Enhancing Meningococcal Genomic Surveillance in the Meningitis Belt Using High-Resolution Culture-Free Whole-Genome Sequencing.

Rollout of meningococcal serogroup A conjugate vaccine in Africa started in 2010, aiming to eliminate meningitis outbreaks, in meningitis belt countries. Since then, studies have been conducted, primarily using isolates, to assess the vaccine impact on the distribution of meningococcal strains in the region. Here, we implemented an innovative, culture-free whole-genome sequencing approach on almost 400 clinical specimens collected between 2017 and 2019 from meningococcal meningitis cases in 6 African countries. About 50% of specimens provided high-quality whole-genome sequence data for comprehensive molecular profiling of the meningococcal pathogen. Three major clonal complexes were identified: CC11 associated with serogroup W, CC181 associated with serogroup X, and CC10217 associated with serogroup C, which continues to rise as a predominant clonal complex in the region. Genomic surveillance for meningococcal meningitis can be significantly improved using culture-free methods to increase data representativeness and monitor changes in epidemiological landscape, especially for countries with low culture rate.

Modeling Optimal Laboratory Testing Strategies for Bacterial Meningitis Surveillance in Africa.

Since 2010, the introduction of an effective serogroup A meningococcal conjugate vaccine has led to the near-elimination of invasive Neisseria meningitidis serogroup A disease in Africa's meningitis belt. However, a significant burden of disease and epidemics due to other bacterial meningitis pathogens remain in the region. High-quality surveillance data with laboratory confirmation is important to monitor circulating bacterial meningitis pathogens and design appropriate interventions, but complete testing of all reported cases is often infeasible. Here, we use case-based surveillance data from 5 countries in the meningitis belt to determine how accurately estimates of the distribution of causative pathogens would represent the true distribution under different laboratory testing strategies. Detailed case-based surveillance data was collected by the MenAfriNet surveillance consortium in up to 3 seasons from participating districts in 5 countries. For each unique country-season pair, we simulated the accuracy of laboratory surveillance by repeatedly drawing subsets of tested cases and calculating the margin of error of the estimated proportion of cases caused by each pathogen (the greatest pathogen-specific absolute error in proportions between the subset and the full set of cases). Across the 12 country-season pairs analyzed, the 95% credible intervals around estimates of the proportion of cases caused by each pathogen had median widths of ±0.13, ±0.07, and ±0.05, respectively, when random samples of 25%, 50%, and 75% of cases were selected for testing. The level of geographic stratification in the sampling process did not meaningfully affect accuracy estimates. These findings can inform testing thresholds for laboratory surveillance programs in the meningitis belt.

Full Molecular Typing of Neisseria meningitidis Directly from Clinical Specimens for Outbreak Investigation.

Neisseria meningitidis is a leading cause of bacterial meningitis and sepsis worldwide and an occasional cause of meningococcal urethritis. When isolates are unavailable for surveillance or outbreak investigations, molecular characterization of pathogens needs to be performed directly from clinical specimens, such as cerebrospinal fluid (CSF), blood, or urine. However, genome sequencing of specimens is challenging because of low bacterial and high human DNA abundances. We developed selective whole-genome amplification (SWGA), an isothermal multiple-displacement amplification-based method, to efficiently enrich, sequence, and de novo assemble N. meningitidis DNA from clinical specimens with low bacterial loads. SWGA was validated with 12 CSF specimens from invasive meningococcal disease cases and 12 urine specimens from meningococcal urethritis cases. SWGA increased the mean proportion of N. meningitidis reads by 2 to 3 orders of magnitude, enabling identification of at least 90% of the 1,605 N. meningitidis core genome loci for 50% of the specimens. The validated method was used to investigate two meningitis outbreaks recently reported in Togo and Burkina Faso. Twenty-seven specimens with low bacterial loads were processed by SWGA before sequencing, and 12 of 27 were successfully assembled to obtain the full molecular typing and vaccine antigen profile of the N. meningitidis pathogen, thus enabling thorough characterization of outbreaks. This method is particularly important for enhancing molecular surveillance in regions with low culture rates. SWGA produces enough reads for phylogenetic and allelic analysis at a low cost. More importantly, the procedure can be extended to enrich other important human bacterial pathogens.

Neisseria meningitidis Serogroup W Meningitis Epidemic in Togo, 2016.

BACKGROUND: During 2014, 4 regions in Togo within the African meningitis belt implemented vaccination campaigns with meningococcal serogroup A conjugate vaccine (MACV). From January to July 2016, Togo experienced its first major Neisseria meningitidis serogroup W (NmW) outbreak. We describe the epidemiology, response, and management of the outbreak. METHODS: Suspected, probable, and confirmed cases were identified using World Health Organization case definitions. Through case-based surveillance, epidemiologic and laboratory data were collected for each case. Cerebrospinal fluid specimens were analyzed by polymerase chain reaction, culture, or latex agglutination. Vaccination campaigns were conducted in affected districts. RESULTS: From January 11 to July 5, 2016, 1995 suspected meningitis cases were reported, with 128 deaths. Among them, 479 (24.0%) were confirmed by laboratory testing, and 94 (4.7%) and 1422 (71.3%) remained as probable and suspected cases, respectively. Seven epidemic districts had cumulative attack rates greater than 100 per 100 000 population. Of the confirmed cases, 91.5% were NmW; 39 of 40 available NmW isolates were sequence type-11/clonal complex-11. CONCLUSIONS: This outbreak demonstrates that, although high coverage with MACV has reduced serogroup A outbreaks, large meningococcal meningitis outbreaks due to other serogroups may continue to occur; effective multivalent meningococcal conjugate vaccines could improve meningococcal disease prevention within meningitis belt populations.

Bacterial Meningitis Epidemiology in Five Countries in the Meningitis Belt of Sub-Saharan Africa, 2015-2017.

BACKGROUND: The MenAfriNet Consortium supports strategic implementation of case-based meningitis surveillance in key high-risk countries of the African meningitis belt: Burkina Faso, Chad, Mali, Niger, and Togo. We describe bacterial meningitis epidemiology in these 5 countries in 2015-2017. METHODS: Case-based meningitis surveillance collects case-level demographic and clinical information and cerebrospinal fluid (CSF) laboratory results. Neisseria meningitidis, Streptococcus pneumoniae, or Haemophilus influenzae cases were confirmed and N. meningitidis/H. influenzae were serogrouped/serotyped by real-time polymerase chain reaction, culture, or latex agglutination. We calculated annual incidence in participating districts in each country in cases/100 000 population. RESULTS: From 2015-2017, 18 262 suspected meningitis cases were reported; 92% had a CSF specimen available, of which 26% were confirmed as N. meningitidis (n = 2433; 56%), S. pneumoniae (n = 1758; 40%), or H. influenzae (n = 180; 4%). Average annual incidences for N. meningitidis, S. pneumoniae, and H. influenzae, respectively, were 7.5, 2.5, and 0.3. N. meningitidis incidence was 1.5 in Burkina Faso, 2.7 in Chad, 0.4 in Mali, 14.7 in Niger, and 12.5 in Togo. Several outbreaks occurred: NmC in Niger in 2015-2017, NmC in Mali in 2016, and NmW in Togo in 2016-2017. Of N. meningitidis cases, 53% were NmC, 30% NmW, and 13% NmX. Five NmA cases were reported (Burkina Faso, 2015). NmX increased from 0.6% of N. meningitidis cases in 2015 to 27% in 2017. CONCLUSIONS: Although bacterial meningitis epidemiology varied widely by country, NmC and NmW caused several outbreaks, NmX increased although was not associated with outbreaks, and overall NmA incidence remained low. An effective low-cost multivalent meningococcal conjugate vaccine could help further control meningococcal meningitis in the region.

Improving Case-Based Meningitis Surveillance in 5 Countries in the Meningitis Belt of Sub-Saharan Africa, 2015-2017.

BACKGROUND: The MenAfriNet consortium was established in 2014 to support implementation of case-based meningitis surveillance in 5 countries in the meningitis belt of sub-Saharan Africa: Burkina Faso, Chad, Mali, Niger, and Togo. Assessing surveillance performance is critical for interpretation of the collected data and implementation of future surveillance-strengthening initiatives. METHODS: Detailed epidemiologic and laboratory data were collected on suspected meningitis cases through case-based meningitis surveillance in participating districts in 5 countries. Performance of case-based surveillance was evaluated through sensitivity of case ascertainment in case-based versus aggregate meningitis surveillance and an analysis of surveillance indicators. RESULTS: From 2015 to 2017, 18 262 suspected meningitis cases were identified through case-based surveillance and 16 262 were identified through aggregate surveillance, for a case ascertainment sensitivity of 112.3%. Among suspected cases, 16 885 (92.5%) had a cerebrospinal fluid (CSF) specimen collected, 13 625 (80.7%) of which were received at a national reference laboratory. Among these, 13 439 (98.6%) underwent confirmatory testing, and, of those tested, 4371 (32.5%) were confirmed for a bacterial pathogen. CONCLUSIONS: Overall strong performance for case ascertainment, CSF collection, and laboratory confirmation provide evidence for the quality of MenAfriNet case-based surveillance in evaluating epidemiologic trends and informing future vaccination strategies.

Description of Antimicrobial Resistance Patterns at the National Institute of Hygiene in Lome, Togo.

The monitoring of antimicrobial resistance has become a priority, mainly in developing countries, to control the emergence of multidrug-resistant bacterial strains. This study evaluated the antibiotic resistance profiles of bacteria isolated at the National Institute of Hygiene in Togo. The laboratory records from January 2013 to December 2015 were examined, which showed that a total of 33,147 samples had been analyzed. Among them, vaginal swabs and urine samples were predominant, amounting to 38.17% and 33.24%, respectively. In total, 3,497 Gram-positive and 1,970 Gram-negative bacterial strains were cultured. Enterobacteriaceae (57.6%), Staphylococcus spp. (21.1%), and Streptococcus spp. (10.5%) were primarily isolated. The records showed that over 3 years, Escherichia coli was resistant to piperacillin (79.66%, 75.1%, and 83%), trimethoprim/sulfamethoxazole (79.3%, 82%, and 82.8%), ticarcillin (89%, 89.7%, and 93%), and amoxicillin (94.1%, 94%, and 96.09%), whereas Staphylococcus aureus was resistant to penicillin G. Streptococcaceae isolates were resistant to trimethoprim/sulfamethoxazole (78.11-87.1%), tetracycline (82.2-91.16%), and norfloxacin (86.16-94.3%). Escherichia coli and S. aureus isolated from urine were more resistant to antibiotics than those isolated from vaginal swabs. There is a need to develop new strategies to fight the emergence of multi-resistant bacteria in Togo.

On-site verification of the CBEU method using UriSelect4 chromogenic medium at the National Institute of Hygiene (INH) of Lomé.

PURPOSE: The Cytobacteriological Examination of Urine (CBEU) is one of the most requested microbiological analyses in the medical bacteriology laboratory. According to the ISO 15189 standard, Medical Biology Laboratory (MBL) must validate the techniques before their use. It is in this perspective that, within the Medical Bacteriology Laboratory of the INH of Lome, we have carried out the on-site verification of the CBEU method using UriSelect4 chromogenic medium at the INH of Lomé. MATERIAL AND METHODS: The biological material was composed of reference bacterial strains and clinical bacterial strains isolated at INH. Failure Modes Effects and Criticality Analysis (FMECA) with 5 â€‹M model was used to perform risk analysis and performances as repeatability, intermediate fidelity/reproducibility, sensitivity and inter-operator variability were evaluated using references strains and different operators. "Comité Français d'Accréditation" (COFRAC) SH GTA 04 was used as reference. Single-factor ANOVA was used to analyze the data. RESULTS: Operating procedures, Patients preparation, Samples and Preparation of culture media were identified as main critical points with a criticality index of 8, 9, 9 and 12 respectively. The use of uriselect4 to perform ECBU was shown to be repeatable and reproducible. The sensibility of Uriselect4 to detect urinary infection was 100% with negligible inter-operator variability. CONCLUSION: This study performed on-site verification of Uriselect4 at the bacteriology lab of INH of Lome and identified some critical points to master. The overall performance criteria from COFRAC SH GTA 04 were conform.

Neisseria meningitidis Serogroup C Clonal Complex 10217 Outbreak in West Kpendjal Prefecture, Togo 2019.

Togo has reported seasonal meningitis outbreaks caused by non-Neisseria meningitidis serogroup A (NmA) pathogens since the introduction of meningococcal serogroup A conjugate vaccine (MACV, MenAfriVac) in 2014. From 2016 to 2017, NmW caused several outbreaks. In early 2019, a NmC outbreak was detected in the Savanes region of Togo and its investigation is described here. Under case-based surveillance, epidemiological and clinical data, and cerebrospinal fluid specimens were collected for every suspected case of meningitis. Specimens were tested for meningitis pathogens using confirmatory microbiological and molecular methods. During epidemic weeks 9 to 15, 199 cases were reported, with 179 specimens being available for testing and 174 specimens (97.2%) were tested by at least one confirmatory method. The NmC was the predominant pathogen confirmed (93.9%), belonging to sequence type (ST)-9367 of clonal complex (CC) 10217. All NmC cases were localized to the West Kpendjal district of the Savanes region with attack rates ranging from 4.1 to 18.8 per 100,000 population and case fatality rates ranging up to 2.2% during weeks 9 to 15. Of the 93 NmC confirmed cases, 63.4% were males and 88.2% were in the 5 to 29 age group. This is the first report of a NmC meningitis outbreak in Togo. The changing epidemiology of bacterial meningitis in the meningitis belt post-MACV highlights the importance of monitoring of emerging strain and country preparedness for outbreaks in the region. IMPORTANCE The recent emergence of an invasive NmC strain in Togo is an example of the changing bacterial meningitis epidemiology in the meningitis belt post-MACV. The current epidemiology includes the regional circulation of various non-NmA serogroups, which emphasizes the need for effective molecular surveillance, laboratory diagnosis, and a multivalent vaccine that is effective against all serogroups in circulation.

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.

Dynamics of cholera epidemics from Benin to Mauritania.

BACKGROUND: The countries of West Africa are largely portrayed as cholera endemic, although the dynamics of outbreaks in this region of Africa remain largely unclear. METHODOLOGY/PRINCIPAL FINDINGS: To understand the dynamics of cholera in a major portion of West Africa, we analyzed cholera epidemics from 2009 to 2015 from Benin to Mauritania. We conducted a series of field visits as well as multilocus variable tandem repeat analysis and whole-genome sequencing analysis of V. cholerae isolates throughout the study region. During this period, Ghana accounted for 52% of the reported cases in the entire study region (coastal countries from Benin to Mauritania). From 2009 to 2015, we found that one major wave of cholera outbreaks spread from Accra in 2011 northwestward to Sierra Leone and Guinea in 2012. Molecular epidemiology analysis confirmed that the 2011 Ghanaian isolates were related to those that seeded the 2012 epidemics in Guinea and Sierra Leone. Interestingly, we found that many countries deemed "cholera endemic" actually suffered very few outbreaks, with multi-year lulls. CONCLUSIONS/SIGNIFICANCE: This study provides the first cohesive vision of the dynamics of cholera epidemics in a major portion of West Africa. This epidemiological overview shows that from 2009 to 2015, at least 54% of reported cases concerned populations living in the three urban areas of Accra, Freetown, and Conakry. These findings may serve as a guide to better target cholera prevention and control efforts in the identified cholera hotspots in West Africa.

Relationship between Distinct African Cholera Epidemics Revealed via MLVA Haplotyping of 337 Vibrio cholerae Isolates.

BACKGROUND: Since cholera appeared in Africa during the 1970s, cases have been reported on the continent every year. In Sub-Saharan Africa, cholera outbreaks primarily cluster at certain hotspots including the African Great Lakes Region and West Africa. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we applied MLVA (Multi-Locus Variable Number Tandem Repeat Analysis) typing of 337 Vibrio cholerae isolates from recent cholera epidemics in the Democratic Republic of the Congo (DRC), Zambia, Guinea and Togo. We aimed to assess the relationship between outbreaks. Applying this method, we identified 89 unique MLVA haplotypes across our isolate collection. MLVA typing revealed the short-term divergence and microevolution of these Vibrio cholerae populations to provide insight into the dynamics of cholera outbreaks in each country. Our analyses also revealed strong geographical clustering. Isolates from the African Great Lakes Region (DRC and Zambia) formed a closely related group, while West African isolates (Togo and Guinea) constituted a separate cluster. At a country-level scale our analyses revealed several distinct MLVA groups, most notably DRC 2011/2012, DRC 2009, Zambia 2012 and Guinea 2012. We also found that certain MLVA types collected in the DRC persisted in the country for several years, occasionally giving rise to expansive epidemics. Finally, we found that the six environmental isolates in our panel were unrelated to the epidemic isolates. CONCLUSIONS/SIGNIFICANCE: To effectively combat the disease, it is critical to understand the mechanisms of cholera emergence and diffusion in a region-specific manner. Overall, these findings demonstrate the relationship between distinct epidemics in West Africa and the African Great Lakes Region. This study also highlights the importance of monitoring and analyzing Vibrio cholerae isolates.