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

Scar of secondary nodule at the back of the right thigh, September 2011.

<p>Scar of secondary nodule at the back of the right thigh, September 2011.</p

Positivity rates.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001228#pntd-0001228-t003" target="_blank">Table 3</a> describes the positivity rates, i.e. the number of positive samples divided by the total number of samples tested, of microscopy and IS<i>2404</i> gel-based polymerase chain reaction per type of lesion and type of sample; diagnostic samples from 202 patients with suspected BUD from three study sites in Togo (CHR Tsévié, CHP Sotouboua, USP Agbetiko) were collected within three years (September 2007 through August 2010);</p>a<p>FNA, fine needle aspiration;</p>b<p>MIC, microscopic examination for the detection of acid fast bacilli;</p>c<p>PCR, IS<i>2404</i>, gel-based polymerase chain reaction;</p>d<p>ND, not done;</p>e<p>NA, not available;</p

Primary nodule at the left costal arch, June 2010.

<p>Primary nodule at the left costal arch, June 2010.</p

Case confirmation rates.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001228#pntd-0001228-t001" target="_blank">Table 1</a> describes the case confirmation rates, i.e. the number of laboratory confirmed BUD cases divided by the total number of patients with suspected BUD (suspected cases) of whom samples were subjected to a certain diagnostic test; diagnostic samples from 202 suspected BUD cases (suspected cases) from three study sites in Togo (CHR Tsévié, CHP Sotouboua, USP Agbetiko) were collected within three years (September 2007 through August 2010);</p>a<p>Non-ulcerative lesions: FNA (fine needle aspiration) samples, punch biopsy samples and surgical biopsy samples were analyzed; ulcerative lesions: swab samples, FNA (fine needle aspiration) samples, punch biopsy samples and surgical biopsy samples were analyzed;</p>b<p>Test: MIC, microscopic examination for the detection of acid fast bacilli; swab samples and FNA samples were analyzed;</p>c<p>Test: PCR, polymerase chain reaction, gel-based IS<i>2404</i> PCR; swab samples, FNA samples, punch biopsy samples and surgical biopsy samples were analyzed;</p>d<p>NA, not available;</p

Case confirmation rate per observation period.

<p>The PCR case confirmation rate was 36/84 (42.86%) in the first observation period (September 2007–August 2008), 37/66 (56.06%) in the second observation period (September 2008–August 2009) and 36/52 (69.23%) in the third observation period (September 2009–August 2010). The case confirmation rate increased during the three observation periods with a definite trend (coefficient of determination, R² = 1).</p

Diagnostic specimens and transport media.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001228#pntd-0001228-t002" target="_blank">Table 2</a> describes diagnostic specimens and transport media according to diagnostic tests, type of lesion and type of treatment.</p>a<p>FNA, fine needle aspiration;</p>b<p>MIC, microscopic examination for the detection of acid fast bacilli;</p>c<p>NA, not applicable;</p>d<p>PCR, IS<i>2404</i> gel-based polymerase chain reaction;</p>e<p>CLS, cell lysis solution (Qiagen, Germany).</p