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

Buruli ulcer disease (BUD) is an emerging disease particularly affecting children under the age of 15 years. Due to scarring and contractures BUD may lead to severe functional disability. Introduction of antimycobacterial treatment necessitated the laboratory confirmation of BUD, and WHO recommends confirmation of at least 50% of patients with suspected BUD by polymerase chain reaction (PCR). In Togo, cases have been reported since the early 1990s. However, less than five percent were laboratory confirmed. Since 2007, the German Leprosy and Tuberculosis Relief Organization (DAHW) has supported the Togolese National Buruli Ulcer Control Program in the area of training, treatment and laboratory confirmation of BUD. In close collaboration of DAHW and the Department for Infectious Diseases and Tropical Medicine, University Hospital, Munich (DITM), diagnostic samples from Togolese patients with suspected BUD were subjected to PCR. Out of 202 suspected BUD cases 109 BUD patients (54%) were PCR confirmed over a period of three years. Whereas the PCR case confirmation rate initially was below 50%, intensified training measures for health staff in the field of clinical diagnosis and collection of diagnostic samples ultimately resulted in 69% PCR confirmed cases. Our findings confirm the prevalence of BUD in Maritime Region

Risk factors for Mycobacterium ulcerans infection (Buruli Ulcer) in Togo ─ a case-control study in Zio and Yoto districts of the maritime region

Abstract Background Buruli ulcer (BU) is a neglected mycobacterial skin infection caused by Mycobacterium ulcerans. This disease mostly affects poor rural populations, especially in areas with low hygiene standards and sanitation coverage. The objective of this study was to identify these risk factors in the districts of Zio and Yoto of the Maritime Region in Togo. Methods We conducted a case-control study in Zio and Yoto, two districts proved BU endemic from November 2014 to May 2015. BU cases were diagnosed according to the WHO clinical case definition at the Centre Hospitalier Régional de Tsévié (CHR Tsévié) and confirmed by Ziehl-Neelsen (ZN) microscopy and IS2404 polymerase chain reaction (PCR). For each case, up to two controls matched by sex and place of residence were recruited. Socio-demographic, environmental or behavioral data were collected and conditional logistic regression analysis was used to identify and compare risk factors between BU cases and controls. Results A total of 83 cases and 128 controls were enrolled. The median age was 15 years (range 3–65 years). Multivariate conditional logistic regression analysis after adjustment for potential confounders identified age (< 10 years (OR =11.48, 95% CI = 3.72–35.43) and 10–14 years (OR = 3.63, 95% CI = 1.22–10.83)), receiving insect bites near a river (OR = 7.8, 95% CI = 1.48–41.21) and bathing with water from open borehole (OR = 5.77, (1.11–29.27)) as independent predictors of acquiring BU infection. Conclusions This study identified age, bathing with water from open borehole and receiving insect bites near a river as potential risk of acquiring BU infection in Zio and Yoto districts of the Maritime Region in south Togo

Geographical distribution of <i>M</i>. <i>ulcerans</i> profiles detected in the environment of districts of Zio and Yoto, maritime region, Togo May 19–30, 2015.

<p>The circles in red correspond to the <i>M</i>. <i>ulcerans</i> strains with three sequences (<i>IS2404/IS2606</i>/KR). This profile is found in the villages around the Haho river with some cases at the neighbourhood of Zio river.</p

Comparison of <i>M</i>. <i>ulcerans</i> profiles detected in clinical and environmental samples by Ct value of <i>IS2404/IS2606</i> and KR-B sequences detected, districts of Zio and Yoto, maritime region, Togo, May 19–30, 2015.

<p>Comparison of <i>M</i>. <i>ulcerans</i> profiles detected in clinical and environmental samples by Ct value of <i>IS2404/IS2606</i> and KR-B sequences detected, districts of Zio and Yoto, maritime region, Togo, May 19–30, 2015.</p

Molecular detection of <i>Mycobacterium ulcerans</i> in the environment and its relationship with Buruli ulcer occurrence in Zio and Yoto districts of maritime region in Togo

<div><p>Background</p><p>Buruli Ulcer (BU) is a neglected tropical skin infection caused by <i>Mycobacterium ulcerans</i>. Residence near aquatic areas has been identified as an important source of transmission of <i>M</i>. <i>ulcerans</i> with increased risk of contracting Buruli ulcer. However, the reservoir and the mode of transmission are not yet well known. The aim of this study was to identify the presence of <i>M</i>. <i>ulcerans</i> in the environment and its relationship with Buruli ulcer occurrence in Zio and Yoto districts of the maritime region in south Togo.</p><p>Methods</p><p>A total of 219 environmental samples including soil (n = 119), water (n = 65), biofilms/plants (n = 29) and animals’ feces (n = 6) were collected in 17 villages of Zio and Yoto districts of the maritime region in Togo. DNA of <i>M</i>. <i>ulcerans</i> including <i>IS2404</i> and <i>IS2606</i> insertions sequences and mycolactone ketoreductase-B gene (KR-B) was detected using real time PCR amplification (qPCR) technique. In parallel, clinical samples of patients were tested to establish a comparison of the genetic profile of <i>M</i>. <i>ulcerans</i> between the two types of samples. A calibration curve was generated for <i>IS2404</i> from a synthetic gene of <i>M</i>. <i>ulcerans</i> Transposase pMUM001, the plasmid of virulence.</p><p>Results</p><p>In the absence of inhibition of the qPCR, 6/219 (2.7%) samples were tested positive for <i>M</i>. <i>ulcerans</i> DNA containing three sequences (<i>IS2404/IS2606/</i>KR-B). Positive samples of <i>M</i>. <i>ulcerans</i> were consisting of biofilms/plants (3/29; 10.3%), water (1/65; 1.7%) and soil (2/119; 1.5%). Comparative analysis between DNA detected in environmental and clinical samples from BU patients showed the same genetic profile of <i>M</i>. <i>ulcerans</i> in the same environment. All these samples were collected in the environment of Haho and Zio rivers in the maritime region.</p><p>Conclusion</p><p>This study confirms the presence of <i>M</i>. <i>ulcerans</i> in the environment of the Zio and Yoto districts of the maritime region of Togo. This may explain partially, the high rates of Buruli ulcer patients in this region. Also, water, plants and soil along the rivers could be possible reservoirs of the bacterium. Therefore, Haho and Zio rivers could be potential sources of infection with <i>M</i>. <i>ulcerans</i> in humans in these districts.</p></div

Real-time PCR results of environmental samples tested positive by Ct values of <i>IS2404</i> and <i>IS2606</i> insertions and KR-B gene, Zio and Yoto districts, maritime region, Togo, May 19–30, 2015.

<p>Real-time PCR results of environmental samples tested positive by Ct values of <i>IS2404</i> and <i>IS2606</i> insertions and KR-B gene, Zio and Yoto districts, maritime region, Togo, May 19–30, 2015.</p

List of primers and probes sequences used for real time PCR (qPCR) targeting <i>IS2404</i> and <i>IS2606</i> insertions sequences and Ketoreductase-B domain gene, KR-B.

<p>List of primers and probes sequences used for real time PCR (qPCR) targeting <i>IS2404</i> and <i>IS2606</i> insertions sequences and Ketoreductase-B domain gene, KR-B.</p