The Study of Microbiome of the Female Genital Area in Relation to Pelvic Floor Dysfunction: A Systematic Review.

INTRODUCTION AND HYPOTHESIS: The aim of this article is to present a systematic literature review focused on microbiome diversity in women experiencing pelvic floor dysfunction. METHODS: Utilizing PubMed/MedLine and Scopus, 25 pertinent studies were meticulously selected for this review. RESULTS: A key theme identified is the potential of microbiomes as diagnostic tools. The findings consistently highlight Lactobacillus as recurrent microbiota. Additionally, Gardnerella, Streptococcus, Prevotella, Aerococcus, Staphylococcus, Proteus, and Bifidobacterium species were frequently observed. This suggests the influential role of these microorganisms in shaping female urological and reproductive health. A deeper understanding of these predominant bacterial genera could offer invaluable insights into healthy physiological states and various disorders. The complex relationship between microbial compositions and diverse health conditions paves the way for novel diagnostic and therapeutic approaches. As we further explore the complexities of microbiomes, their role becomes increasingly crucial in transforming women's health care. CONCLUSIONS: These findings emphasize the need for personalized care, integrating the microbiome into a comprehensive health assessment and treatment framework. This review lays the groundwork for future medical strategies where the microbiome is a pivotal element in both preventive and therapeutic care.

Professionalization of the public health workforce: scoping review and call to action.

BACKGROUND: The 'WHO-ASPHER Roadmap to Professionalizing the Public Health Workforce in the European Region' provides recommendations for strategic and systematic workforce planning around professionalization levers including: (i) competencies, (ii) training and education, (iii) formal organization, (iv) professional credentialing and (v) code of ethics and professional conduct as well as taxonomy and enumeration. It was based on a literature review till 2016. This scoping review aims to explore how the professionalization was documented in the literature between 2016 and 2022. METHODS: Following the Joanna Briggs Institute guidelines, we searched Medline via PubMed, Web of Science, ERIC via EBSCO and Google Scholar and included studies on professionalization levers. Four critical appraisal tools were used to assess qualitative, quantitative, mixed methods studies and grey literature. The PRISMA Extension for Scoping Reviews (PRISMA-ScR) was used for reporting. RESULTS: Eleven articles included in this review spanned 61 countries, targeting undergraduate, master's, doctoral degrees and continuing professional development. Most of these documents were reviews. About half provided a definition of the public health workforce; more than half covered the taxonomy and included information about competences, but the use of frameworks was sporadic and inconsistent. Formal organization and the necessity of a code of conduct for the public health workforce were acknowledged in only two studies. CONCLUSIONS: In spite of some efforts to professionalize the public health workforce, this process is fragmented and not fully recognized and supported. There is an urgent need to engage policymakers and stakeholders to prioritize investments in strengthening the public health workforce worldwide.

Addressing misclassification bias in vaccine effectiveness studies with an application to Covid-19.

Safe and effective vaccines are crucial for the control of Covid-19 and to protect individuals at higher risk of severe disease. The test-negative design is a popular option for evaluating the effectiveness of Covid-19 vaccines. However, the findings could be biased by several factors, including imperfect sensitivity and/or specificity of the test used for diagnosing the SARS-Cov-2 infection. We propose a simple Bayesian modeling approach for estimating vaccine effectiveness that is robust even when the diagnostic test is imperfect. We use simulation studies to demonstrate the robustness of our method to misclassification bias and illustrate the utility of our approach using real-world examples.

|tPRiors |: a tool for prior elicitation and obtaining posterior distributions of true disease prevalence.

BACKGROUND: Tests have false positive or false negative results, which, if not properly accounted for, may provide misleading apparent prevalence estimates based on the observed rate of positive tests and not the true disease prevalence estimates. Methods to estimate the true prevalence of disease, adjusting for the sensitivity and the specificity of the diagnostic tests are available and can be applied, though, such procedures can be cumbersome to researchers with or without a solid statistical background. This manuscript introduces a web-based application that integrates statistical methods for Bayesian inference of true disease prevalence based on prior elicitation for the accuracy of the diagnostic tests. This tool allows practitioners to simultaneously analyse and visualize results while using interactive sliders and output prior/posterior plots. METHODS - IMPLEMENTATION: Three methods for prevalence prior elicitation and four core families of Bayesian methods have been combined and incorporated in this web tool. |tPRiors| user interface has been developed with R and Shiny and may be freely accessed on-line. RESULTS: |tPRiors| allows researchers to use preloaded data or upload their own datasets and perform analysis on either single or multiple population groups clusters, allowing, if needed, for excess zero prevalence. The final report is exported in raw parts either as.rdata or.png files and can be further analysed. We utilize a real multiple-population and a toy single-population dataset to demonstrate the robustness and capabilities of |tPRiors|. CONCLUSIONS: We expect |tPRiors| to be helpful for researchers interested in true disease prevalence estimation and who are keen on accounting for prior information. |tPRiors| acts both as a statistical tool and a simplified step-by-step statistical framework that facilitates the use of complex Bayesian methods. The application of |tPRiors| is expected to aid standardization of practices in the field of Bayesian modelling on subject and multiple group-based true prevalence estimation.

Application of Bayesian modeling for diagnostic assays of Mycobacterium avium subsp. paratuberculosis in sheep and goats flocks.

BACKGROUND: This study aimed to screen the sera of goats and sheep from flocks suspected of Mycobacterium avium subsp. paratuberculosis (MAP) infection by a newly standardized Mce-truncated ELISA (Mt-ELISA) kit for the detection of antibodies against MAP. Four diagnostic applied tests were evaluated including Indigenous plate-ELISA (IP-ELISA), Mt-ELISA, fecal Polymerase Chain Reaction (f-PCR) and fecal culture (FC). MATERIALS AND METHODS: Assuming the absence of a gold standard, latent-class models in a Bayesian framework were used to estimate the diagnostic accuracy of the four tests for MAP. RESULTS: Mt-ELISA had higher Sensitivity (Se) in sheep (posterior median: 0.68 (95% Probability Interval (PI): 0.43-0.95), while IP-ELISA recorded the highest Se in goats as 0.83 (95% PI, 0.61-0.97). The f-PCR Se estimate slightly differed between species [sheep 0.36 (0.19-0.58), goats 0.19 (0.08-0.35)], while the Se of FC was similar between species [sheep 0.29 (0.15-0.51), goats 0.27 (0.13-0.45)]. The specificity estimates for all tests were high, close to unity, and similar between species. CONCLUSION: Overall, the results showed that the Mt-ELISA method can be used for MAP detection in small ruminants' flocks.

Diagnostic Accuracy Estimates for COVID-19 Real-Time Polymerase Chain Reaction and Lateral Flow Immunoassay Tests With Bayesian Latent-Class Models.

Our objective was to estimate the diagnostic accuracy of real-time polymerase chain reaction (RT-PCR) and lateral flow immunoassay (LFIA) tests for coronavirus disease 2019 (COVID-19), depending on the time after symptom onset. Based on the cross-classified results of RT-PCR and LFIA, we used Bayesian latent-class models, which do not require a gold standard for the evaluation of diagnostics. Data were extracted from studies that evaluated LFIA (immunoglobulin G (IgG) and/or immunoglobulin M (IgM)) assays using RT-PCR as the reference method. The sensitivity of RT-PCR was 0.68 (95% probability interval (PrI): 0.63, 0.73). IgG/M sensitivity was 0.32 (95% PrI :0.23; 0.41) for the first week and increased steadily. It was 0.75 (95% PrI: 0.67; 0.83) and 0.93 (95% PrI: 0.88; 0.97) for the second and third weeks after symptom onset, respectively. Both tests had a high to absolute specificity, with higher point median estimates for RT-PCR specificity and narrower probability intervals. The specificity of RT-PCR was 0.99 (95% PrI: 0.98; 1.00). and the specificity of IgG/IgM was 0.97 (95% PrI: 0.92, 1.00), 0.98 (95% PrI: 0.95, 1.00) and 0.98 (95% PrI: 0.94, 1.00) for the first, second, and third weeks after symptom onset. The diagnostic accuracy of LFIA varies with time after symptom onset. Bayesian latent-class models provide a valid and efficient alternative for evaluating the rapidly evolving diagnostics for COVID-19, under various clinical settings and different risk profiles.

Toxoplasma gondii Infection in Swine: Implications for Public Health.

Toxoplasmosis, due to Toxoplasma gondii, is a parasitic disease with global importance. Among livestock, chronic T. gondii infection has been reported in higher rates in pigs and small ruminants, but with subclinical infections in case commonly encountered in pigs. Seroprevalence in the global pig population ranges according to the age or species of pigs, geographical distribution, production programs, and systems. Generally, T. gondii infections are noticed in low prevalence rates in conventional pig farms with high hygiene standards. In contrast, higher prevalence is common on free-ranging farms, outdoor or backyard small pig fams, as well as in farmed or hunted wild boars. The T. gondii average worldwide seroprevalence in pigs is reported to be 13% in Europe, 21% in Africa, 25% in North America, 21% in Asia, and 23% in South America. Human toxoplasmosis outbreaks have been correlated with the consumption of raw or undercooked meat, especially from infected pigs or wild boars, as well as of contaminated drinking water. The risk of infection in processed pork products is lower compared with fresh pork, as meat processing can reduce or inactivate T. gondii tissue cysts. Hence, the prevalence of T. gondii in the pig population may be a useful indicator of the risk of human toxoplasmosis associated with the consumption of pork products. The lack of obligatory screening methods at farm level for the detection of antibodies in farmed animals or the viable T. gondii in carcasses at slaughterhouse level increases the risk of contaminated pork or meat products. For this reason, the application of biosecurity and surveillance programs at farm level is very important to prevent a T. gondii infection.

Control of paratuberculosis: who, why and how. A review of 48 countries.

Paratuberculosis, a chronic disease affecting ruminant livestock, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It has direct and indirect economic costs, impacts animal welfare and arouses public health concerns. In a survey of 48 countries we found paratuberculosis to be very common in livestock. In about half the countries more than 20% of herds and flocks were infected with MAP. Most countries had large ruminant populations (millions), several types of farmed ruminants, multiple husbandry systems and tens of thousands of individual farms, creating challenges for disease control. In addition, numerous species of free-living wildlife were infected. Paratuberculosis was notifiable in most countries, but formal control programs were present in only 22 countries. Generally, these were the more highly developed countries with advanced veterinary services. Of the countries without a formal control program for paratuberculosis, 76% were in South and Central America, Asia and Africa while 20% were in Europe. Control programs were justified most commonly on animal health grounds, but protecting market access and public health were other factors. Prevalence reduction was the major objective in most countries, but Norway and Sweden aimed to eradicate the disease, so surveillance and response were their major objectives. Government funding was involved in about two thirds of countries, but operations tended to be funded by farmers and their organizations and not by government alone. The majority of countries (60%) had voluntary control programs. Generally, programs were supported by incentives for joining, financial compensation and/or penalties for non-participation. Performance indicators, structure, leadership, practices and tools used in control programs are also presented. Securing funding for long-term control activities was a widespread problem. Control programs were reported to be successful in 16 (73%) of the 22 countries. Recommendations are made for future control programs, including a primary goal of establishing an international code for paratuberculosis, leading to universal acknowledgment of the principles and methods of control in relation to endemic and transboundary disease. An holistic approach across all ruminant livestock industries and long-term commitment is required for control of paratuberculosis.

The effect of butorphanol on the incidence of dexmedetomidine-induced emesis in cats.

OBJECTIVE: Τo evaluate the antiemetic effect of butorphanol (BUT) when co-administered with dexmedetomidine (DEX) in cats. STUDY DESIGN: Double-blind, randomized controlled cross-over experimental study. ANIMALS: Fourteen purpose-bred healthy Domestic Short Hair cats, seven females and seven males, aged median (range) 14-84 (78) months and weighing 1.7-5.5 (4.0) kg. METHODS: Each cat received five different treatment protocols intramuscularly (IM): (A) 25 µg kg(-1) DEX; (B) 20 µg kg(-1) DEX and 0.2 mg kg(-1) BUT; (C) 20 µg kg(-1) DEX and 0.1 mg kg(-1) BUT; (D) 25 µg kg(-1) DEX and 0.2 mg kg(-1) BUT; and (E) 20 µg kg(-1) DEX. Episodes of emesis, incidence and severity of nausea, and time to lateral recumbency were recorded for a period of 8 minutes after treatment administration, and the sedation was scored at the end of this period. The Friedman test and the Cochran's Q-test were used to analyse the data. Significance was evaluated at the 5% level. RESULTS: The proportion of cats that vomited was significantly lower with the treatment protocols that included BUT (B, C and D) compared with the protocols that included only DEX (A and E). The proportion of cats that had nausea was significantly higher with the protocols that included only DEX (A and E) compared with protocols B and D. Time to lateral recumbency (p=0.09) and sedation score (p=0.07) was not statistically different between the treatment protocols. CONCLUSIONS AND CLINICAL RELEVANCE: Butorphanol can be used to prevent emesis and reduce the incidence and the severity of nausea caused by DEX in cats. It seems that the combination of BUT and DEX is very useful not only when emesis could result in serious complications, but also to provide comfort and well-being in cats sedated for minor procedures.

Bayesian true prevalence estimation of brucellosis in sheep, goats, cattle and camels in southeast regions of Iran.

AIMS AND METHODS: Brucellosis is worldwide one of the most prevalent zoonotic diseases with serious public health hazard affecting domestic livestock and causing economic losses. Objective of this study is to estimate the true prevalence of brucellosis in livestock, specifically cattle, sheep, goats and camels, using a novel Bayesian latent class model, adjusting for the imperfect sensitivity and specificity of the applied tests, where the second test was restricted only to first test-positive samples. RESULTS: Brucellosis seems more prevalent in goats and sheep, while the posterior medians and 95% probability intervals (95% PI) for the average true prevalence for sheep, goats, cattle and camels are 18% (4%-43%), 19% (7%-37%), 16% (5%-34%) and 18% (1%-48%) respectively. CONCLUSIONS: The study results indicate that brucellosis is highly endemic in Iran and crucial steps are needed to control and raise awareness about the high public health concern of the disease.

Determining the conclusiveness of a meta-analysis.

The pursuit of conclusive evidence related to an unanswered foreground (decision-making) question has been the driving factor behind multiple ongoing and planned randomized controlled trials as well as meta-analyses. However, a fundamental challenge lies in establishing robust methods for ascertaining whether a collection of synthesized trials has yielded a definitive answer to that foreground question through the process of meta-analysis. This article explores the evolution of methods that attempt to address this challenge. These methods have primarily focused on defining and measuring the sufficiency and stability of evidence within a meta-analytic context. Cumulative meta-analysis and trial sequential analysis are the tools currently used, but they both come with limitations and challenges. We further discuss methods aimed at evaluating the evolution of effects over time more directly, such as the recursive cumulative meta-analysis. The latter method can be considered a better alternative, as it serves to demonstrate whether there is a true underlying treatment effect to which the meta-analysis is converging. However, recursive cumulative meta-analysis falls short of a specific indicator that establishes whether convergence has been reached. We coin the term exit for a meta-analysis where convergence can be demonstrated. Developing methods to determine the exit status of a meta-analysis is the next priority in research synthesis methods, as it will indicate that the research journey has concluded on a particular foreground question with no expectation of a different result with the addition of future trials.

Estimates of sensitivity and specificity of serological tests for SARS-CoV-2 specific antibodies using a Bayesian latent class model approach.

OBJECTIVES: Assessing the accuracy of serological tests for SARS-CoV-2 was challenging due to the lack of a gold standard. This study aimed to estimate the accuracy of SARS-CoV-2-specific serological tests using Bayesian latent class models (BLCM) and compare methods with and without a gold standard. STUDY DESIGN AND SETTING: In this study, we analyzed 356 samples-254 positives, ie, from individuals with a previous SARS-CoV-2 infection diagnosis, and 102 negatives, ie, prepandemic samples-using six different rapid serological tests and one laboratory assay. A BLCM was employed to concurrently estimate the sensitivity and specificity of all serological tests for the immunoglobulin (Ig) M and IgG antibodies specific for SARS-CoV-2. Noninformative priors were used. A sensitivity analysis was conducted considering three methods: 1) reverse transcription-polymerase chain reaction test (RT-PCR) as the gold standard, 2) BLCM with RT-PCR as an imperfect gold standard, and 3) frequentist latent class model (LCM). All analyses used software R version 4.3.0, and BLCM were fitted using package runjags using the software JAGS (Just Another Gibbs Sampler). RESULTS: The BLCM-derived sensitivity for IgM varied from 10.7% [95% credibility interval (CrI):1.9-24.6] to 96.9% (95% CrI: 91.0-100.0), with specificities ranging from 48.3% (95% CrI: 39.0-57.6) to 98.9% (95% CrI: 96.2-100.0). Sensitivity for IgG varied between 76.9% (95% CrI: 68.2-84.7) and 99.1% (95% CrI: 96.1-100.0), and specificity ranged from 49.9% (95% CrI: 19.4-95.8) to 99.3% (95% CrI: 97.2-100.0). LCM results were comparable to BLCM. Considering the RT-PCR as a gold standard underestimated the tests' sensitivity, particularly for IgM. CONCLUSION: BLCM-derived results deviated from those using a gold standard, which underestimated the tests' characteristics, particularly sensitivity. Although Bayesian and frequentist LCM approaches yielded comparable results, BLCM had the benefit of enabling credibility interval computation even when sample power is limited.

Animal Health Discourse during Ecological Crises in the Media-Lessons Learnt from the Flood in Thessaly from the One Health Perspective.

Due to the increasing risk of extreme events caused by climate change (i.e., floods, fires and hurricanes) or wars, European veterinary public health may need some improvement. Utilizing a mix of qualitative (participatory observation) and quantitative methods (Internet mining), we analyzed the Greek media's responses to the millennial flood in Thessaly (September 2023), focusing on animal health (including wild, companion animals and livestock) and public sentiment towards epizootic/epidemic threats. The study revealed a gap in crisis management plans regarding veterinary-related issues, emphasizing the need for comprehensive emergency response strategies. Our findings show how (i) the lay referral system is projecting the perception of epidemic threats into the population; (ii) the emotional load of images of animal carcasses is misused by media creators aiming for a big audience; and (iii) pets' owners are creating online communities for the searching and treatment of their pets. Our results stress the importance of integrating crisis communication in consecutive phases of the discourse, such as the following: (i) weather change; (ii) acute flood; (iii) recovery; and (iv) outbreaks, into veterinary practices to better prepare for such disasters.

Bayesian latent class modelling of true prevalence in animal subgroups with application to bovine paratuberculosis infection.

The prevalence of an infectious disease of animals living in separate groups (e.g. herds) is naturally analyzed using a Bayesian hierarchical latent class model. We propose an extension to this methodology by including subgroup level prevalence measures within the groups of animals. As an application illustrating the merits of our methodology, we reassessed the prevalence of bovine paratuberculosis (PTBC) infection in Hungarian commercial dairy farms. Our aim was to consolidate previous findings using a large amount of recent data and priors based on historical data. To model the subgroup level infection prevalence within animal groups, we considered correlated prevalences following beta distributions derived from independent normally distributed random herd effects. In the application, infection status of herds was handled as latent classes, multiparous and primiparous cows as within-herd subgroups. The novel methodology allows us to estimate both the mean and median conditional within-herd true prevalence (CWHP) related to each animal subgroup as well as other measures characterizing the interrelation of subgroups. The results of the application aligned with the findings of the former PTBC study, while the more recent and considerably larger dataset and the use of historical priors increased the reliability of the results. The STAN and JAGS codes of the application are available in Supplementary material.

Real-world evidence of rabies post-exposure prophylaxis in Serbia: Nation-wide observational study (2017-2019).

BACKGROUND: Rabies remains a deadly zoonotic disease, primarily prevalent in Eastern European countries, with a significant global burden in Asia and Africa. Post-exposure prophylaxis (PEP) is critical to prevent clinical rabies. Serbia, a country with a relatively low animal rabies incidence, has been implementing a 4-dose Essen PEP regimen for 13 years. This real-world study aimed to assess the effectiveness of the 4-dose Essen regimen, considering demographic and clinical factors, after WHO Category III exposure. METHOD: The study included 601 patients who received the 4-dose Essen PEP and 79 who received an additional 5th dose. RESULTS: Age emerged as a critical factor influencing seroconversion rates after the 4-dose regimen, with older individuals exhibiting lower RVNA titers. Logistic regression indicated a 3.18% decrease in seroconversion odds for each added year of age. The Cox proportional hazards mixed model highlighted age-related risks, with age groups 45-60 and 75-92 at the highest risk of non-seroconversion. Human Rabies Immune Globulin (HRIG) administration was associated with lower RVNA values after the 4-dose regimen, suggesting interference with vaccine immunogenicity among people who received larger doses of HRIG. CONCLUSIONS: This study provides valuable real-world evidence for rabies PEP in a non-homogeneous population with potential comorbidities. The results underscore the importance of optimizing PEP strategies, particularly in older individuals, and reconsidering HRIG dosing to improve seroconversion rates.

Early warning of potential epidemics: A pilot application of an early warning tool to data from the pulmonary clinic of the university hospital of Thessaly, Greece.

BACKGROUND & METHODS: This paper describes a pilot application of the Epidemic Volatility Index (EVI) to data from the pulmonary clinic of the University Hospital of Thessaly, Greece, for monitoring respiratory infections, COVID-19, and flu cases. EVI, a simple and easily implemented early warning method based on the volatility of newly reported cases, exhibited consistent and stable performance in detecting new waves of epidemics. The study highlights the importance of implementing early warning tools to address the effects of epidemics, including containment of outbreaks, timely intervention strategies, and resource allocation within real-world clinical settings as part of a broader public health strategy. RESULTS: The results presented in the figures demonstrate the association between successive early warnings and the onset of new waves, providing valuable insights for proactive decision-making. A web-based application enabling real-time monitoring and informed decision-making by healthcare professionals, public health officials, and policymakers was developed. CONCLUSIONS: This study emphasizes the significant role of early warning methods in managing epidemics and safeguarding public health. Future research may explore extensions and combinations of multiple warning systems for optimal outbreak interventions and application of the methods in the context of personalized medicine.

Assessment of the effect of long-term serum storage for retrospective serologic diagnosis of canine monocytic ehrlichiosis (Ehrlichia canis).

There is currently sparse information on the possible effect of long-term storage of serum specimens for the retrospective serodiagnosis of canine monocytic ehrlichiosis (CME). The aim of this study was to assess the agreement between the original serologic outcome and the results of a repeat indirect fluorescent antibody (IFA) assay for the detection of IgG antibodies against E. canis. A secondary aim was to compare the diagnostic performance of two commercially available point-of-care (POC) immunochromatographic (IC) assays. Archived serum samples originally tested as positive (n=66) or negative (n=19) for E. canis IgG antibodies and kept frozen at -20°C for a median of 22 years, were retrospectively examined by IFA and by two POC IC assays. Cohen's Kappa coefficient (0.748, p < 0.0001), indicated a substantial agreement between the original and repeat serologic testing results. An almost identical high sensitivity and moderate specificity were established for the two POC IC assays. Canine serum specimens on long-term storage may still be of value for seroepidemiologic surveys investigating the exposure to E. canis.

Effects of a Curcumin/Silymarin/Yeast-Based Mycotoxin Detoxifier on Redox Status and Growth Performance of Weaned Piglets under Field Conditions.

The aim of this in vivo study was to investigate the effects of a novel mycotoxin detoxifier whose formulation includes clay (bentonite and sepiolite), phytogenic feed additives (curcumin and silymarin) and postbiotics (yeast products) on the health, performance and redox status of weaned piglets under the dietary challenge of fumonisins (FUMs). The study was conducted in duplicate in the course of two independent trials on two different farms. One hundred and fifty (150) weaned piglets per trial farm were allocated into two separate groups: (a) T1 (control group): 75 weaned piglets received FUM-contaminated feed and (b) T2 (experimental group): 75 weaned piglets received FUM-contaminated feed with the mycotoxin-detoxifying agent from the day of weaning (28 days) until 70 days of age. Thiobarbituric acid reactive substances (TBARSs), protein carbonyls (CARBs) and the overall antioxidant capacity (TAC) were assessed in plasma as indicators of redox status at 45 and 70 days of age. Furthermore, mortality and performance parameters were recorded at 28, 45 and 70 days of age, while histopathological examination was performed at the end of the trial period (day 70). The results of the present study reveal the beneficial effects of supplementing a novel mycotoxin detoxifier in the diets of weaners, including improved redox status, potential hepatoprotective properties and enhanced growth performance.

Review state-of-the-art of output-based methodological approaches for substantiating freedom from infection.

A wide variety of control and surveillance programmes that are designed and implemented based on country-specific conditions exists for infectious cattle diseases that are not regulated. This heterogeneity renders difficult the comparison of probabilities of freedom from infection estimated from collected surveillance data. The objectives of this review were to outline the methodological and epidemiological considerations for the estimation of probabilities of freedom from infection from surveillance information and review state-of-the-art methods estimating the probabilities of freedom from infection from heterogeneous surveillance data. Substantiating freedom from infection consists in quantifying the evidence of absence from the absence of evidence. The quantification usually consists in estimating the probability of observing no positive test result, in a given sample, assuming that the infection is present at a chosen (low) prevalence, called the design prevalence. The usual surveillance outputs are the sensitivity of surveillance and the probability of freedom from infection. A variety of factors influencing the choice of a method are presented; disease prevalence context, performance of the tests used, risk factors of infection, structure of the surveillance programme and frequency of testing. The existing methods for estimating the probability of freedom from infection are scenario trees, Bayesian belief networks, simulation methods, Bayesian prevalence estimation methods and the STOC free model. Scenario trees analysis is the current reference method for proving freedom from infection and is widely used in countries that claim freedom. Bayesian belief networks and simulation methods are considered extensions of scenario trees. They can be applied to more complex surveillance schemes and represent complex infection dynamics. Bayesian prevalence estimation methods and the STOC free model allow freedom from infection estimation at the herd-level from longitudinal surveillance data, considering risk factor information and the structure of the population. Comparison of surveillance outputs from heterogeneous surveillance programmes for estimating the probability of freedom from infection is a difficult task. This paper is a 'guide towards substantiating freedom from infection' that describes both all assumptions-limitations and available methods that can be applied in different settings.