Distribution of quinolone resistance gene (qnr) in ESBL-producing Escherichia coli and Klebsiella spp. in Lomé, Togo.

Background: Qnr genes are known to confer a low-level resistance to fluoroquinolone in Enterobacteriaceae. They are often found on the same resistance plasmids as extended spectrum ß-lactamase (ESBL) and constitute the most common antibiotic resistance mechanism. This study aimed to detect the presence of qnr genes in ESBL-producing E. coli and Klebsiella spp. Methods: From May 2013 to July 2015, 91 E. coli and 64 Klebsiella spp. strains with phenotypic resistance to quinolone were collected from several specimens and analyzed for the detection of qnrA, qnrB, qnrS genes and the ß-lactamase resistance genes (blaCTX-M, blaTEM, blaSHV) using simplex and multiplex PCR. Results: In the present study, 107 (69%; 61 E. coli and 46 Klebsiella spp.) of 155 bacterial strains tested were found harboring at least one qnr gene consisting of 74 (47.74%) qnrB, 73 (47.10%) qnrS and 4 (2.58%) qnrA. Of the 107 strains encoding qnr genes, 102, 96 and 52 carried CTX-M1, TEM and SHV type ESBL respectively. Conclusion: This study identified quinolone resistance (qnr) gene in ESBL-producing E. coli and Klebsiella spp. in Togo. These finding which suggest a possible resistance to quinolone are of high interest for better management of patients and control of antimicrobial resistance in Togo.

Sensitivity, Specificity, and Public-Health Utility of Clinical Case Definitions Based on the Signs and Symptoms of Cholera in Africa.

During 2014, Africa reported more than half of the global suspected cholera cases. Based on the data collected from seven countries in the African Cholera Surveillance Network (Africhol), we assessed the sensitivity, specificity, and positive and negative predictive values of clinical cholera case definitions, including that recommended by the World Health Organization (WHO) using culture confirmation as the gold standard. The study was designed to assess results in real-world field situations in settings with recent cholera outbreaks or endemicity. From June 2011 to July 2015, a total of 5,084 persons with suspected cholera were tested for Vibrio cholerae in seven different countries of which 35.7% had culture confirmation. For all countries combined, the WHO case definition had a sensitivity = 92.7%, specificity = 8.1%, positive predictive value = 36.1%, and negative predictive value = 66.6%. Adding dehydration, vomiting, or rice water stools to the case definition could increase the specificity without a substantial decrease in sensitivity. Future studies could further refine our findings primarily by using more sensitive methods for cholera confirmation.

The African cholera surveillance network (Africhol) consortium meeting, 10-11 June 2015, Lomé, Togo.

The fifth annual meeting of the African cholera surveillance network (Africhol) took place on 10-11 June 2015 in Lomé, Togo. Together with international partners, representatives from the 11 member countries -Cameroon, Côte d'Ivoire, Democratic Republic of Congo, Guinea, Kenya, Mozambique, Nigeria, Tanzania, Togo, Uganda, Zimbabwe- and an invited country (Malawi) shared their experience. The meeting featured three sessions: i) cholera surveillance, prevention and control in participating countries, ii) cholera surveillance methodology, such as cholera mapping, cost-effectiveness studies and the issue of overlapping epidemics from different diseases, iii) cholera laboratory diagnostics tools and capacity building. The meeting has greatly benefitted from the input of technical expertise from participating institutions and the observations emerging from the meeting should enable national teams to make recommendations to their respective governments on the most appropriate and effective measures to be taken for the prevention and control of cholera. Recommendations for future activities included collecting precise burden estimates in surveillance sites; modeling cholera burden for Africa; setting up cross-border collaborations; strengthening laboratory capacity for the confirmation of suspected cholera cases and for vaccine impact assessment in settings where oral cholera vaccine would be used; adapting cholera surveillance to concurrent issues (e.g., Ebola); and developing national cholera control plans including rationale vaccination strategies together with other preventive and control measures such as improvements in water, sanitation and hygiene (WASH).

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.

Treatment Outcome of Patients with Buruli Ulcer Disease in Togo.

BACKGROUND: Following introduction of antimycobacterial treatment of Buruli ulcer disease (BUD), several clinical studies evaluated treatment outcomes of BUD patients, in particular healing times, secondary lesions and functional limitations. Whereas recurrences were rarely observed, paradoxical reactions and functional limitations frequently occurred. Although systematic BUD control in Togo was established as early as 2007, treatment outcome has not been reviewed to date. Therefore, a pilot project on post-treatment follow-up of BUD patients in Togo aimed to evaluate treatment outcomes and to provide recommendations for optimization of treatment success. METHODOLOGY/PRINCIPAL FINDINGS: Out of 199 laboratory confirmed BUD patients, 129 could be enrolled in the study. The lesions of 109 patients (84.5%) were completely healed without any complications, 5 patients (3.9%) had secondary lesions and 15 patients (11.6%) had functional limitations. Edema, category III ulcers >15 cm, healing times >180 days and a limitation of movement at time of discharge constituted the main risk factors significantly associated with BUD related functional limitations (P<0.01). Review of all BUD related documentation revealed major shortcomings, in particular concerning medical records on adjuvant surgical and physiotherapeutic treatment. CONCLUSIONS/SIGNIFICANCE: This study presents the first systematic analysis of treatment outcome of BUD patients from Togo. Median times to healing and the absence of recurrences were in line with findings reported by other investigators. The percentage of functional limitations of 11.6% was lower than in other studies, and edema, category III ulcers, healing time >180 days and limitation of movement at discharge constituted the main risk factors for functional limitations in Togolese BUD patients. Standardized treatment plans, patient assessment and follow-up, as well as improved management of medical records are recommended to allow for intensified monitoring of disease progression and healing process, to facilitate implementation of therapeutic measures and to optimize treatment success.

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.

National surveillance data on the epidemiology of cholera in Togo.

BACKGROUND: Togo is a cholera-endemic country bordered by other countries where this disease is endemic. We describe the epidemiology of cholera in Togo, using national surveillance data. METHODS: We reviewed national surveillance data housed in the National Ministry of Health. Districts submitted reports of summary weekly case counts and deaths at the national level. Data were available at the district level during 2008-2010 and at the national level from 1996 onward. Microbiological confirmation usually was not performed, and case identification was based on clinical suspicion. RESULTS: From 1996 through 2010, Togo had 12 676 reported cholera cases and 554 deaths. Annual national cholera incidence varied from 0.9 to 66 cases per 100 000 population, with little variation except for 2 large epidemics during 1998 and 2001. The case-fatality ratio declined from 12%-17% during 1996-1997 to <1% during 2008-2010. During 2008-2010, 85% of 26 district-level outbreaks occurred in the capital Lomé or the coastal Maritime Region. The average outbreak duration was 6 weeks, and only 2 lasted >15 weeks. DISCUSSION: While cholera control remains elusive in Togo, reductions in case-fatality ratios have occurred, possibly due to improvements in case management. The short duration of outbreaks may preclude reactive vaccination; however, the restricted geographic location may make preventive immunization attractive.

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.