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

Augmentation de la résistance aux antibiotiques des Entérobactéries isolées à l’Institut National d’Hygiène de Lomé de 2010 à 2017: Increase in antibiotic resistance of Enterobacteriaceae isolated at the National Institute of Hygiene of Lomé from 2010 to 2017

Introduction: La résistance des Entérobactéries aux antibiotiques est un problème d’importance croissante en pratique médicale. L’objectif de cette étude était de déterminer le profil de résistance aux antibiotiques des Entérobactéries isolées à l’institut national d’hygiène (INH) de Lomé et d’analyser son évolution dans le temps. Méthodes: Il s’agissait d’une analyse rétrospective, sur une période de huit ans (2010-2017), portant sur l’ensemble des souches d’Entérobactéries isolées des prélèvements pathologiques analysés au laboratoire de bactériologie de l’INH. Résultats: Au total, 5910 Entérobactéries ont été isolées majoritairement des urines (59,59%), avec une prédominance d’Escherichia coli (63,93%) suivie de Klebsiella spp (22,86%). Entre 2010 et 2017, le taux de résistance des souches d’Escherichia coli a augmenté significativement de 18,69% à 39,26% (p< 0,0001) à la Ceftazidime ; de 1,68% à 40,22% à la Ceftriaxone (p< 0,0001) et de 42,37% à 63,23% (p< 0,0001) à la Ciprofloxacine. La résistance des souches de Klebsiella spp à la Ceftazidime a augmenté significativement de 25,26% à 42,54% (p< 0,0001) et celle à la Ceftriaxone de 2,17% à 41,79% (p< 0,0001) respectivement de 2010 à 2017. Conclusion: L’augmentation de la résistance des Entérobactéries aux antibiotiques et surtout l’évolution des résistances aux Céphalosporines de 3e Génération et aux Fluoroquinolones est un phénomène réel. Ceci exposera à des difficultés de prise en charge thérapeutique et nécessite la mise en place des dispositions idoines. Background: Antibiotic resistance in Enterobacteriaceae is a growing problem in medical practice. The objective of this study was to determine the antibiotic resistance profile of Enterobacteriaceae isolated at the National Institute of Hygiene (INH) of Lomé and to analyse its evolution over time. Method: This was a retrospective analysis, over a period of eight years (2010-2017), of all strains of Enterobacteriaceae isolated from pathological samples analysed in the bacteriology laboratory of the INH. Results: A total of 5910 Enterobacteriaceae were isolated mainly from urine (59.59%), with a predominance of Escherichia coli (63.93%) followed by Klebsiella spp (22.86%). Between 2010 and 2017, the resistance rate of Escherichia coli strains increased significantly from 18.69% to 39.26% (p<0.0001) to Ceftazidime; from 1.68% to 40.22% to Ceftriaxone (p<0.0001) and from 42.37% to 63.23% (p<0.0001) to Ciprofloxacin. Resistance of Klebsiella spp strains to Ceftazidime increased significantly from 25.26% to 42.54% (p< 0.0001) and to Ceftriaxone from 2.17% to 41.79% (p< 0.0001) respectively from 2010 to 2017. Conclusion: The increase in antibiotic resistance in Enterobacteriaceae and especially the evolution of resistance to 3rd generation cephalosporins and fluoroquinolones is a real phenomenon. This will lead to difficulties in therapeutic management and requires the implementation of appropriate measures

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-Guerin (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

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-Guerin (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

Age distribution of laboratory confirmed BUD patients.

<p>Age distribution of 64 laboratory confirmed BUD patients recruited from September 2010 through April 2012. The age of all patients was known and 48.4% (31/64) were in age group 5–14 years. The age range was 2–68 years with a mean of 18.1 years and a median of 13 years.</p

Geographic origin, type and classification of lesions of clinically suspected and laboratory confirmed BUD patients.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002011#pntd-0002011-t001" target="_blank">Table 1</a> shows the geographic origin of all suspected and confirmed BUD patients, and type/category of lesions of confirmed BUD patients who presented from September 2010 through April 2012 in Togo. More than 85% of confirmed BUD patients originated from the districts Yoto and Zio of region “Maritime”.</p>a<p>Patients were confirmed by dry-reagent-based IS<i>2404</i>, standard gel-based IS<i>2404</i> PCR and/or IS<i>2404</i> quantitative real-time PCR. BUD, Buruli ulcer disease.</p>b<p>Number of confirmed BUD patients per district.</p>c<p>Category I, single lesion <50 mm in diameter.</p>d<p>Category II, single lesion between 50 and 150 mm in diameter.</p>e<p>Category III, single lesion >150 mm in diameter or multiple lesions, osteomyelitis or lesions at critical sites.</p>f<p>Laboratory confirmed BUD patients were referred to CHR, Tsévié, for antimycobacterial treatment.</p

Results of EQA for PCR.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002011#pntd-0002011-t005" target="_blank">Table 5</a> shows results of external quality assurance for PCR. During the initial phase (phase I) PCR samples were analyzed at DITM by IS<i>2404</i> standard PCR and IS<i>2404</i> quantitative real-time PCR (qPCR). During the transitional phase (phase II) diagnostic sample pairs where analyzed in parallel at INH (IS<i>2404</i> dry-reagent-based [DRB] PCR) and DITM as described for phase I. Positivity rates and case confirmation rates are provided for IS<i>2404</i> DRB- and standard PCR, and additional diagnostic yields were calculated for IS<i>2404</i> qPCR. N/A, not applicable.</p>a<p>Swab samples, DNA extract were prepared from swab samples.</p>b<p>FNA samples, DNA extract were prepared from fine-needle aspirate samples.</p>c<p>Punch samples, DNA extract were prepared from 3-mm punch biopsy samples.</p>d<p>Total per phase and laboratory.</p>e<p>INH applied IS<i>2404</i>-DRB-PCR. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002011#pntd.0002011-Siegmund1" target="_blank">[21]</a></p>f<p>DITM applied standard, gel-based, IS<i>2404</i> PCR <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002011#pntd.0002011-Stinear1" target="_blank">[17]</a> and IS<i>2404</i> qPCR <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002011#pntd.0002011-Fyfe1" target="_blank">[27]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002011#pntd.0002011-World7" target="_blank">[42]</a> on all DNA extracts tested negative with standard PCR. For qPCR the additional diagnostic yield (i.e. the deviation of total final result from total result of standard PCR) were 5.7% (phase I) and 6.1% (phase II).</p>g<p>Final result of standard PCR and qPCR.</p>h<p>Phase I, initial phase of implementation of the national reference laboratory at INH from September through December 2010.</p>i<p>Positivity rate, number of positive samples divided by the total number of samples tested.</p>j<p>Case confirmation rate, number of laboratory confirmed BUD patients divided by the total number of suspected BUD cases.</p>k<p>Rate of false negative results at INH as determined by re-testing of DNA extracts at DITM by standard PCR.</p>l<p>Rate of concordant results from sample pairs at INH and DITM.</p>m<p>Phase II, transitional phase of implementation of the national reference laboratory at INH from January 2011 through April 2012.</p>n<p>Total results of the initial and the transitional phase.</p

Results of external quality assurance for microscopy.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002011#pntd-0002011-t003" target="_blank">Table 3</a> shows results of Ziehl-Neelsen microscopy conducted at CHR and corresponding results of EQA conducted at INH and DITM during the initial (phase I, September 2010 through December 2010) and transitional (phase II, January 2011 through April 2012) phases. N/A, not applicable.</p>a<p>Swab, slides were prepared as direct smears from swab samples.</p>b<p>FNA, slides were prepared as direct smears from fine-needle aspirate samples.</p>c<p>Positivity rate, number of positive samples divided by the total number of samples tested.</p>d<p>Rate of false negative results at CHR and INH, respectively, compared to DITM results.</p>e<p>Rate of false positive results at CHR and INH, respectively, compared to DITM results.</p>f<p>Rate of false negative and false positive results at CHR and INH, respectively, compared to DITM results.</p>g<p>Rate of concordant results between CHR/INH, CHR/DITM and INH/DITM.</p>h<p>Confirmation rate, number of laboratory confirmed BUD patients divided by the total number of suspected BUD cases.</p

Clinical samples analyzed by microscopy for <i>M. ulcerans</i>.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002011#pntd-0002011-t002" target="_blank">Table 2</a> indicates all slides prepared from swab or FNA samples and subjected to Ziehl-Neelsen staining at “Centre Hospitalier Régional” (CHR) for the detection of acid fast bacilli. Slides were analyzed consecutively at CHR and the Department of Infectious Diseases and Tropical Medicine (DITM), Ludwig-Maximilians-University during initial phase (phase I) or CHR, at the “Institut National d'Hygiène” (INH) and DITM during transitional phase (phase II). N/A, not applicable.</p>a<p>MIC, microscopic detection of acid fast bacilli.</p>b<p>Total, number of slides prepared from swab and FNA samples and subjected to reading at CHR/DITM or CHR/INH/DITM.</p>c<p>Phase I, initial phase of implementation of the national reference laboratory at INH from September 2010 through December 2010; slides were read at CHR and forwarded via DAHWT to DITM for EQA.</p>d<p>Phase II, transitional phase of implementation of the national reference laboratory at INH from January 2011 through April 2012; slides were read at CHR, followed by blinded re-reading at INH and DITM.</p

Impact of local reference laboratory and external quality assurance measures on BUD control.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002011#pntd-0002011-t006" target="_blank">Table 6</a> shows analyses of clinical parameters (i.e. “type of lesion” and “duration of disease before clinical diagnosis”) among PCR confirmed BUD new cases to assess impact of the local reference laboratory and external quality assurance measures on BUD control in Togo. Therefore, data from a previous study (September 2007 through December 2010) prior to implementation of the national reference laboratory at INH were analyzed and compared with data obtained in the present study (January 2011 through April 2012). Analysis for linear trends in proportions revealed a significant (<i>p</i><0.01) increase of patients presenting with non-ulcerative lesions from 37.0% (2007–2010) to 50.0% (2011–2012). The mean duration of disease among patients with non-ulcerative lesions before presentation and establishment of clinical diagnosis decreased not significantly from 51.8 (95% CI: 19.0–84.7) to 35.0 (95% CI: 23.5–46.5) days during the two observation periods. However, the mean duration of disease among patients with ulcerative lesions before presentation of patients and establishment of clinical diagnosis decreased significantly from 182.6 (95% CI: 119.2–245.9) to 82.1 (95% CI: 51.3–112.8) days during the two observation periods. Furthermore, analysis of the development of categories of lesions showed a statistically non significant decrease from 30.3% (95% CI: 22.0–38.5) to 19.2% (95% CI: 8.5–29.9) of category III lesions. BUD, Buruli ulcer disease; CI, confidence interval.</p>a<p>Number of confirmed BUD patients, laboratory confirmation was conducted by standard IS<i>2404</i> PCR, IS<i>2404</i> DRB-PCR and/or IS<i>2404</i> qPCR.</p>b<p>Rate of confirmed BUD patients with non-ulcerative lesions among all confirmed BUD patients per observation period.</p>c<p>Category I, single lesion <50 mm in diameter.</p>d<p>Category II, single lesion between 50 and 150 mm in diameter.</p>e<p>Category III, single lesion >150 mm in diameter or multiple lesions, osteomyelitis or lesions at critical sites.</p>f<p>Mean duration of disease in days based on the time from first recognition of clinical symptoms by patients and availability of the clinical diagnosis “BUD”. Only data from PCR confirmed BUD patients were analyzed.</p