On-site verification of the CBEU method using UriSelect4 chromogenic medium at the National Institute of Hygiene (INH) of Lomé.

PURPOSE: The Cytobacteriological Examination of Urine (CBEU) is one of the most requested microbiological analyses in the medical bacteriology laboratory. According to the ISO 15189 standard, Medical Biology Laboratory (MBL) must validate the techniques before their use. It is in this perspective that, within the Medical Bacteriology Laboratory of the INH of Lome, we have carried out the on-site verification of the CBEU method using UriSelect4 chromogenic medium at the INH of Lomé. MATERIAL AND METHODS: The biological material was composed of reference bacterial strains and clinical bacterial strains isolated at INH. Failure Modes Effects and Criticality Analysis (FMECA) with 5 â€‹M model was used to perform risk analysis and performances as repeatability, intermediate fidelity/reproducibility, sensitivity and inter-operator variability were evaluated using references strains and different operators. "Comité Français d'Accréditation" (COFRAC) SH GTA 04 was used as reference. Single-factor ANOVA was used to analyze the data. RESULTS: Operating procedures, Patients preparation, Samples and Preparation of culture media were identified as main critical points with a criticality index of 8, 9, 9 and 12 respectively. The use of uriselect4 to perform ECBU was shown to be repeatable and reproducible. The sensibility of Uriselect4 to detect urinary infection was 100% with negligible inter-operator variability. CONCLUSION: This study performed on-site verification of Uriselect4 at the bacteriology lab of INH of Lome and identified some critical points to master. The overall performance criteria from COFRAC SH GTA 04 were conform.

Description of Antimicrobial Resistance Patterns at the National Institute of Hygiene in Lome, Togo.

The monitoring of antimicrobial resistance has become a priority, mainly in developing countries, to control the emergence of multidrug-resistant bacterial strains. This study evaluated the antibiotic resistance profiles of bacteria isolated at the National Institute of Hygiene in Togo. The laboratory records from January 2013 to December 2015 were examined, which showed that a total of 33,147 samples had been analyzed. Among them, vaginal swabs and urine samples were predominant, amounting to 38.17% and 33.24%, respectively. In total, 3,497 Gram-positive and 1,970 Gram-negative bacterial strains were cultured. Enterobacteriaceae (57.6%), Staphylococcus spp. (21.1%), and Streptococcus spp. (10.5%) were primarily isolated. The records showed that over 3 years, Escherichia coli was resistant to piperacillin (79.66%, 75.1%, and 83%), trimethoprim/sulfamethoxazole (79.3%, 82%, and 82.8%), ticarcillin (89%, 89.7%, and 93%), and amoxicillin (94.1%, 94%, and 96.09%), whereas Staphylococcus aureus was resistant to penicillin G. Streptococcaceae isolates were resistant to trimethoprim/sulfamethoxazole (78.11-87.1%), tetracycline (82.2-91.16%), and norfloxacin (86.16-94.3%). Escherichia coli and S. aureus isolated from urine were more resistant to antibiotics than those isolated from vaginal swabs. There is a need to develop new strategies to fight the emergence of multi-resistant bacteria in Togo.

Estimation of yellow fever incidence in Togo between 2010 and 2020.

BACKGROUND: Yellow fever virus is an arbovirus transmitted to humans by Aedes and Haemogogus mosquito species. To date, there is no specific treatment for yellow fever. However, an effective vaccine is available for the prevention. After a decline in yellow fever cases in Africa between 2004 and 2015, large-scale transmission of the virus was observed in Africa during 2019, with outbreaks recorded in West Africa. The objective of this study was to estimate the incidence of yellow fever cases recorded in the national reference laboratory of Togo from 2010 to 2020. METHOD: Data were extracted from the National Institute of Hygiene database from 2010 to 2020 with an Excel sheet and descriptive analyses were performed. RESULTS: A total of 4350 samples were collected between 2010 and 2020 in Togo from yellow fever suspected cases. These cases had a median age of 12 years (IQR: 5-24), and 21% of them were from the Maritime region. Among them, 30 cases were reported by national laboratory, with a global incidence of 0.7% (confidence interval 95%: [0.4-1.0]). At the yellow fever regional laboratory, 14 cases were confirmed with an incidence of 0.33% (confidence interval 95%: [0.18-0.55]). In this population, 37.7% had been immunized against yellow fever. CONCLUSION: This study shows that Togo presents cases of yellow fever. Identification of the vectors and implementation of efficient vector control measures could help prevent this disease, as well as other diseases transmitted by the same vectors. Yellow fever vaccination should be a priority in vaccination programs.

Onchocerca volvulus-specific antibody and cellular responses in onchocerciasis patients treated annually with ivermectin for 30 years and exposed to parasite transmission in central Togo.

BACKGROUND: Annual mass drug administrations (MDA) of ivermectin will strongly reduce Onchocerca volvulus microfilariae (mf) in the skin and in the onchocerciasis patients' eyes. Ivermectin treatment will also affect the expression of immunity in patients, such that activated immune defenses may help control and contribute to clearance of mf of O. volvulus. Longitudinal surveys are a prerequisite to determining the impact of ivermectin on the status of anti-parasite immunity, notably in risk zones where parasite transmission and active O. volvulus infections persist. METHODOLOGY/PRINCIPAL FINDINGS: Onchocerciasis patients were treated annually with ivermectin and their Onchocerca volvulus antigen (OvAg) specific IgG and cellular responses were investigated before and at 30 years post initial ivermectin treatment (30yPT). Repeated annual ivermectin treatments eliminated persisting O. volvulus microfilariae (mf) from the skin of patients and abrogated patent infections. The OvAg-specific IgG1 and IgG4 responses were diminished at 30yPT to the levels observed in endemic controls. Prior to starting ivermectin treatment, OvAg-induced cellular productions of IL-10, IFN-γ, CCL13, CCL17 and CCL18 were low in patients, and at 30yPT, cellular cytokine and chemokine responses increased to the levels observed in endemic controls. In contrast, mitogen(PHA)- induced IL-10, IFN-γ, CCL17 and CCL18 cellular production was diminished. This divergent response profile thus revealed increased parasite antigen-specific but reduced polyclonal cellular responsiveness in patients. The transmission of O. volvulus continued at the patients' location in the Mô river basin in central Togo 2018 and 2019 when 0.58% and 0.45%, respectively, of Simulium damnosum s.l. vector blackflies carried O. volvulus infections. CONCLUSIONS/SIGNIFICANCE: Repeated annual ivermectin treatment of onchocerciasis patients durably inhibited their patent O. volvulus infections despite ongoing low-level parasite transmission in the study area. Repeated MDA with ivermectin affects the expression of immunity in patients. O. volvulus parasite-specific antibody levels diminished to levels seen in infection-free endemic controls. With low antibody levels, antibody-dependent cellular cytotoxic responses against tissue-dwelling O. volvulus larvae will weaken. O. volvulus antigen inducible cytokine and chemokine production increased in treated mf-negative patients, while their innate responsiveness to mitogen declined. Such lower innate responsiveness in elderly patients could contribute to reduced adaptive immune responses to parasite infections and vaccines. On the other hand, increased specific cellular chemokine responses in mf-negative onchocerciasis patients could reflect effector cell activation against tissue invasive larval stages of O. volvulus. The annual Simulium damnosum s.l. biting rate observed in the Mô river basin was similar to levels prior to initiation of MDA with ivermectin, and the positive rtPCR results reported here confirm ongoing O. volvulus transmission.

African National Public Health Institutes Responses to COVID-19: Innovations, Systems Changes, and Challenges

National public health institutes (NPHIs)-science-based governmental agencies typically part of, or closely aligned with, ministries of health-have played a critical part in many countries' responses to the COVID-19 pandemic. Through listening sessions with NPHI leadership, we captured the experiences of NPHIs in Africa. Our research was further supplemented by a review of the literature. To address issues related to COVID-19, NPHIs in Africa developed a variety of innovative approaches, such as working with the private sector to procure and manage vital supplies and address key information needs. Creative uses of technology, including virtual training and messaging from drones, contributed to sharing information and battling misinformation. Positive impacts of the pandemic response include increased laboratory capacity in many countries, modernized surveillance systems, and strengthened public-private partnerships; much of this enhanced capacity is expected to persist beyond the pandemic. However, several challenges remain, including the lack of staff trained in areas like bioinformatics (essential for genomic analysis) and the need for sustained relationships and data sharing between NPHIs and agencies not traditionally considered public health (eg, those related to border crossings), as well as the impact of the pandemic on prevention and control of non-COVID-19 conditions-both infectious and noncommunicable. Participants in the listening sessions also highlighted concerns about inequities in access to, and quality of, the public health services and clinical care with resultant disproportionate impact of the pandemic on certain populations. COVID-19 responses and challenges highlight the need for continued investment to strengthen NPHIs and public health infrastructure to address longstanding deficiencies and ensure preparedness for the next public health crisis...

African National Public Health Institutes Responses to COVID-19: Innovations, Systems Changes, and Challenges.

National public health institutes (NPHIs)-science-based governmental agencies typically part of, or closely aligned with, ministries of health-have played a critical part in many countries' responses to the COVID-19 pandemic. Through listening sessions with NPHI leadership, we captured the experiences of NPHIs in Africa. Our research was further supplemented by a review of the literature. To address issues related to COVID-19, NPHIs in Africa developed a variety of innovative approaches, such as working with the private sector to procure and manage vital supplies and address key information needs. Creative uses of technology, including virtual training and messaging from drones, contributed to sharing information and battling misinformation. Positive impacts of the pandemic response include increased laboratory capacity in many countries, modernized surveillance systems, and strengthened public-private partnerships; much of this enhanced capacity is expected to persist beyond the pandemic. However, several challenges remain, including the lack of staff trained in areas like bioinformatics (essential for genomic analysis) and the need for sustained relationships and data sharing between NPHIs and agencies not traditionally considered public health (eg, those related to border crossings), as well as the impact of the pandemic on prevention and control of non-COVID-19 conditions-both infectious and noncommunicable. Participants in the listening sessions also highlighted concerns about inequities in access to, and quality of, the public health services and clinical care with resultant disproportionate impact of the pandemic on certain populations. COVID-19 responses and challenges highlight the need for continued investment to strengthen NPHIs and public health infrastructure to address longstanding deficiencies and ensure preparedness for the next public health crisis.

Assessment of the usefulness of anti-Wb123 antibody for post-elimination surveillance of lymphatic filariasis.

BACKGROUND: The World Health Organization has targeted lymphatic filariasis (LF) for elimination as a public health problem and recommends, among other measures, post-elimination surveillance of LF. The identification of sensitive and specific surveillance tools is therefore a research priority. The Wuchereria bancrofti-specific antigen Wb123-based enzyme-linked immunosorbent assay (Wb123 ELISA) detects antibodies to the recombinant Wb123 antigen of W. bancrofti and may be useful as a surveillance tool for LF. Six years after stopping mass drug administration to eliminate LF and recording successful results on two post-treatment transmission assessment surveys, a study was conducted in Togo aimed at helping to identify the role of the Wb123 ELISA in post-validation surveillance of LF. METHODS: This was a cross-sectional study in eight previously LF-endemic districts and one non-endemic district in Togo. In each sub-district of these nine districts, two schools were selected and 15 children aged 6 to 9 years old at each school provided finger-stick blood for testing for antibodies to Wb123 using the Filaria Detect™ IgG4 ELISA kit® (InBios, International, Inc., Seattle, WA, USA). RESULTS: A total of 2654 children aged 6 to 9 years old were tested in 134 schools in the nine districts. Overall, 4.7% (126/2654) children tested positive for antibodies to the Wb123 antigen of W. bancrofti. The prevalence of Wb123 antibodies varied across the eight previously endemic LF districts, from 1.56 to 6.62%. The highest prevalence, 6.99%, was found in the non-endemic district, but this was not significantly different from the average of all the LF districts (4.49%, P = 0.062). CONCLUSIONS: The Wb123 ELISA was positive in 4.7% of Togolese school-age children who were almost certainly unexposed to LF. This apparent lack of specificity in the Togo context makes it difficult to establish a seroprevalence threshold that could serve to signal LF resurgence in the country, precluding the use of this test for post-validation surveillance in Togo. There remains a need to develop a useful and reliable test for post-elimination surveillance for LF in humans.

Prevalence of SARS-CoV-2 among high-risk populations in Lomé (Togo) in 2020.

BACKGROUND: In December 2019, the COVID-19 outbreak began in China and quickly spread throughout the world and was reclassified as a pandemic in March 2020. The first case of COVID-19 was declared in Togo on March 5. Two months later, few data were available to describe the circulation of the new coronavirus in the country. OBJECTIVE: This survey aimed to estimate the prevalence of SARS-CoV-2 in high-risk populations in Lomé. MATERIALS AND METHODS: From April 23, 2020, to May 8, 2020, we recruited a sample of participants from five sectors: health care, air transport, police, road transport and informal. We collected oropharyngeal swabs for direct detection through real-time reverse transcription polymerase chain reaction (rRT-PCR) and blood for antibody detection by serological tests. The overall prevalence (current and past) of infection was defined by positivity for both tests. RESULTS: A total of 955 participants with a median age of 36 (IQR 32-43) were included, and 71.6% (n = 684) were men. Approximately 22.1% (n = 212) were from the air transport sector, 20.5% (n = 196) were from the police sector, and 38.7% (n = 370) were from the health sector. Seven participants (0.7%, 95% CI: 0.3-1.6%) had a positive rRT-PCR test result at the time of recruitment, and nine (0.9%, 95% CI: 0.4-1.8%) were seropositive for IgM or IgG against SARS-CoV-2. We found an overall prevalence of 1.6% (n = 15), 95% CI: 0.9-2.6%. CONCLUSION: The prevalence of SARS-CoV-2 infection among high-risk populations in Lomé was relatively low and could be explained by the various measures taken by the Togolese government. Therefore, we recommend targeted screening.

Risk factors of hepatitis B virus surface antigen carriage and serological profile of HBsAg carriers in Lomé Togo, 2016.

BACKGROUND: In Togo, the prevalence of Hepatitis B Virus Surface Antigen (HBsAg) among young people aged 15-24 years was estimated at 16.4% in 2010; however, risk factors for HBsAg carriage are poorly documented. We sought to identify risk factors for HBsAg carriage and the serological profile of HBsAg carriers in Lomé (capital city of Togo). METHOD: We conducted a case control study from October 2016 to March 2017 in Lomé. Cases and controls were randomly selected from a database of Institut National d'Hygiène (INH) of Lomé during a free screening campaign for hepatitis B. We calculated means, frequencies, proportions, odds ratios (OR), and 95% confidence interval (CI) and performed logistic regression. RESULTS: We included 83 confirmed cases and 249 controls. The median age was 31 years among cases and 30 years among the controls. The sex ratios (M/F) were 11/6 among cases and 4/3 for the controls. The independent risk factors for HBsAg carriage were the awareness of hepatitis B serological status (OR = 3.56, 95% CI [1.80-7.04]) and Kabyè-tem ethnic group (OR = 3.56, 95% CI [1.98-6.39]). Among HBsAg carriers, 13.3% were at the viral replication stage (all of whom were between 30 and 45 years of age) and 1.2% were at the acute stage of the disease. The prevalence of co-infection with hepatitis B and C was 4.80%. All co-infections were in women aged 24-28 years. CONCLUSION: The Kabyè-tem ethnic group is at risk of HBsAg carriage in Lomé. Of note, most HBsAg carriers in this ethnic group are aware of their HBsAg serological status. Furthermore, the prevalence of Hepatitis among adults of reproductive age is high and is cause for concern. We therefore recommend screening and vaccination campaigns at subsidized prices among people aged 30 years and older.

Impact of community-based integrated mass drug administration on schistosomiasis and soil-transmitted helminth prevalence in Togo.

BACKGROUND: Togo has conducted annual, integrated, community-based mass drug administration (MDA) for soil-transmitted helminths (STH) and schistosomiasis since 2010. Treatment frequency and target populations are determined by disease prevalence, as measured by baseline surveys in 2007 and 2009, and WHO guidelines. Reported programmatic treatment coverage has averaged over 94%. Togo conducted a cross-sectional survey in 2015 to assess the impact of four to five years of MDA on these diseases. METHODOLOGY/PRINCIPAL FINDINGS: In every sub-district in the country outside the capital, the same schools were visited as at baseline and a sample of fifteen children age 6 to 9 years old was drawn. Each child submitted urine and a stool sample. Urine samples were tested by dipstick for the presence of blood as a proxy measure of Schistosoma haematobium infection. Stool samples were analyzed by the Kato-Katz method for STH and Schistosoma mansoni. At baseline, 17,100 children were enrolled at 1,129 schools in 562 sub-districts; in 2015, 16,890 children were enrolled at the same schools. The overall prevalence of both STH and schistosomiasis declined significantly, from 31.5% to 11.6% for STH and from 23.5% to 5.0% for schistosomiasis (p<0.001 in both instances). Egg counts from both years were available only for hookworm and S. mansoni; intensity of infection decreased significantly for both infections from 2009 to 2015 (p<0.001 for both infections). In areas with high baseline prevalence, rebound of hookworm infection was noted in children who had not received albendazole in the past 6 months. CONCLUSIONS/SIGNIFICANCE: After four to five years of MDA in Togo, the prevalence and intensity of STH and schistosomiasis infection were significantly reduced compared to baseline. Data on STH indicate that stopping MDA in areas with high baseline prevalence may result in significant rebound of infection. Togo's findings may help refine treatment recommendations for these diseases.

Prevalence of syphilis among female sex workers and their clients in Togo in 2011.

BACKGROUND: During the last ten years, a resurgence of syphilis has occurred in many countries worldwide, including Togo. Previous studies have shown a wide range of syphilis infection among the female sex workers (FSWs), from 1.5 to 42.1%. In Togo, Key populations, including FSWs, are rarely involved in the sentinel surveillance programs to determine the prevalence of HIV and syphilis. The aim of this study was to determine the prevalence of syphilis among female sex workers (FSWs) and their clients in Togo. METHODS: We conducted a cross-sectional study in December 2011 targeting FSWs and their clients in Togo. Among participant who consented, we collected blood samples for syphilis and HIV testing. RESULTS: In total, 1,836 participants (1,106 FSWs and 730 clients) were included in the survey. Their mean age was 28.6 ± 9 years. The prevalence of syphilis was 2.2% (2.2% among FSWs compare to 2.3% among their clients, p = 0.82). This prevalence was higher among FSWs over 30 years old compare to those less than 30 years old (Odd Ratio (OR) =5.03; 95% CI [1.95-13.49]). Single FSWs were three times less likely to have syphilis than those living in couple or married (OR = 3.11; CI 95% [1.16-8.83]). Brothel based or declared FSWs were 4 times more likely to be infected by syphilis than secret ones (OR = 3.89; CI 95% [1.60-9.54]). Out of the 1,836 participants of the survey, 165 (8.9%) were HIV positive. Having syphilis was associated with HIV infection (OR = 3.41; IC 95% [1.53-7.41]). CONCLUSION: This study showed that: i) the prevalence of syphilis among FSWs and their clients was high; ii) syphilis was significantly associated with HIV infection. It is necessary to increase awareness campaigns and emphasize on condom use among this key population group.

Emergence of Lassa Fever Disease in Northern Togo: Report of Two Cases in Oti District in 2016.

BACKGROUND: Lassa fever belongs to the group of potentially fatal hemorrhagic fevers, never reported in Togo. The aim of this paper is to report the first two cases of Lassa fever infection in Togo. CASE PRESENTATION: The two first Lassa fever cases occurred in two expatriate's health professionals working in Togo for more than two years. The symptoms appeared among two health professionals of a clinic located in Oti district in the north of the country. The absence of clinical improvement after antimalarial treatment and the worsening of clinical symptoms led to the medical evacuation. The delayed diagnosis of the first case led to a fatal outcome. The second case recovered under ribavirin treatment. CONCLUSION: The emergence of this hemorrhagic fever confirms the existence of Lassa fever virus in Togo. After a period of intensive Ebola virus transmission from 2013 to 2015, this is an additional call for the establishment and enhancement of infection prevention and control measures in the health care setting in West Africa.

Effectiveness of routine BCG vaccination on buruli ulcer disease: a case-control study in the Democratic Republic of Congo, Ghana and Togo.

BACKGROUND: The only available vaccine that could be potentially beneficial against mycobacterial diseases contains live attenuated bovine tuberculosis bacillus (Mycobacterium bovis) also called Bacillus Calmette-Guérin (BCG). Even though the BCG vaccine is still widely used, results on its effectiveness in preventing mycobacterial diseases are partially contradictory, especially regarding Buruli Ulcer Disease (BUD). The aim of this case-control study is to evaluate the possible protective effect of BCG vaccination on BUD. METHODOLOGY: The present study was performed in three different countries and sites where BUD is endemic: in the Democratic Republic of the Congo, Ghana, and Togo from 2010 through 2013. The large study population was comprised of 401 cases with laboratory confirmed BUD and 826 controls, mostly family members or neighbors. PRINCIPAL FINDINGS: After stratification by the three countries, two sexes and four age groups, no significant correlation was found between the presence of BCG scar and BUD status of individuals. Multivariate analysis has shown that the independent variables country (p = 0.31), sex (p = 0.24), age (p = 0.96), and presence of a BCG scar (p = 0.07) did not significantly influence the development of BUD category I or category II/III. Furthermore, the status of BCG vaccination was also not significantly related to duration of BUD or time to healing of lesions. CONCLUSIONS: In our study, we did not observe significant evidence of a protective effect of routine BCG vaccination on the risk of developing either BUD or severe forms of BUD. Since accurate data on BCG strains used in these three countries were not available, no final conclusion can be drawn on the effectiveness of BCG strain in protecting against BUD. As has been suggested for tuberculosis and leprosy, well-designed prospective studies on different existing BCG vaccine strains are needed also for BUD.

First observation in a non-endemic country (Togo) of Penicillium marneffei infection in a human immunodeficiency virus-infected patient: a case report.

BACKGROUND: Infection with Penicillium marneffei is a common opportunistic infection in Southeast Asia where it is endemic. We report a case of Penicillium marneffei infection with fatal outcome in a Togolese woman infected with Human Immunodeficiency Virus (HIV). CASE PRESENTATION: A 45-years-old patient, infected with Human Immunodeficiency Virus had consulted for ongoing febrile pneumonia since two weeks. Clinical examination revealed fever of 38.5°C, dyspnea, pulmonary syndrome condensation and papulo-nodular of "molluscum contagiosum" like lesions located on the face, arms, neck and trunk. Sputum smear was negative for tuberculosis. The chest radiograph showed reticulonodular opacities in the right upper and middle lobes and two caves in the right hilar region. The CD4 count was 6 cells/mm3 after a year of antiretroviral treatment (Zidovudine-Lamivudine-Efavirenz). She was treated as smear negative pulmonary tuberculosis after a lack of gentamicin and amoxicillin plus clavulanic acid response. Culture of skin samples and sputum had revealed the presence of P. marneffei. A treatment with ketoconazole 600 mg per day was initiated. After two weeks of treatment, there was a decrease in the size and number of papules and nodules, without any new lesions. We noted disappearance of cough and fever. The chest X-ray showed a decrease of pulmonary lesions. There was no reactivation of P. marneffei infection but the patient died from AIDS after two years of follow up. CONCLUSION: We report a case of P. marneffei infection in a HIV-infected patient in a non-endemic country. Clinicians should think of P. marneffei infection in all HIV-infected patients with "molluscum contagiosum" like lesions.

Implementation of a national reference laboratory for Buruli ulcer disease in Togo.

BACKGROUND: In a previous study PCR analysis of clinical samples from suspected cases of Buruli ulcer disease (BUD) from Togo and external quality assurance (EQA) for local microscopy were conducted at an external reference laboratory in Germany. The relatively poor performance of local microscopy as well as effort and time associated with shipment of PCR samples necessitated the implementation of stringent EQA measures and availability of local laboratory capacity. This study describes the approach to implementation of a national BUD reference laboratory in Togo. METHODOLOGY: Large scale outreach activities accompanied by regular training programs for health care professionals were conducted in the regions "Maritime" and "Central," standard operating procedures defined all processes in participating laboratories (regional, national and external reference laboratories) as well as the interaction between laboratories and partners in the field. Microscopy was conducted at regional level and slides were subjected to EQA at national and external reference laboratories. For PCR analysis, sample pairs were collected and subjected to a dry-reagent-based IS2404-PCR (DRB-PCR) at national level and standard IS2404 PCR followed by IS2404 qPCR analysis of negative samples at the external reference laboratory. PRINCIPAL FINDINGS: The inter-laboratory concordance rates for microscopy ranged from 89% to 94%; overall, microscopy confirmed 50% of all suspected BUD cases. The inter-laboratory concordance rate for PCR was 96% with an overall PCR case confirmation rate of 78%. Compared to a previous study, the rate of BUD patients with non-ulcerative lesions increased from 37% to 50%, the mean duration of disease before clinical diagnosis decreased significantly from 182.6 to 82.1 days among patients with ulcerative lesions, and the percentage of category III lesions decreased from 30.3% to 19.2%. CONCLUSIONS: High inter-laboratory concordance rates as well as case confirmation rates of 50% (microscopy), 71% (PCR at national level), and 78% (including qPCR confirmation at external reference laboratory) suggest high standards of BUD diagnostics. The increase of non-ulcerative lesions, as well as the decrease in diagnostic delay and category III lesions, prove the effect of comprehensive EQA and training measures involving also procedures outside the laboratory.