Effect of previous heterologous flavivirus vaccinations on human antibody responses in tick-borne encephalitis and dengue virus infections.

Arthropod-borne flaviviruses include a number of medically relevant human pathogens such as the mosquito-borne dengue (DEN), Zika, and yellow fever (YF) viruses as well as tick-borne encephalitis virus (TBEV). All flaviviruses are antigenically related and anamnestic responses due to prior immunity can modulate antibody specificities in subsequent infections or vaccinations. In our study, we analyzed the induction of broadly flavivirus cross-reactive antibodies in tick-borne encephalitis (TBE) and DEN patients without or with prior flavivirus exposure through TBE and/or YF vaccination, and determined the contribution of these antibodies to TBE and dengue virus (DENV) neutralization. In addition, we investigated the formation of cross-reactive antibodies in TBE-vaccination breakthroughs (VBTs). A TBEV infection without prior YF or TBE vaccination induced predominantly type-specific antibodies. In contrast, high levels of broadly cross-reactive antibodies were found in samples from TBE patients prevaccinated against YF as well as in DEN patients prevaccinated against TBE and/or YF. While these cross-reactive antibodies did not neutralize TBEV, they were effective in neutralizing DENV. This discrepancy points to structural differences between the two viruses and indicates that broadly cross-reactive epitopes are less accessible in TBEV than in DENV. In TBE VBT infections, type-specific antibodies dominated the antibody response, thus revealing no difference from that of unvaccinated TBE patients. Our results emphasize significant differences in the structural properties of different flaviviruses that have an impact on the induction of broadly cross-reactive antibodies and their functional activities in virus neutralization.

Report on an unusual case of leprosy from Germany: just an exception of the rule?

CASE PRESENTATION: We report on a German leprosy patient originating from Pakistan who had a relapse more than 5 years after completion of multi-drug therapy (MDT) of his first episode of multibacillary (MB) leprosy. State-of-the-art laboratory techniques (histopathology, PGL-I serology, microscopy and DNA/RNA qPCR) were applied for laboratory confirmation and monitoring of treatment outcome. Serology indicated the relapse long before the presence of unambiguous clinical signs. At the time of diagnosis of the relapse the patient had a remarkably high bacterial load suggesting increased risk for a second relapse. Furthermore, unexpectedly prolonged excretion of viable bacilli through the upper respiratory tract for more than 3 months after onset of MDT was shown. Therefore, MDT was administered for 2 years. DISCUSSION AND CONCLUSIONS: The clinical course of the patient, as well as the prolonged excretion of viable bacilli, underlines the usefulness of laboratory assessment. Laboratory tools including up-to-date molecular assays facilitate rapid diagnosis, timely MDT, identification of individuals excreting viable bacilli and patients at risk for relapses, monitoring of treatment outcome and respective adaptation of treatment where appropriate.

Plasmodium knowlesi infection in a returning German traveller from Thailand: a case report on an emerging malaria pathogen in a popular low-risk travel destination.

BACKGROUND: Thailand is a major destination for German travellers with more than 760,000 arrivals in 2015. At the same time, malaria is a concern in travel recommendations with regard to this destination. The World Malaria Report of 2016 mentions only P. falciparum and P. vivax as prevalent species for Thailand, however, P. knowlesi infections have been occasionally reported in Thailand. In German travellers, only five cases of P. knowlesi infections have been reported to date. CASE PRESENTATION: A 45-year-old German male tourist travelled to Thailand from 25 December 2016 to 13 January 2017. On 14 January he developed fever with no other symptoms, and presented on 17 January at the Division for Tropical Medicine and Infectious Diseases in Munich, Germany. Malaria was diagnosed, primarily based on a single parasite in the thin smear microscopy, while commercial rapid diagnostic testing remained negative. Only the result of a differential PCR assay revealed P. knowlesi infection. CONCLUSIONS: P. knowlesi has to be considered in travellers returning from Thailand. Cases may present with an extremely low parasitaemia. This is in contrast to the assumption that P. knowlesi was likely to cause high parasitaemia due to its short replication cycle.

Long-term kinetics of Zika virus RNA and antibodies in body fluids of a vasectomized traveller returning from Martinique: a case report.

BACKGROUND: The magnitude of the current Zika virus (ZIKV) epidemic has led to a declaration of a Public Health Emergency of International Concern by the WHO. Findings of viable viral particles in semen for several weeks are corroborating reports of sexual transmission of ZIKV. Serious consequences of a positive diagnostic result particularly in the pregnant patient are calling for precise diagnostic tools also at later time points after infection. Currently, recommendations suggest a diagnostic period of direct viral detection of 5 to 7 days after onset of symptoms in serum or plasma, and up to 3 weeks in urine samples. CASE PRESENTATION: A vasectomized 41-year-old German returning from Martinique presented at the outpatient clinic of the Department for Infectious Diseases and Tropical Medicine, Munich, with subfebrile temperature, rash, malaise, severe retro-orbital pain and occipital lymphadenopathy. The main complaints resolved after ten days without specific treatment. We are reporting on clinical course and results of direct and indirect detection methods of ZIKV in different sample types including whole blood, ejaculate, urine, serum, plasma and saliva samples up to 119 days post symptom onset. Ejaculate samples remained PCR positive for ZIKV until day 77, whole blood samples until day 101. CONCLUSIONS: The case presentation adds to the still limited knowledge of kinetics of detection of ZIKV by direct as well as indirect methods. Here, a complete data set including results from PCR, serology and cell culture is provided allowing an improved evaluation of optimum diagnostic periods for testing a variety of sample types. Moreover, a high viral load of ZIKV RNA was detected in ejaculate of the vasectomized patient. This finding sheds new light on the possible localizations of ZIKV replication in the human male reproductive tract.

Comparison of two assays for molecular determination of rifampin resistance in clinical samples from patients with Buruli ulcer disease.

This study evaluates a novel assay for detecting rifampin resistance in clinical Mycobacterium ulcerans isolates. Although highly susceptible for PCR inhibitors in 50% of the samples tested, the assay was 100% M. ulcerans specific and yielded >98% analyzable sequences with a lower limit of detection of 100 to 200 copies of the target sequence.

Epidemiological and clinical characteristics of patients with monkeypox in the GeoSentinel Network: a cross-sectional study.

BACKGROUND: The early epidemiology of the 2022 monkeypox epidemic in non-endemic countries differs substantially from the epidemiology previously reported from endemic countries. We aimed to describe the epidemiological and clinical characteristics among individuals with confirmed cases of monkeypox infection. METHODS: We descriptively analysed data for patients with confirmed monkeypox who were included in the GeoSentinel global clinical-care-based surveillance system between May 1 and July 1 2022, across 71 clinical sites in 29 countries. Data collected included demographics, travel history including mass gathering attendance, smallpox vaccination history, social history, sexual history, monkeypox exposure history, medical history, clinical presentation, physical examination, testing results, treatment, and outcomes. We did descriptive analyses of epidemiology and subanalyses of patients with and without HIV, patients with CD4 counts of less than 500 cells per mm3 or 500 cells per mm3 and higher, patients with one sexual partner or ten or more sexual partners, and patients with or without a previous smallpox vaccination. FINDINGS: 226 cases were reported at 18 sites in 15 countries. Of 211 men for whom data were available, 208 (99%) were gay, bisexual, or men who have sex with men (MSM) with a median age of 37 years (range 18-68; IQR 32-43). Of 209 patients for whom HIV status was known, 92 (44%) men had HIV infection with a median CD4 count of 713 cells per mm3 (range 36-1659; IQR 500-885). Of 219 patients for whom data were available, 216 (99%) reported sexual or close intimate contact in the 21 days before symptom onset; MSM reported a median of three partners (IQR 1-8). Of 195 patients for whom data were available, 78 (40%) reported close contact with someone who had confirmed monkeypox. Overall, 30 (13%) of 226 patients were admitted to hospital; 16 (53%) of whom had severe illness, defined as hospital admission for clinical care rather than infection control. No deaths were reported. Compared with patients without HIV, patients with HIV were more likely to have diarrhoea (p=0·002), perianal rash or lesions (p=0·03), and a higher rash burden (median rash burden score 9 [IQR 6-21] for patients with HIV vs median rash burden score 6 [IQR 3-14] for patients without HIV; p<0·0001), but no differences were identified in the proportion of men who had severe illness by HIV status. INTERPRETATION: Clinical manifestations of monkeypox infection differed by HIV status. Recommendations should be expanded to include pre-exposure monkeypox vaccination of groups at high risk of infection who plan to engage in sexual or close intimate contact. FUNDING: US Centers for Disease Control and Prevention, International Society of Travel Medicine.

FluoRNT: A robust, efficient assay for the detection of neutralising antibodies against yellow fever virus 17D.

There is an urgent need for better diagnostic and analytical methods for vaccine research and infection control in virology. This has been highlighted by recently emerging viral epidemics and pandemics (Zika, SARS-CoV-2), and recurring viral outbreaks like the yellow fever outbreaks in Angola and the Democratic Republic of Congo (2016) and in Brazil (2016-2018). Current assays to determine neutralising activity against viral infections in sera are costly in time and equipment and suffer from high variability. Therefore, both basic infection research and diagnostic population screenings would benefit from improved methods to determine virus-neutralising activity in patient samples. Here we describe a robust, objective, and scalable Fluorescence Reduction Neutralisation Test (FluoRNT) for yellow fever virus, relying on flow cytometric detection of cells infected with a fluorescent Venus reporter containing variant of the yellow fever vaccine strain 17D (YF-17D-Venus). It accurately measures neutralising antibody titres in human serum samples within as little as 24 h. Samples from 32 vaccinees immunised with YF-17D were tested for neutralising activity by both a conventional focus reduction neutralisation test (FRNT) and FluoRNT. Both types of tests proved to be equally reliable for the detection of neutralising activity, however, FluoRNT is significantly more precise and reproducible with a greater dynamic range than conventional FRNT. The FluoRNT assay protocol is substantially faster, easier to control, and cheaper in per-assay costs. FluoRNT additionally reduces handling time minimising exposure of personnel to patient samples. FluoRNT thus brings a range of desirable features that can accelerate and standardise the measurement of neutralising anti-yellow fever virus antibodies. It could be used in applications ranging from vaccine testing to large cohort studies in systems virology and vaccinology. We also anticipate the potential to translate the methodology and analysis of FluoRNT to other flaviviruses such as West Nile, Dengue and Zika or to RNA viruses more generally.

Switch detection in preschoolers' cognitive flexibility.

The current study addressed the role of switch detection in cognitive flexibility by testing the effect of transition cues (i.e., cues that directly signal the need to switch or maintain a given task goal) in a cued set-shifting paradigm at 5 years of age. Children performed better, especially on switch trials, when transition cues were combined with traditional task cues (i.e., cues that directly signal the relevant task on a given trial) relative to conditions without transition cues. This effect was not influenced by explicit knowledge of transition cues or transition cue transparency, suggesting that transition cues did not need to be semantically processed to be beneficial. These findings reveal that young children's difficulties in set-shifting situations stem partially from failures to monitor for the need to switch.

Efficiency of fine-needle aspiration compared with other sampling techniques for laboratory diagnosis of Buruli ulcer disease.

In accordance with recent WHO recommendations, this study evaluates the sensitivities of PCR and microscopy for fine-needle aspiration (FNA) versus techniques involving swabs and punch biopsy specimens and suggests that FNA can replace punch biopsies for nonulcerative lesions and may serve as an alternative for ulcerative lesions in cases where scarred edges prevent the collection of swabs.

Impact of flavivirus vaccine-induced immunity on primary Zika virus antibody response in humans.

BACKGROUND: Zika virus has recently spread to South- and Central America, causing congenital birth defects and neurological complications. Many people at risk are flavivirus pre-immune due to prior infections with other flaviviruses (e.g. dengue virus) or flavivirus vaccinations. Since pre-existing cross-reactive immunity can potentially modulate antibody responses to Zika virus infection and may affect the outcome of disease, we analyzed fine-specificity as well as virus-neutralizing and infection-enhancing activities of antibodies induced by a primary Zika virus infection in flavivirus-naïve as well as yellow fever- and/or tick-borne encephalitis-vaccinated individuals. METHODOLOGY: Antibodies in sera from convalescent Zika patients with and without vaccine-induced immunity were assessed by ELISA with respect to Zika virus-specificity and flavivirus cross-reactivity. Functional analyses included virus neutralization and infection-enhancement. The contribution of IgM and cross-reactive antibodies to these properties was determined by depletion experiments. PRINCIPAL FINDINGS: Pre-existing flavivirus immunity had a strong influence on the antibody response in primary Zika virus infections, resulting in higher titers of broadly flavivirus cross-reactive antibodies and slightly lower levels of Zika virus-specific IgM. Antibody-dependent enhancement (ADE) of Zika virus was mediated by sub-neutralizing concentrations of specific IgG but not by cross-reactive antibodies. This effect was potently counteracted by the presence of neutralizing IgM. Broadly cross-reactive antibodies were able to both neutralize and enhance infection of dengue virus but not Zika virus, indicating a different exposure of conserved sequence elements in the two viruses. CONCLUSIONS: Our data point to an important role of flavivirus-specific IgM during the transient early stages of infection, by contributing substantially to neutralization and by counteracting ADE. In addition, our results highlight structural differences between strains of Zika and dengue viruses that are used for analyzing infection-enhancement by cross-reactive antibodies. These findings underscore the possible impact of specific antibody patterns on flavivirus disease and vaccination efficacy.

Zika beyond the Americas: Travelers as sentinels of Zika virus transmission. A GeoSentinel analysis, 2012 to 2016.

BACKGROUND: Zika virus (ZIKV) was first isolated in Africa; decades later, caused large outbreaks in the Pacific, and is considered endemic in Asia. We aim to describe ZIKV disease epidemiology outside the Americas, the importance of travelers as sentinels of disease transmission, and discrepancies in travel advisories from major international health organizations. METHODS AND FINDINGS: This descriptive analysis using GeoSentinel Surveillance Network records involves sixty-four travel and tropical medicine clinics in 29 countries. Ill returned travelers with a confirmed or probable diagnosis of ZIKV disease acquired in Africa, Asia and the Pacific seen between 1 January 2012 and 31 December 2016 are included, and the frequencies of demographic, trip, and diagnostic characteristics described. ZIKV was acquired in Asia (18), the Pacific (10) and Africa (1). For five countries (Indonesia, Philippines, Thailand, Vietnam, Cameroon), GeoSentinel patients were sentinel markers of recent Zika activity. Additionally, the first confirmed ZIKV infection acquired in Kiribati was reported to GeoSentinel (2015), and a probable case was reported from Timor Leste (April 2016), representing the only case known to date. Review of Zika situation updates from major international health authorities for country risk classifications shows heterogeneity in ZIKV country travel advisories. CONCLUSIONS: Travelers are integral to the global spread of ZIKV, serving as sentinel markers of disease activity. Although GeoSentinel data are collected by specialized clinics and do not capture all imported cases, we show that surveillance of imported infections by returned travelers augments local surveillance system data regarding ZIKV epidemiology and can assist with risk categorization by international authorities. However, travel advisories are variable due to risk uncertainties.

Elevated Values of C-Reactive Protein Induced by Imported Infectious Diseases: A Controlled Cross-Sectional Study of 11,079 Diseased German Travelers Returning from the Tropics and Subtropics.

The present controlled cross-sectional study aimed to assess elevated values of C-reactive protein (CRP), a positive acute-phase protein, induced by imported infectious diseases (IDs) seen in patients consulting the University of Munich (1999-2015) after being in the tropics/subtropics. The analysis investigated data sets from 11,079 diseased German travelers (cases) returning from Latin America (1,986), Africa (3,387), and Asia (5,706), and from 714 healthy Germans who had not recently traveled (controls). The proportions of elevated values of CRP (> 0.5 mg/dL) were significantly larger among cases (44.3%) than among controls (20.7%). Among cases, this proportion was largest among males (49.2%) in comparison to females (39.9%), among travelers with short travel duration of 1-14 days (49.6%) in comparison to travelers with a travel duration of > 180 days (30.8%), and with travel destination in Africa (47.0%) in comparison to Asia (44.2%) and Latin America (39.9%), among all-inclusive travelers (47.4%) in comparison to business travelers (46.7%) and backpackers (44.1%), and among patients presenting with fever (70.9%) and arthralgia (54.3%). The study identified various imported IDs with significantly larger proportions of elevated values of CRP including viral (cytomegalovirus infection [94.7%], influenza [88.9%], infectious mononucleosis [71.8%]), bacterial (typhoid fever [100%], paratyphoid fever [92.9%], shigellosis [76.8%], rickettsiosis [74.2%], Salmonella enteritis [71.3%], Campylobacter infection [68.7%]), and protozoan (vivax malaria [100%], ovale malaria [100%], falciparum malaria [95.4%], noninvasive Entamoeba infection [65.9%]) IDs. This study demonstrates that elevated values of CRP can be a useful laboratory finding for travelers returning from the tropics/subtropics, as these findings are typically caused mainly by certain imported bacterial IDs, but also by viral and protozoan IDs.

Buruli ulcer in Nigeria: results of a pilot case study in three rural districts.

BACKGROUND: Buruli ulcer (BU), also known as Mycobacterium ulcerans disease, is the third most common mycobacterial disease worldwide. Although BU disease has been diagnosed among Nigerians in neighbouring West African countries, data on the burden of the disease in Nigeria itself are scanty. This study aimed to assess the magnitude and epidemiology of BU in the South South region of Nigeria. METHODS: We conducted a cross-sectional survey in the Ogoja territory (comprising 31 communities). We undertook sensitisation programmes centred on BU in 10 of the communities. Participants were asked to identify community members with long-standing ulcers, who were then invited for evaluation. We also contacted traditional healers to refer their clients who had non-healing ulcers. All suspected cases had a full clinical evaluation and laboratory testing. Confirmed cases were given treatment in a referral hospital in the territory. RESULTS: We diagnosed 41 clinical BU cases; 36 (87.8 %) of which were confirmed by quantitative polymerase chain reaction (qPCR). These 36 PCR-confirmed cases were diagnosed in a total population of 192,169 inhabitants. Therefore, the estimated crude prevalence of BU was 18.7 per 100,000 population, varying from 6.0 to 41.4 per 100,000 in the districts surveyed. The majority (66.7 %) of the cases were females. About 92 % of the BU lesions were located on the patients' extremities. No differences were observed between the sexes in terms of the location of the lesions. The age of the patients ranged from four to 60 years, with a median age of 17 years. All 35 (100 %) patients who consented to treatment completed chemotherapy as prescribed. Of the treated cases, 29 (82.9 %) needed and received surgery. All cases healed, but 29 (82.9 %) had some limitations in movement. Healing with limitations in movement occurred in 18/19 (94.7 %) and 8/10 (80.0 %) of patients with lesions >15 cm (Category III) and 6-15 cm in diameter (Category II), respectively. The median duration of treatment was 130 (87-164) days for children and 98 (56-134) days for adults (p = 0.15). CONCLUSIONS: In Nigeria, BU is endemic but its severity is underestimated-at least in the study setting. There is a need to identify and map BU endemic regions in Nigeria. A comprehensive BU control programme is also urgently needed.

Loop-Mediated Isothermal Amplification for Laboratory Confirmation of Buruli Ulcer Disease-Towards a Point-of-Care Test.

BACKGROUND: As the major burden of Buruli ulcer disease (BUD) occurs in remote rural areas, development of point-of-care (POC) tests is considered a research priority to bring diagnostic services closer to the patients. Loop-mediated isothermal amplification (LAMP), a simple, robust and cost-effective technology, has been selected as a promising POC test candidate. Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagent-based LAMP assay (DRB-LAMP) employing lyophilized reagents. METHODOLOGY/PRINCIPAL FINDINGS: Following the design of an IS2404 based conventional LAMP (cLAMP) assay suitable to apply lyophilized reagents, a lyophylization protocol for the DRB-LAMP format was developed. Clinical performance of cLAMP was validated through testing of 140 clinical samples from 91 suspected BUD cases by routine assays, i.e. IS2404 dry-reagent-based (DRB) PCR, conventional IS2404 PCR (cPCR), IS2404 qPCR, compared to cLAMP. Whereas qPCR rendered an additional 10% of confirmed cases and samples respectively, case confirmation and positivity rates of DRB-PCR or cPCR (64.84% and 56.43%; 100% concordant results in both assays) and cLAMP (62.64% and 52.86%) were comparable and there was no significant difference between the sensitivity of the assays (DRB PCR and cPCR, 86.76%; cLAMP, 83.82%). Likewise, sensitivity of cLAMP (95.83%) and DRB-LAMP (91.67%) were comparable as determined on a set of 24 samples tested positive in all routine assays. CONCLUSIONS/SIGNIFICANCE: Both LAMP formats constitute equivalent alternatives to conventional PCR techniques. Provided the envisaged availability of field friendly DNA extraction formats, both assays are suitable for decentralized laboratory confirmation of BUD, whereby DRB-LAMP scores with the additional advantage of not requiring cold-chains. As validation of the assays was conducted in a third-level laboratory environment, field based evaluation trials are necessary to determine the clinical performance at peripheral health care level.

Qualitative change in executive control during childhood and adulthood.

Executive control development typically has been conceptualized to result from quantitative changes in the efficiency of the underlying processes. In contrast, the present study addressed the possibility of qualitative change with age by examining how children and adults detect task switches. Participants in three age groups (5- and 10-year-old children, young adults) completed two conditions of a cued task-switching paradigm where task cues were presented either in isolation or in conjunction with transition cues. Five-year-olds performed better with transition cues, whereas the reverse effect was observed at age 10 and with adults. Unlike 5-year-olds who detect switches after semantically processing cues, older participants strategically detect switches based on perceptual processing only. Age-related qualitative changes promote increasingly optimal adjustment of executive resources with age.

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.

Laboratory confirmation of Buruli ulcer disease in Togo, 2007-2010.

BACKGROUND: Since the early 1990s more than 1,800 patients with lesions suspicious for Buruli ulcer disease (BUD) have been reported from Togo. However, less than five percent of these were laboratory confirmed. Since 2007, the Togolese National Buruli Ulcer Control Program has been supported by the German Leprosy and Tuberculosis Relief Association (DAHW). Collaboration with the Department for Infectious Diseases and Tropical Medicine (DITM), University Hospital, Munich, Germany, allowed IS2404 PCR analysis of diagnostic samples from patients with suspected BUD during a study period of three years. METHODOLOGY/PRINCIPAL FINDINGS: The DAHW integrated active BUD case finding in the existing network of TB/Leprosy Controllers and organized regular training and outreach activities to identify BUD cases at community level. Clinically suspected cases were referred to health facilities for diagnosis and treatment. Microscopy was carried out locally, external quality assurance (EQA) at DITM. Diagnostic samples from 202 patients with suspected BUD were shipped to DITM, 109 BUD patients (54%) were confirmed by PCR, 43 (29.9%) by microscopy. All patients originated from Maritime Region. EQA for microscopy resulted in 62% concordant results. CONCLUSIONS/SIGNIFICANCE: This study presents a retrospective analysis of the first cohort of clinically suspected BUD cases from Togo subjected to systematic laboratory analysis over a period of three years and confirms the prevalence of BUD in Maritime Region. Intensified training in the field of case finding and sample collection increased the PCR case confirmation rate from initially less than 50% to 70%. With a PCR case confirmation rate of 54% for the entire study period the WHO standards (case confirmation rate ≥50%) have been met. EQA for microscopy suggests the need for intensified supervision and training. In January 2011 the National Hygiene Institute, Lomé, has assumed the role of a National Reference Laboratory for PCR confirmation and microscopy.