraichtal, Germany), 2 μl primer/probe mix (final concentration 400 nM of each of the primers, 200 nM of BLV probe), and 3 μl extracted genomic DNA. Amplification was performed in the Rotor-Gene Q system (Qiagen) with an initial denaturation step and polymerase activation at 95 °C for 3 min, followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s. As a standard, plasmid pBLV1 (NVRI, Pulawy, PL) containing a BLV pol fragment was used. Tenfold dilutions of plasmid DNA were made from 1 × 10 10 copies to 1 × 10 1 copies per reaction and used to generate the standard curve and estimate BLV copy number per 100 ng.

Method qPCR8

Proviral load quantification was assessed by SYBR Green real-time quantitative PCR (qPCR) using the pol gene as the target sequence [ 36 ]. Briefly, 12-μl PCR mixture contained Fast Start Universal SYBR Green Master Mix (Roche), 800 nM each BLV pol primers and 1 µl DNA as template. The reactions were incubated at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, 55 °C for 15 s and 60 °C for 1 min. All samples were tested in duplicate on a StepOne Plus machine (Applied Biosystems). A positive and negative control, as well as a no-template control, were included in each plate. After the reaction was completed, the specificity of the amplicons was checked by analyzing the individual dissociation curves. As a standard, plasmid pBLV1 (NVRI, Pulawy, PL) containing a BLV pol fragment was used. Tenfold dilutions of plasmid DNA were made from 1 × 10 6 to 10 copies per µl and used to generate the standard curve and estimate BLV copy number per 100 ng.

Method qPCR9

This qPCR method is a modified option of widely available quantitative TaqMan qPCR described by Rola-Łuszczak et al. [ 11 ], using the same primers and standards. The detection of BLV genome was combined with an endogenous control system (Toussaint 2007) in a duplex assay. Briefly, 20-µl qPCR reaction contained AhPath ID™ One-Step RT-PCR Reagents with ROX (Applied Biosystems, CA, USA) – 10 µl of 2 × RT-PCR buffer and 0.8 µl of 25 × RT-PCR enzyme mix, 400 nM each primer for pol gene, 100 nM BLV specific probe, 40 nM each β-actin primer, 40 nM β-actin specific probe and 2 µl DNA sample. All samples were tested in ABI7500 Real-Time PCR System (Applied Biosystems) according to the following protocol: 10 min at 48 °C (reverse transcription), 10 min at 95 °C (inactivation reverse transcriptase / activation Taq polymerase) followed by 45 cycles (15 s at 95 °C and 60 s at 60 °C). As a standard, plasmid pBLV1 (NVRI, Pulawy, PL) containing a BLV pol fragment was used. Tenfold dilutions of plasmid DNA were made from 1 × 10 4 copies to 0.1 copies per μl and used to generate the standard curve and estimate BLV copy number per 100 ng.

Method qPCR10

The BLV qPCR was performed as published previously [ 11 ]. A quantitative TaqMan PCR was carried out in a 25-μl PCR mixture containing 12.5 μl of 2 × QuantiTect Multiplex PCR NoROX master mix (Qiagen), 0.4 μM each primer, 0.2 μM specific BLV probe, and 500 ng of extracted genomic DNA. Amplification was performed in the Rotor-Gene Q system (Qiagen) using an initial denaturation step and polymerase activation at 95 °C for 15 min, followed by 50 cycles of 94 °C for 60 s and 60 °C for 60 s. All samples were amplified in duplicate. As a standard, the pBLV1 plasmid (NVRI, Pulawy, PL), containing a 120-bp BLV pol fragment, was used. Tenfold dilutions of this standard were made from 1 × 10 6 copies per μl to 100 copies per μl and were used to estimate the BLV copy numbers per 100 ng.

Method qPCR11

This qPCR method for the BLV pol gene is a modified option of widely available quantitative TaqMan qPCR described by Rola-Łuszczak et al. [ 11 ], using the same primers and standards. The reaction mixture contained 400 nM of each primer, 200 nM of probe, 10 µl of 2 × SsoFast probes supermix (Bio-Rad), 5 µl of DNA sample and H 2 O up to 20 µl of the final volume. PCR assays were carried out on a CFX96 thermocycler (Bio-Rad) under the following amplification profile: 98 °C for 3 min, followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s. As a standard, plasmid pBLV1 (NVRI, Pulawy, PL) containing a BLV pol fragment was used. Tenfold dilutions of plasmid DNA were used to generate the standard curve and estimate BLV copy number per 100 ng.

Analysis of BLV pol, env and LTR sequences targeted by particular qPCR/ddPCR assays

In order to assess full-length pol , env and LTR sequence variability among BLV genotypes, all BLV sequences ( n  = 2191) available on 30 September 2023 in GenBank ( https://www.ncbi.nlm.nih.gov/GenBank/ ) repository were retrieved. From the collected sequences, 100 pol , env and LTR sequences, which were characterized by the highest level of sequence variability and divergence, were selected for the further analysis. A pol -based, env -based and LTR-based maximum likelihood (ML) phylogenetic trees (see Additional file 6) was constructed to assign genotypes to the unassigned BLV genomes [ 37 , 38 , 39 ]. For all genes and LTR region the Tamura-Nei model and Bootstrap replications (1,000) were applied. In this analysis, pol sequences were assigned to 7 BLV genotypes (G1, G2, G3, G4, G6, G9, and G10), while env and LTR sequences were assigned to 10 BLV genotypes (G1, G2, G3, G4, G5, G6, G7, G8, G9, and G10). Phylogeny of the same isolates assigned to particular genotypes by ML method was confirmed by Mr. Bayes analysis [ 40 , 41 , 42 ] (data not shown). From this analysis, a total of 100 full-length pol, env and LTR sequences were used for multiple-sequence alignment (MSA) using ClustalW algorithm, implemented in MEGA X. For all sequences, nucleotide diversity (π), defined as the average number of nucleotide differences per site between two DNA sequences in all possible pairs in the sample population, was estimated using MEGA X. To measure the relative variation in different positions of aligned genes and LTR region the Shannon’s entropy (a quantitative measure of diversity in the alignment, where H = 0 indicates complete conservation) was estimated using BioEdit v. 7.2.5 software 64. The statistical analyses were performed using DATAtab e.U. Graz, Austria and GraphPad Software by Dotmatics, Boston.

Examination of the quality and stability of DNA samples

To test the quality of DNA samples, the H3F3A copy number of each individual sample was assessed by qPCR at the NVRI. Copy numbers were normalized to DNA mass input and results were expressed as copy numbers per 300 ng of total DNA. The respective values were tested by Grubbs' test. The results for 43 DNA samples (sample ID: 42 with BLV genome plasmid was excluded) followed a normal distribution (Shapiro–Wilk 0.97; P  = 0.286), with a mean value of 35,626 copies (95% confidence interval [ 43 ] 33,843 to 37,408 copies), a minimum value of 19,848 copies and a maximum value of 46,951 copies (see Additional file 2). Despite a low value for sample ID: 40 no significant outlier was detected in the dataset ( P  > 0.05). Therefore, it can be assumed that the DNA quality was acceptable for all samples present in the panel. Next, DNA stability was assessed by retesting the H3F3A copy numbers in each sample ( n  = 43) after a combined storage consisting in 10 days at RT and 10 days at + 4°C. A Mann–Whitney U-test was used to compare the median values between fresh and stored samples (time 0 and time 1, respectively), and no significant difference was observed at the 5% level ( P  = 0.187) (Fig.  1 A).

Assessment of the stability of DNA samples. A Shown are copy numbers of the H3F3A housekeeping gene in 43 DNA samples that were stored in 10 days at RT and 10 days at + 4°C and tested twice with a 21-day interval. A Mann–Whitney U-test was used to compare the median values between two groups ( P  = 0.187); B Shown are GQN values ( n  = 43) tested twice with a 21-day interval: `before freeze-drying` and `after freeze-drying`. A Mann–Whitney U-test results between two groups ( P  = 0.236)

In addition, the quality of DNA samples after lyophilization was analyzed. DNA from individual samples ( n  = 43) was assessed with the genomic DNA quality number on the Fragment Analyzer system. The GQN from all lyophilized samples ranged from 4.0 to 9.7—that represented undegraded DNA. There was no significant difference in GQN values between `before freeze-drying` and `after freeze-drying` groups with respect to the corresponding DNA samples ( P  = 0.236) (Fig.  1 B). Altogether, these results suggested that sample storage, lyophilization and shipping has a minimal impact in DNA stability and further testing during the interlaboratory trial.

Detection of BLV proviral DNA by different qPCR assays

A total of 44 DNA samples, including two positive (ID: 42 and 43) and one negative (ID: 32) controls, were blinded and independently tested by eleven laboratories using their own qPCR methods (Table  2 ). All laboratories measured the concentration of DNA in samples (Additional file 3). BLV provirus copy number was normalized to DNA concentration and expressed per 100 ng of genomic DNA for each test.

Except for the positive (pBLV344 and FLK cell line) and the negative controls, all samples had previously shown detectable levels of BLV-specific antibodies (BLV-Abs) by enzyme-linked immunosorbent assays (ELISA). During the current interlaboratory study, both the positive and negative controls were assessed adequately by all eleven PCR tests. Of all 43 positive samples, 43, 35, 37, 36, 40, 32, 40, 42, 42, 42 and 41 samples were detected as positive by the qPCR1, qPCR2, qPCR3, qPCR4, qPCR5, ddPCR6, qPCR7, qPCR8, qPCR9, qPCR10 and qPCR11 methods, respectively. Based on these observations, the most sensitive method was the qPCR1, and the method with the lowest sensitivity was the ddPCR6. Twenty-nine out of 44 samples were identified correctly by all qPCRs. The remaining 15 samples gave discordant results. Comparison of qualitative results (positive versus negative) from all eleven methods revealed 87.33% overall agreement and a kappa value of 0.396 (Cohen's kappa method adapted by Fleiss) [ 44 , 45 ]. The levels of agreement among the results from the eleven methods are represented in Table  3 . The maximum agreement was seen between two methods (qPCR9 and qPCR10 [100% agreement and a Cohen's kappa value of 1.000]) that used similar protocols and targeted the same region of BLV pol .

Analysis of BLV pol, env and LTR sequences targeted by particular PCR assays

Due to differences in performance observed among the pol -based qPCR assays (the qPCR1, qPCR2, qPCR4, qPCR5 and qPCR7- qPCR11 methods), and considering that the env -based ddPCR6 and LTR-based qPCR3 assay showed the lowest sensitivity and the poorest agreement with the other assays, the degree of sequence variability between the pol , env and LTR genes was addressed. From the MSAs for pol , env and LTR, the nucleotide diversity (π) was calculated. The π value for pol gene was lower than that for LTR and env gene (π pol , 0.023 [standard deviation {SD}, 0.018]; π LTR , 0.024 [SD, 0.011]; π env , 0.037 [SD, 0.013]). From this analysis, pol sequences appeared to be less variable than env and LTR sequences. In addition, we performed a Shannon entropy-based per-site variability profile of the pol , env and LTR sequences used in this study (Fig.  2 A-C).

Sequence variability measured as per-site entropy. A Multiple alignment of the pol gene showing the locations of qPCR fragments in regions of the pol gene for the qPCR1 (highlighted in pink), qPCR4 (highlighted in yellow) and for the qPCR7, qPCR8, qPCR9, qPCR10 and qPCR11 assays (highlighted in orange). B Multiple alignment of the env gene targeted by ddPCR6 (highlighted by blue rectangle). C Multiple alignment of the LTR region by qPCR3 (highlighted in mint)

The all-observed entropy plots were homogeneous along the whole sequences. Considering the three regions of pol gene, the highest entropy (4.67) occurred in the region targeted by the qPCR1 primers, whereas the entropy for qPCR7—qPCR11 and qPCR4 primers were 1.57 and 0.38, respectively. For the LTR region targeted by qPCR3 primers and for env gene targeted by ddPCR6, the total entropy was equal to 4.46 and 7.85, respectively. This analysis showed a marked region of variability for LTR and env fragments. Interestingly, we noted that the qPCR7—qPCR11 targeted the most conserved regions of reverse transcriptase and qPCR4 primers targeted the most-conserved region of virus integrase (Fig.  2 A-C; see also Additional file 7).

Quantitation of BLV proviral DNA by different qPCR/ddPCR assays

To analyze whether the range of copy numbers detected by each qPCR was comparable to those of the others, Kruskal–Wallis one-way analysis of variance (ANOVA) was used. The violin plots were used to visualize the ANOVA results (Fig.  3 A-B).

Comparison of detection of BLV proviral DNA copy numbers by eleven testing methods. Shown is a box plot of data from Kruskal–Wallis ANOVA, a rank test. The DNA copy numbers for 41 samples, determined independently by each of the 11 qPCRs, were used for the variance analysis. In this analysis, the positive controls (sample ID 42 and ID 43) and negative control (sample ID 32) were excluded. A Violin plot for graphical presentation of the ANOVA of proviral copy number values. B Violin plot for ANOVA analysis of variance, copy number values are presented on a logarithmic scale (Log1.2) for better illustration of copy number differences between PCR methods

The grouping variable revealed significant differences among the distributions of proviral DNA copy numbers with the various qPCRs ( P  < 0.001). These results showed that the abilities of qPCRs/ddPCR to determine the proviral DNA copy number differed. A Dunn-Bonferroni test was used to compare the groups in pairs to find out which was significantly different. The Dunn-Bonferroni test revealed that the pairwise group comparisons of qPCR2—qPCR4, qPCR3—ddPCR6, qPCR4—qPCR5, qPCR4—ddPCR6, qPCR4—qPCR9, qPCR4—qPCR10, qPCR5—qPCR11, ddPCR6—qPCR11 and qPCR9—qPCR11 have an adjusted P value less than 0.05 and thus, it can be assumed that these groups were significantly different in each pair (see Additional file 4). The Pareto chart was used to show the average copy number values of all methods in descending order. These Pareto charts were prepared based on 80–20 rule, which states that 80% of effects come from 20% of the various causes [ 46 ]. The methods that generated the highest copy numbers was qPCR3 and qPCR4, on the other hand the lowest copy numbers and/or highest negative results were generated by ddPCR6 (Fig.  4 ).

A Pareto chart with the proviral BLV copy mean values for eleven PCR assay arranged in descending order. Pareto charts was prepared based on 80–20 rule, which states that 80% of effects come from 20% of the various causes

The correlations between copy numbers detected by different qPCRs and ddPCR assays were calculated. The Kendall's Tau correlation coefficient measured between each pair of the assays was shown in the Additional file 5 and in Fig.  5 as a correlation heatmap. The average correlation for all qPCRs and ddPCR assays was strong (Kendall's tau = 0.748; P  < 0.001).

The heatmap of Kendall’s tau correlation coefficients between copy numbers detected by ten qPCRs and one ddPCR. Statistically significant differences in the distribution of copy numbers, a moderate, strong and very strong correlation between particular qPCRs/ddPCR was observed. The strength of the association, for absolute values of r, 0–0.19 is regarded as very weak, 0.2–0.39 as weak, 0.40–0.59 as moderate, 0.6–0.79 as strong and 0.8–1 as very strong correlation

Since the differences between PCR tests may be influenced by the number of BLV proviral copies present in each sample, we compared the average number of BLV copies between a group of genomic DNA samples that gave concordant results (group I [ n  = 28]) and a group that gave discordant results (group II [ n  = 15]). The mean number of copies was 73,907 (minimum, 0; maximum, 4,286,730) in group I, and 3,479 (minimum, 0; maximum, 218,583) in group II, and this difference was statistically significant ( P  < 0.001 by a Mann–Whitney U- test) (Fig.  6 ).

Impact of BLV proviral copy numbers on the level of agreement. Violin plot for graphical presentation of Mann–Whitney U test. The test was performed to compare BLV provirus copy number in two groups of samples: 28 samples with fully concordant results from all eleven qPCR/ddPCR assays (left) and 15 samples with discordant results from different qPCR/ddPCR assays (right) ( P  < 0.001). Sample ID 42 was excluded from the statistical analysis

The results show that the concordant results group had considerably higher copy numbers (median, 5,549.0) than the discordant results group (median, 6.3).

BLV control and eradication programs consist of correct identification and subsequent segregation/elimination of BLV-infected animals [ 47 ]. Detection of BLV- infected cows by testing for BLV-specific antibodies in serum by agar gel immunodiffusion and ELISA is the key step and standard to be implemented of EBL eradication programs according to WOAH ( https://www.woah.org/en/disease/enzootic-bovine-leukosis/) [ 9 ]. Despite the low cost and high throughput of serological tests, there are several scenarios where highly specific and sensitive molecular assays for the detection of BLV DNA might improve detection and program efficiency.

In this perspective, qPCR assays can detect small quantities of proviral DNA during acute infection, in which animals show very low levels of anti-BLV antibodies [ 43 , 48 , 49 , 50 ]. qPCR methods can also work as confirmatory tests to clarify ambiguous and inconsistent serological test results [ 11 ]. Such quantitative features of qPCRs are crucial when eradication programs progress and prevalence decreases. Moreover, qPCR allows not only the detection of BLV infection but also estimation of the BLV PVL, which directly correlates with the risk of disease transmission [ 51 , 52 ]. This feature of qPCR allows for a rational segregation of animals based on the stratified risk of transmission. These considerations allow for greater precision in the management of BLV within large herds with a high prevalence of BLV ELISA-positive animals to effectively reduce herd prevalence [ 13 , 53 ]. BLV is a global burden and the lack of technical standardization of molecular detection systems remains a huge obstacle to compare surveillance data globally based on the first interlaboratory trial performed in 2018 [ 15 ]. In the 2018 study we observed an adjusted level of agreement of 70% comparing qualitative qPCR results; however, inconsistencies amongst methods were larger when low number of copies of BLV DNA were compared. Samples with low copies of BLV DNA (< 20 copies per 100 ng) accounted for the higher variability and discrepancies amongst tests. We concluded from the first interlaboratory trial that standardizing protocols to improve sensitivity of assays with lower detection rates was necessary.

In this follow up study, we re-tested the TaqMan BLV qPCR developed and validated by NVRI (acting as reference WOAH laboratory) and the one adapted from this original protocol to be used with SYBR Green dye, allowing a significant reduction in costs [ 11 ]. Another 3 laboratories also performed NVRI´s qPCR with slight modifications (i.e., Spain performed a multiplex assay for internal normalization). The remaining 6 labs introduced novel methodologies to the trial including one ddPCR (UY).

To compare different qPCR methods, a more comprehensive sample panel, accounting for a more geographical diversification was used in this trial. The amounts of BLV DNA in these samples were representative of the different BLV proviral loads found in field samples (from 1 to > 10,000 copies of BLV proviral DNA). Of note, 34% of reference samples had less than 100 copies of BLV DNA per 100 ng; samples were lyophilized to grant better preservation and reduced variability during distribution to participants around the globe.

The panel included a single negative control and two positive controls. Diagnostic sensitivity (DxSn) was estimated for each qPCR. Considering the 43 positive samples, the DxSn for the different qPCRs were: qPCR1 = 100%, qPCR2 = 82%, qPCR3 = 86%, qPCR4 = 84%, qPCR5 = 93%, ddPCR6 = 74%, qPCR7 = 93%, qPCR8 = 98%, qPCR9 = 98%, qPCR10 = 98% and qPCR11 = 95%. The most sensitive method was the qPCR1, and the method with the lowest sensitivity was the ddPCR6 method. Twenty-nine out of 44 samples were identified correctly by all qPCRs. The remaining 15 samples gave discordant results. The comparison of qualitative qPCR results among all raters revealed an overall observed agreement of 87%, indicating strong interrater reliability (Cohen´s kappa = 0.396) [ 54 , 55 ].

There are several factors that contribute to variability in qPCR results (i.e., number of copies of target input, sample acquisition, processing, storage and shipping, DNA purification, target selection, assay design, calibrator, data analysis, etc.). For that reason and as expected, the level of agreement among sister qPCRs (qPCR7, qPCR9-11) sharing similar protocols was higher compared to the rest of assays; this was also true for qPCR8 which targets the same region of BLV pol gene (shares same primers) but has a particular set-up to be used with SYBR Green chemistry. Oppositely, lower sensitivity and larger discrepancy against other tests was observed for the ddPCR6 and qPCR2-4.

Based on these observations we investigated which factors might have accounted for larger assessment variability amongst tests. In the first place, we observed that the use of different chemistries was not detrimental for the sensitivity and agreement among tests; similar DxSn and comparable level of agreement were obtained comparing TaqMan (qPCR7, 10, 11) vs SYBR Green (qPCR8) chemistries while targeting identical BLV sequence and using same standards. Also, when a multiplex qPCR (TaqMan) targeting the same BLV sequence and using the same standard was compared to previous ones, agreement was kept high, indicating that the lower sensitivity described for some multiplex qPCRs did not take place in this comparison. The use of an international calibrator and the efficiency estimation (standard curve) might inform variability associated with different chemistries. In contrast, another multiplex assay targeting another region of BLV pol (qPCR2) showed much lower sensitivity and agreement. As qPCR2 is performed as service by private company and oligonucleotide sequences were not available, we were not able to investigate in which proportion each of these two variables contributed to the lower performance of this assay, but we note the addition of 4 µl genomic DNA to this assay that would have an impact the DxSn. In this regard, there is substantial evidence showing that the variability of target sequence among strains from different geographical areas, might affect the sensitivity of BLV qPCRs. Previous studies comparing the pol , gag , tax and env genes reported that the pol gene was the most suitable region to target for diagnostic purposes, since it provided the most-sensitive assays [ 11 , 15 , 56 , 57 , 58 , 59 ]. This might be due in part to higher sequence conservation of pol among strains from different geographical areas. Supporting this observation, it is noticeable how JPN qPCR improved their performance in the current trial, by targeting pol in place of tax , as it did in the previous interlaboratory trial. Since it is a commercial test, we cannot exclude other factors contributing for the performance upgrade observed for this qPCR. In the current study, qPCR3 and ddPCR6 targeting LTR and env sequences, showed lower performances than other assays. Standardization of DNA input into each qPCR would have likely resulted in higher concordance in results. For instance, qPCR1 added 10 µl of genomic DNA per reaction and ddPCR6 added 1 µl of genomic DNA, impacting the resulting sensitivity differences.

Since the sensitivity of each assay and, consequently, the level of agreement among assays might also be influenced by the number of BLV DNA copies present in each sample [ 48 ], we compared the average number of BLV DNA copies between a group of genomic DNA samples that gave concordant results and a group that gave discordant results, and observed that samples that gave discordant results had significantly lower numbers of BLV DNA copies than samples that gave concordant results. Related to this point, the degradation of target DNA during lyophilization, shipment and resuspension, could have been more significant in low-copy compared to high-copy samples. Consequently, the degradation of target DNA in samples with low copies of BLV DNA might have accounted for the greater level of discrepancy within this subset of samples. The rational of adding a large proportion of such samples (34% samples with less than 100 BLV copies per 100 ng of total DNA) was to mimic what is frequently observed in surveillance programs (i.e., hyperacute infection, chronic asymptomatic infection, etc.).

Quantitative methods for the detection of BLV DNA copies are important for segregation programs based on animal level of BLV PVL, as well as for scientific research and the study of BLV dynamics. When the numbers of copies of BLV DNA detected by different assays were compared, in the present study, we observed that although the ability to quantify BLV DNA differed among qPCRs/ddPCR and there were statistically significant differences in the distribution of copy numbers among assays, a strong average correlation was found for the eleven qPCRs/ddPCR. In this regard, the lack of an international calibrator (standard curve) could be a major contributor to the increment of quantitative variation amongst laboratories. For that reason, plasmid pBLV1 containing pol 120 bp sequence was originally constructed for use as standard for quantification and shared with some collaborators (i.e., qPCR7, qPCR8, qPCR 9, qPCR10 and qPCR11). Remarkably, the laboratories used pBLV1 standard in the current trial obtained the most comparable results, indicating that the use of an international standard may have significant impact on the convergence of results; such standard reference material should be prepared under identical conditions. To avoid further variability a detailed protocol for lyophilized DNA sample resuspension, quantitation and template input into each qPCR should be shared with all participants.

Conclusions

BLV DNA was detected with different level of sensitivity in serologically positive samples from different origin and classified into different BLV genotypes. Overall agreement was high; however, we found significant differences in results for the samples with low BLV DNA copy numbers. This second interlaboratory study demonstrated that differences in target sequence, DNA input and calibration curve standards can increase interlaboratory variability considerably. Next steps should focus on (i) standard unification (international gold standard) to estimate individual test efficiency and improve quantitative accuracy amongst tests; (ii) building a new panel of samples with low BLV DNA copy numbers to re-evaluate sensitivity and quantitation of molecular methods. Since no variation was observed in samples from different genotypes, all samples will be collected in Poland to standardize the collection, purification, lyophilization and shipping steps with precise instructions for suspension and constant input volume for the PCR reaction. Finally, we believe that following this standardization approach we will be able to improve overall agreement amongst tests, improving the diagnostic of BLV around the world.

Availability of data and materials

Not applicable.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

One-way analysis of variance

Bovine leukemia virus

BLV-specific antibodies

Digital PCR

Diagnostic sensitivity

Enzootic bovine leukosis

Enzyme-linked immunosorbent assays

Real-time fluorescence resonance energy transfer PCR

Genomic quality number

Histone H3 family 3A housekeeping gene

Maximum likelihood phylogenetic tree

Multiple-sequence alignment

Peripheral blood leukocytes

Phosphate-buffered saline

Proviral load

Quantitative real-time PCR

Room temperature

World Organisation for Animal Health

Coffin JM, Hughes SH, Varmus HE. (Eds.). 1650–1655 (1997). Retroviruses. Cold Spring Harbor Laboratory Press.

Ghysdael J, Bruck C, Kettmann R, Burny A. Bovine leukemia virus. Curr Top Microbiol Immunol. 1984;112:1–19.

CAS   PubMed   Google Scholar  

Ott SL, Johnson R, Wells SJ. Association between bovine-leukosis virus seroprevalence and herd-level productivity on US dairy farms. Prev Vet Med. 2003;61:249–62.

Article   CAS   PubMed   Google Scholar  

Bartlett PC, et al. Options for the control of bovine leukemia virus in dairy cattle. J Am Vet Med Assoc. 2014;244:914–22.

Article   PubMed   Google Scholar  

Kuczewski A, et al. Economic evaluation of 4 bovine leukemia virus control strategies for Alberta dairy farms. J Dairy Sci. 2019;102:2578–92.

Frie MC, Coussens PM. Bovine leukemia virus: a major silent threat to proper immune responses in cattle. Vet Immunol Immunopathol. 2015;163:103–14.

Panel, E.A. Scientific opinion on enzootic bovine leukosis. EFSA J. 2015;13:4188.

Google Scholar  

OIE. World Animal Health Information Database - Version: 1.4. World Animal Health Information Database. Paris, France: World Organisation for Animal Health; 2009. Available from: http://www.oie.int . Accessed 16 Aug 2024.

Health, W.O.f.A. Manual of diagnostic tests and vaccines for terrestrial animals. Infect Bursal Dis. 2012;12:549–65.

Hutchinson HC, et al. Bovine leukemia virus detection and dynamics following experimental inoculation. Res Vet Sci. 2020;133:269–75.]

Rola-Luszczak M, Finnegan C, Olech M, Choudhury B, Kuzmak J. Development of an improved real time PCR for the detection of bovine leukaemia provirus nucleic acid and its use in the clarification of inconclusive serological test results. J Virol Methods. 2013;189:258–64.

Nakada S, Kohara J, Makita K. Estimation of circulating bovine leukemia virus levels using conventional blood cell counts. J Dairy Sci. 2018;101:11229–36.

Ruggiero VJ, Bartlett PC. Control of Bovine Leukemia Virus in Three US Dairy Herds by Culling ELISA-Positive Cows. Vet Med Int. 2019;2019:3202184.

Article   PubMed   PubMed Central   Google Scholar  

Kobayashi T, et al. Increasing Bovine leukemia virus (BLV) proviral load is a risk factor for progression of Enzootic bovine leucosis: A prospective study in Japan. Prev Vet Med. 2020;178: 104680.

Article   Google Scholar  

Jaworski JP, Pluta A, Rola-Łuszczak M, McGowan SL, Finnegan C, Heenemann K, Carignano HA, Alvarez I, Murakami K, Willems L, Vahlenkamp TW, Trono KG, Choudhury, B, Kuźmak J. Interlaboratory Comparison of Six Real-Time PCR Assays for Detection of Bovine Leukemia Virus Proviral DNA.  J Clin Microbiol. 2018;56(7):e00304-18. https://doi.org/10.1128/JCM.00304-18 .

Pluta A, Rola-Luszczak M, Douville RN, Kuzmak J. Bovine leukemia virus long terminal repeat variability: identification of single nucleotide polymorphisms in regulatory sequences. Virol J. 2018;15:165.

Article   CAS   PubMed   PubMed Central   Google Scholar  

Pluta A, Willems L, Douville RN, Kuźmak J. Effects of Naturally Occurring Mutations in Bovine Leukemia Virus 5'-LTR and Tax Gene on Viral Transcriptional Activity. Pathog. 2020;9(10):836. https://doi.org/10.3390/pathogens9100836 .

Pluta A, et al. Molecular characterization of bovine leukemia virus from Moldovan dairy cattle. Arch Virol. 2017;162:1563–76.

Rola-Łuszczak M, Sakhawat A, Pluta A, Ryło A, Bomba A, Bibi N, Kuźmak J. Molecular Characterization of the env Gene of Bovine Leukemia Virus in Cattle from Pakistan with NGS-Based Evidence of Virus Heterogeneity. Pathogens (Basel, Switzerland). 2021;10(7):910. https://doi.org/10.3390/pathogens10070910 .

Rola-Luszczak M, et al. The molecular characterization of bovine leukaemia virus isolates from Eastern Europe and Siberia and its impact on phylogeny. PLoS ONE. 2013;8: e58705.

Pinheiro de Oliveira TF, et al. Detection of contaminants in cell cultures, sera and trypsin. Biologicals. 2013;41:407–14.

Pluta A, Blazhko NV, Ngirande C, Joris T, Willems L, Kuźmak J. Analysis of Nucleotide Sequence of Tax, miRNA and LTR of Bovine Leukemia Virus in Cattle with Different Levels of Persistent Lymphocytosis in Russia. Pathogens. 2021;10(2):246. https://doi.org/10.3390/pathogens10020246 .

Yang Y, et al. Bovine leukemia virus infection in cattle of China: Association with reduced milk production and increased somatic cell score. J Dairy Sci. 2016;99:3688–97.

DeGraves FJ, Gao D, Kaltenboeck B. High-sensitivity quantitative PCR platform. Biotechniques. 2003;34(106–110):112–105.

Fonseca Junior AA, et al. Evaluation of three different genomic regions for detection of bovine leukemia virus by real-time PCR. Braz J Microbiol. 2021;52:2483–8.

Farias MVN, et al. Toll-like receptors, IFN-gamma and IL-12 expression in bovine leukemia virus-infected animals with low or high proviral load. Res Vet Sci. 2016;107:190–5.

Holland PM, Abramson RD, Watson R, Gelfand DH. Detection of specific polymerase chain reaction product by utilizing the 5’––3’ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A. 1991;88:7276–80.

De Brun ML, et al. Development of a droplet digital PCR assay for quantification of the proviral load of bovine leukemia virus. J Vet Diagn Invest. 2022;34:439–47.

Rola-Łuszczak M, Finnegan C, Olech M, Choudhury B, Kuźmak J. Development of an improved real time PCR for the detection of bovine leukaemia provirus nucleic acid and its use in the clarification of inconclusive serological test results. J Virol Methods. 2013;189:258–64.

Petersen MI, Alvarez I, Trono KG, Jaworski JP. Quantification of bovine leukemia virus proviral DNA using a low-cost real-time polymerase chain reaction. J Dairy Sci. 2018;101:6366–74.

Toussaint JF, Sailleau C, Breard E, Zientara S, De Clercq K. Bluetongue virus detection by two real-time RT-qPCRs targeting two different genomic segments. J Virol Methods. 2007;140:115–23.

John EE, et al. Development of a predictive model for bovine leukemia virus proviral load. J Vet Intern Med. 2022;36:1827–36.

Farias MVN, et al. Toll-like receptors, IFN-γ and IL-12 expression in bovine leukemia virus-infected animals with low or high proviral load. Res Vet Sci. 2016;107:190–5.

Yoneyama S, et al. Comparative Evaluation of Three Commercial Quantitative Real-Time PCRs Used in Japan for Bovine Leukemia Virus. Viruses. 2022;14:1182.

Polat M, Takeshima SN, Aida Y. Epidemiology and genetic diversity of bovine leukemia virus. Virol J. 2017;14:209.

Lee E, et al. Molecular epidemiological and serological studies of bovine leukemia virus (BLV) infection in Thailand cattle. Infect Genet Evol. 2016;41:245–54.

Duran-Yelken S, Alkan F. Molecular analysis of the env, LTR, and pX regions of bovine leukemia virus in dairy cattle of Türkiye. Virus Genes. 2024;60:173–85.

Lv G, Wang J, Lian S, Wang H, Wu R. The Global Epidemiology of Bovine Leukemia Virus: Current Trends and Future Implications. Animals. 2024;14(2):297. https://doi.org/10.3390/ani14020297 .

Úsuga-Monroy C, Díaz FJ, Echeverri-Zuluaga JJ, González-Herrera LG, López-Herrera A. Presence of bovine leukemia virus genotypes 1 and 3 in Antioquia, Colombia. Revista UDCA Actualidad & Divulgación Científica. 2018;21:119–26.

Úsuga-Monroy C, Díaz FJ, González-Herrera LG, Echeverry-Zuluaga JJ, López-Herrera A. Phylogenetic analysis of the partial sequences of the env and tax BLV genes reveals the presence of genotypes 1 and 3 in dairy herds of Antioquia. Colombia VirusDisease. 2023;34:483–97.

Martin D, et al. Comparative study of PCR as a direct assay and ELISA and AGID as indirect assays for the detection of bovine leukaemia virus. J Vet Med B Infect Dis Vet Public Health. 2001;48:97–106.

Cohen J. A Coefficient of Agreement for Nominal Scales. Educ Psychol Measur. 1960;20:37–46.

Feinstein AR, Cicchetti DV. High agreement but low kappa: I. The problems of two paradoxes. J Clin Epidemiol. 1990;43:543–9.

Woolhouse MEJ, et al. Heterogeneities in the transmission of infectious agents: Implications for the design of control programs. Proc Natl Acad Sci. 1997;94:338–42.

Ohshima K, Okada K, Numakunai S, Kayano H, Goto T. An eradication program without economic loss in a herd infected with bovine leukemia virus (BLV). Nihon Juigaku Zasshi. 1988;50:1074–8.

Juliarena MA, Gutierrez SE, Ceriani C. Determination of proviral load in bovine leukemia virus-infected cattle with and without lymphocytosis. Am J Vet Res. 2007;68:1220–5.

Mirsky ML, Olmstead CA, Da Y, Lewin HA. The prevalence of proviral bovine leukemia virus in peripheral blood mononuclear cells at two subclinical stages of infection. J Virol. 1996;70:2178–83.

Eaves FW, Molloy JB, Dimmock CK, Eaves LE. A field evaluation of the polymerase chain reaction procedure for the detection of bovine leukaemia virus proviral DNA in cattle. Vet Microbiol. 1994;39:313–21.

Juliarena MA, Barrios CN, Ceriani MC, Esteban EN. Hot topic: Bovine leukemia virus (BLV)-infected cows with low proviral load are not a source of infection for BLV-free cattle. J Dairy Sci. 2016;99:4586–9.

Yuan Y, et al. Detection of the BLV provirus from nasal secretion and saliva samples using BLV-CoCoMo-qPCR-2: Comparison with blood samples from the same cattle. Virus Res. 2015;210:248–54.

Taxis TM, et al. Reducing bovine leukemia virus prevalence on a large midwestern dairy farm by using lymphocyte counts, ELISA antibody testing, and proviral load. The Bovine Practitioner. 2020;54:136–44.

McHugh ML. Interrater reliability: the kappa statistic. Biochem Med (Zagreb). 2012;22:276–82.

Cicchetti DV, Feinstein AR. High agreement but low kappa: II. Resolving the paradoxes J Clin Epidemiol. 1990;43:551–8.

Heenemann K, et al. Development of a Bovine leukemia virus polymerase gene–based real-time polymerase chain reaction and comparison with an envelope gene–based assay. J Vet Diagn Invest. 2012;24:649–55.

Lew AE, et al. Sensitive and specific detection of proviral bovine leukemia virus by 5′ Taq nuclease PCR using a 3′ minor groove binder fluorogenic probe. J Virol Methods. 2004;115:167–75.

Takeshima SN, Sasaki S, Meripet P, Sugimoto Y, Aida Y. Single nucleotide polymorphisms in the bovine MHC region of Japanese Black cattle are associated with bovine leukemia virus proviral load. Retrovirology. 2017;14:24.

Debacq C, et al. Reduced proviral loads during primo-infection of sheep by Bovine Leukemia virus attenuated mutants. Retrovirology. 2004;1:31.

Kuckleburg CJ, et al. Detection of bovine leukemia virus in blood and milk by nested and real-time polymerase chain reactions. J Vet Diagn Invest. 2003;15:72–6.

Dube S, et al. Degenerate and specific PCR assays for the detection of bovine leukaemia virus and primate T cell leukaemia/lymphoma virus pol DNA and RNA: phylogenetic comparisons of amplified sequences from cattle and primates from around the world. J Gen Virol. 1997;78(Pt 6):1389–98.

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Acknowledgements

The authors thank Luc Willems (University of Liège, Belgium) for plasmid DNA sample pBLV344; Marlena Smagacz and Eliza Czarnecka (National Veterinary Research Institute, Poland) for lyophilizing DNA samples and DNA analysis, respectively; Ali Sakhawat (Animal Quarantine Department, Pakistan), Vitaliy Bolotin (National Scientific Center IECVM, Ukraine), Frank van der Meer and Sulav Shrestha (University of Calgary, Canada) for sharing material.

The APC was funded by the National Veterinary Research Institute, Puławy, Poland.

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Department of Biochemistry, National Veterinary Research Institute, Puławy, 24-100, Poland

Aneta Pluta & Jacek Kuźmak

Instituto de Virología E Innovaciones Tecnológicas (IVIT), Centro de Investigaciones en Ciencias Veterinarias y Agronómicas (CICVyA), Instituto Nacional de Tecnología Agropecuaria (INTA) - CONICET, Buenos Aires, Argentina

Juan Pablo Jaworski & Vanesa Ruiz

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Casey Droscha & Sophie VanderWeele

Department of Animal Science, College of Agriculture and Natural Resources, Michigan State University, East Lansing, Michigan, 48824, USA

Tasia M. Taxis

Niort Laboratory, Unit Pathology and Welfare of Ruminants, French Agency for Food, Environmental and Occupational Health and Safety (Anses), Ploufragan-Plouzané, Niort, France

Stephen Valas

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Contributions

Proposed the conception and design of the study, A.P.; data curation, A.P., J.P.J., C.D., S.V., D.B., A.S., K.M., R.P., G.D., M.F.C. and CH.W.; investigation, A.P., V.R., S.VW., S.V., A.J., M.J.R., K.N., M.L.B., M.L.G., P.L., A.F., A.G. and S.B., formal analysis, A.P.; statistical analysis, A.P.; database analysis, A.P., visualization of the results, A.P.; resources, A.P., T.M.T. and J.K; writing—original draft preparation, A.P., J.P.J.; writing—review and editing, A.P., J.P.J., C.D., S.VW., T.M.T. and J.K; project administration, A.P. All authors read and approved the submitted version.

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Correspondence to Aneta Pluta .

Ethics declarations

Ethics approval and consent to participate.

The study was approved by the Veterinary Sciences Animal Care Committee No. AC21-0210, Canada; the Institutional Animal Care and Use Committee No. PROTO202000096 from 4/13/2020 to 4/14/2023, Michigan State University, United States and the Ethics Review Board, COMSATS Institute of Information Technology, Islamabad, Pakistan, no. CIIT/Bio/ERB/17/26. Blood samples from Polish, Moldovan and Ukrainian cattle, naturally infected with BLV, were selected from collections at local diagnostic laboratories as part of the Enzootic bovine leukosis (EBL) monitoring program between 2012 and 2018 and sent to the National Veterinary Research Institute (NVRI) in Pulawy for confirmation study. The approval for collection of these samples from ethics committee was not required according to Polish regulation (“Act on the Protection of Animals Used for Scientific or Educational Purposes”, Journal of Laws of 2015). All methods were carried out in accordance with relevant guidelines and regulations. The owners of the cattle herds from which the DNA samples originated, the district veterinarians caring for these farms and the ministries of agriculture were informed and consented to the collection of blood from the animals for scientific purposes and the sending of samples to NVRI.

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Supplementary Information

12917_2024_4228_moesm1_esm.pdf.

Additional file 1. Copy of the instruction included with the panel of 44 DNA samples sent to participating laboratories for dilution of the lyophilisates

12917_2024_4228_MOESM2_ESM.png

Additional file 2. Detection of the H3F3A gene copy number in 43 DNA samples; no outlier was found for any samples ( P <0.05) (two-sided).

12917_2024_4228_MOESM3_ESM.docx

Additional file 3. Concentration values of 44 DNA samples measured by the 11 participating laboratories (given in ng per µl)

12917_2024_4228_MOESM4_ESM.pdf

Additional file 4. Post hoc - Dunn-Bonferroni-Tests. The Dunn-Bonferroni test revealed that the pairwise group comparisons of qPCR2 - qPCR4, qPCR3 - ddPCR6, qPCR4 - qPCR5, qPCR4 - ddPCR6, qPCR4 - qPCR9, qPCR4 - qPCR10, qPCR5 - qPCR11, ddPCR6 - qPCR11 and qPCR9 - qPCR11 have an adjusted p-value less than 0,05

12917_2024_4228_MOESM5_ESM.docx

Additional file 5. Kendall's Tau correlation coefficient values measured between each pair of assays. The numbers 1 to 11 in the first column and last row of the table indicate the names of the assays qPCR1-qPCR5, ddPCR6, qPCR7-qPCR11 respectively

12917_2024_4228_MOESM6_ESM.png

Additional file 6. Maximum-likelihood phylogenetic analysis of full-length BLV-pol gene sequences representing 7 BLV genotypes (G1, G2, G3, G4, G6, G9, and G10) (A); (B) env-based sequences assigned to 10 BLV genotypes (G1, G2, G3, G4, G5, G6, G7, G8, G9, and G10); (C) LTR-based sequences representing 10 BLV genotypes (G1-G10). For all genes and LTR region the Tamura-Nei model and Bootstrap replications (1,000) were applied in MEGA X

12917_2024_4228_MOESM7_ESM.pdf

Additional file 7. Multiple sequence alignment of reverse transcriptase, integrase, envelope and LTR sequences in the context of the specific primers used by different qPCR assays. (A) Multiple sequence alignment of reverse transcriptase (pol gene) sequences in the context of qPCR7, qPCR8, qPCR9, qPCR10 and qPCR11 assay primers. (B) Multiple sequence alignment of integrase (pol gene) sequences in the context of qPCR4 assay primers. (C) Multiple sequence alignment of env gene sequences in the context of ddPCR6. (D) Sequence alignment of LTR region sequences in the context of qPCR3 method primers

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Pluta, A., Jaworski, J.P., Droscha, C. et al. Inter-laboratory comparison of eleven quantitative or digital PCR assays for detection of proviral bovine leukemia virus in blood samples. BMC Vet Res 20 , 381 (2024). https://doi.org/10.1186/s12917-024-04228-z

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Received : 24 November 2023

Accepted : 09 August 2024

Published : 26 August 2024

DOI : https://doi.org/10.1186/s12917-024-04228-z

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