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

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

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

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Specificity of 16S rRNA and IS<i>2404</i> qPCR assays.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd-0001756-t002" target="_blank">Table 2</a> shows DNA extracts from closely related mycobacterial species and bacteria potentially contaminating the human skin subjected to the combined 16S rRNA RT/IS<i>2404</i> qPCR viability assay and the corresponding test results. Mycobacterial species were selected according to their respective genetic contiguousness to <i>M. ulcerans</i> Agy99 (GenBank accession no. CP000325.1) within the 16S rRNA gene sequences as determined by BLASTN analysis (GenBank, NCBI) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd.0001756-Benson1" target="_blank">[13]</a>. <i>M.</i>, <i>Mycobacterium</i>; <i>E.</i>, <i>Escherichia</i>; <i>P., Propionibacterium</i>; <i>Staph</i>., <i>Staphylococcus</i>; <i>Str</i>., <i>Streptococcus</i>. While in-silico analysis revealed that the combined 16S rRNA RT/IS<i>2404</i> assay will also amplify mycolactone-producing mycobacteria (MPM) other than <i>M. ulcerans</i> (e.g., <i>M. pseudoshottsii</i>, <i>M. liflandii</i>, and the environmental <i>M. marinum</i> [GenBank accession No. NR_042988.1, AY500838.1, and AF456241.1, respectively]), these MPM species were not included in specificity testing.</p>a<p>DNA extracts that were not available at the DITM were provided by the National Reference Center (NRZ) for Mycobacteria, Borstel, Germany, and the Max von Pettenkofer-Institute (MVP), Ludwig-Maximilians University, Munich, Germany.</p>b<p>The respective primary patient isolates were considered as ^ppathogenic bacteria or as ^ccommensals/contaminants of clinical samples.</p>d<p>Results of the 16S rRNA RT-qPCR of DNA extracts; “+” indicates a positive and “–” a negative test result.</p>e<p>Results of the IS<i>2404</i> qPCR of DNA extracts; “+” indicates a positive and “–” a negative test result.</p

Study participants, clinical information, and diagnostic results.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd-0001756-t006" target="_blank">Table 6</a> shows suspected BUD cases with ulcerative lesions enrolled in the pre-treatment cohort (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd-0001756-g001" target="_blank">Figure 1</a>), clinical information, and diagnostic results. Swab samples from 24 suspected BUD cases were subjected to 16S rRNA RT/IS<i>2404</i> qPCR viability assay (swab 1 in PANTA), microscopic examination and enumeration of acid fast bacilli (AFB) following Ziehl-Neelsen staining (swab 2, direct smear), and conventional IS<i>2404</i> dry-reagent-based (DRB) PCR (swab 3 in Cell Lysis Solution [Qiagen]). 18 patients were laboratory confirmed by IS<i>2404</i> qPCR and 15 out of those were RNA positive; the quantification by IS<i>2404</i> qPCR revealed a bacillary load (1–2 bacilli per sample) below the lower limit of detection of the RNA assay for samples from three RNA negative patients. All samples from six IS<i>2404</i> qPCR negative study participants were also RNA negative. Direct correlation of AFB enumeration with IS<i>2404</i> qPCR quantification is not feasible due to inhomogeneous distribution of <i>M. ulcerans</i> in different clinical samples. NA, not applicable; Neg, negative test result; Pos, positive test result.</p>a<p>Results of the 16S rRNA RT/IS<i>2404</i> qPCR viability assay. Clinical swab samples in PANTA were directly processed at KCCR, and <i>M. ulcerans</i> DNA and cDNA were transported to DITM and subjected to qPCR.</p>b<p>Routine diagnostics were conducted following standardized procedures at KCCR <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd.0001756-Beissner1" target="_blank">[3]</a>.</p>c<p>No., consecutive number of study participants.</p>d<p>Yes, IS<i>2404</i> qPCR confirmed BUD patients; No, IS<i>2404</i> negative study participants.</p>e<p>Duration of disease before presentation of study participants in weeks.</p>f<p>Category of lesion according to the World Health Organization's clinical criteria <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd.0001756-World1" target="_blank">[1]</a>.</p>g<p>Results of the IS<i>2404</i> qPCR with corresponding cycle threshold (Ct)-values.</p>h<p>The bacillary load in the respective swab samples (No. 2) was estimated on the basis of IS<i>2404</i> quantification given an IS<i>2404</i> copy number of 209 copies per <i>M. ulcerans</i> genome <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd.0001756-Fyfe1" target="_blank">[9]</a>. For bacterial numbers <10 ranges were estimated.</p>i<p>Results of the 16S rRNA RT-qPCR.</p>k<p>MIC, microscopic detection and enumeration of AFB was conducted at KCCR including external quality assurance by DITM. The following scale was applied: 0 = negative, +1 = 10–99 AFB/100 fields, +2 = 1–10 AFB/1 field, +3 = more than 10 AFB/1 field.</p>l<p>PCR, conventional, single target gel-based IS<i>2404</i> DRB PCR.</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