(GTG)5-PCR fingerprinting of multi-drug resistant Escherichia coli bacteria isolates from hospital in Ouagadougou, Burkina Faso.

BACKGROUND: Escherichia coli (E. coli) is the most common bacterial species implicated in various types of infections including septicemia, gastroenteritis, urinary tract infections, meningitis and others pathologies. These involve several bacterial clones with multidrug resistance making them difficult to treat. The aims of this study was to perform molecular typing of E. coli strains using universal primer (GTG)5. In this study, 53 E. coli strains were collected from inpatients and outpatients. The test of antimicrobial sensibility was realized using CA-SFM /EUCAST method and strains were identified by conventional microbiological tests. The carbapenemase-producing strains were demonstrated by phenotypic method. Bacterial DNA was extracted by boiling method. (GTG)5-PCR was used for strain subtyping. The DendroUPGMA software was used for grouping of strains from the genetic fingerprints obtained by (GTG)5-PCR. RESULTS: Antibiotic susceptibility test revealed that all strains were multi-drug resistant (MDR). Its strains showed resistance to at least three different families of antibiotics. Of this MDR strains, only one was a metallo-ß-lactamase producer. The dendrogram obtained using genetic fingerprinting allowed the E. coli strains to be grouped into 22 clusters (G1 to G22). CONCLUSION: The (GTG) 5-PCR assay enabled rapid molecular typing of E. coli strains. The strains of E. coli typed in this study would belong to different clones.

Characterization of virulence factors of Salmonella isolated from human stools and street food in urban areas of Burkina Faso.

BACKGROUND: This study was undertaken to identify and functionally characterize virulence genes from Salmonella isolates in street food and stool cultures. From February 2017 to May 2018, clinical and food Salmonella strains were isolated in three regions in Burkina Faso. Salmonella was serotyped according to the White-Kauffmann-Le Minor method, and polymerase chain reaction (PCR) was used to detec invA, spvR, spvC, fimA and stn virulence genes commonly associated with salmonellosis in Sub-Saharan Africa. RESULTS: A total of 106 Salmonella isolates (77 human stools; 14 sandwiches) was analyzed using a serological identification with an O-group test reagent. The presence of Salmonella was confirmed in 86% (91/106) of the samples were reactive (OMA-positive/OMB-positive). Salmonella serogroup O:4,5 was the most common serogroup detected (40%; 36/91). Salmonella Enteritidis and Typhimurium represented 5.5% (5/91) and 3.3% (3/91), respectively and were identified only from clinical isolates. Furthermore, 14 serotypes of Salmonella (12/91 human strains and 2/15 sandwich strains) were evocative of Kentucky/Bargny serotype. For the genetic profile, 66% (70/106) of the Salmonella had invA and stn genes; 77.4% (82/106) had the fimA gene. The spvR gene was found in 36.8% (39/106) of the isolates while 48.1% (51/106) had the spvC gene. Among the identified Salmonella Enteritidis and Salmonella Typhimurium isolated from stools, the virulence genes detected were invA (3/5) versus (2/3), fimA (4/5) versus (3/3), stn (3/5) versus (2/3), spvR (4/5) versus (2/3) and spvC (3/5) versus (2/3), respectively. CONCLUSION: This study reports the prevalence of Salmonella serotypes and virulence genes in clinical isolates and in street foods. It shows that food could be a significant source of Salmonella transmission to humans. Our results could help decision-making by the Burkina Faso health authority in the fight against street food-related diseases, in particular by training restaurateurs in food hygiene.

The genomic epidemiology of multi-drug resistant invasive non-typhoidal Salmonella in selected sub-Saharan African countries.

BACKGROUND: Invasive non-typhoidal Salmonella (iNTS) is one of the leading causes of bacteraemia in sub-Saharan Africa. We aimed to provide a better understanding of the genetic characteristics and transmission patterns associated with multi-drug resistant (MDR) iNTS serovars across the continent. METHODS: A total of 166 iNTS isolates collected from a multi-centre surveillance in 10 African countries (2010-2014) and a fever study in Ghana (2007-2009) were genome sequenced to investigate the geographical distribution, antimicrobial genetic determinants and population structure of iNTS serotypes-genotypes. Phylogenetic analyses were conducted in the context of the existing genomic frameworks for various iNTS serovars. Population-based incidence of MDR-iNTS disease was estimated in each study site. RESULTS: Salmonella Typhimurium sequence-type (ST) 313 and Salmonella Enteritidis ST11 were predominant, and both exhibited high frequencies of MDR; Salmonella Dublin ST10 was identified in West Africa only. Mutations in the gyrA gene (fluoroquinolone resistance) were identified in S. Enteritidis and S. Typhimurium in Ghana; an ST313 isolate carrying blaCTX-M-15 was found in Kenya. International transmission of MDR ST313 (lineage II) and MDR ST11 (West African clade) was observed between Ghana and neighbouring West African countries. The incidence of MDR-iNTS disease exceeded 100/100 000 person-years-of-observation in children aged <5 years in several West African countries. CONCLUSIONS: We identified the circulation of multiple MDR iNTS serovar STs in the sampled sub-Saharan African countries. Investment in the development and deployment of iNTS vaccines coupled with intensified antimicrobial resistance surveillance are essential to limit the impact of these pathogens in Africa.

Meningococcal carriage 7 years after introduction of a serogroup A meningococcal conjugate vaccine in Burkina Faso: results from four cross-sectional carriage surveys.

BACKGROUND: In the first 2 years after a nationwide mass vaccination campaign of 1-29-year-olds with a meningococcal serogroup A conjugate vaccine (MenAfriVac) in Burkina Faso, carriage and disease due to serogroup A Neisseria meningitidis were nearly eliminated. We aimed to assess the long-term effect of MenAfriVac vaccination on meningococcal carriage and herd immunity. METHODS: We did four cross-sectional studies of meningococcal carriage in people aged 9 months to 36 years in two districts of Burkina Faso between May 2, 2016, and Nov 6, 2017. Demographic information and oropharyngeal swabs were collected. Meningococcal isolates were characterised using whole-genome sequencing. FINDINGS: Of 14 295 eligible people, 13 758 consented and had specimens collected and laboratory results available, 1035 of whom were meningococcal carriers. Accounting for the complex survey design, prevalence of meningococcal carriage was 7·60% (95% CI 5·67-9·52), including 6·98% (4·86-9·11) non-groupable, 0·48% (0·01-0·95) serogroup W, 0·10% (0·01-0·18) serogroup C, 0·03% (0·00-0·80) serogroup E, and 0% serogroup A. Prevalence ranged from 5·44% (95% CI 4·18-6·69) to 9·14% (6·01-12·27) by district, from 4·67% (2·71-6·64) to 11·17% (6·75-15·59) by round, and from 3·39% (0·00-8·30) to 10·43% (8·08-12·79) by age group. By clonal complex, 822 (88%) of 934 non-groupable isolates were CC192, all 83 (100%) serogroup W isolates were CC11, and nine (69%) of 13 serogroup C isolates were CC10217. INTERPRETATION: Our results show the continued effect of MenAfriVac on serogroup A meningococcal carriage, for at least 7 years, among vaccinated and unvaccinated cohorts. Carriage prevalence of epidemic-prone serogroup C CC10217 and serogroup W CC11 was low. Continued monitoring of N meningitidis carriage will be crucial to further assess the effect of MenAfriVac and inform the vaccination strategy for future multivalent meningococcal vaccines. FUNDING: Bill & Melinda Gates Foundation and Gavi, the Vaccine Alliance.

Multicountry Distribution and Characterization of Extended-spectrum ß-Lactamase-associated Gram-negative Bacteria From Bloodstream Infections in Sub-Saharan Africa.

BACKGROUND: Antimicrobial resistance (AMR) is a major global health concern, yet, there are noticeable gaps in AMR surveillance data in regions such as sub-Saharan Africa. We aimed to measure the prevalence of extended-spectrum ß-lactamase (ESBL) producing Gram-negative bacteria in bloodstream infections from 12 sentinel sites in sub-Saharan Africa. METHODS: Data were generated during the Typhoid Fever Surveillance in Africa Program (TSAP), in which standardized blood cultures were performed on febrile patients attending 12 health facilities in 9 sub-Saharan African countries between 2010 and 2014. Pathogenic bloodstream isolates were identified at the sites and then subsequently confirmed at a central reference laboratory. Antimicrobial susceptibility testing, detection of ESBL production, and conventional multiplex polymerase chain reaction (PCR) testing for genes encoding for ß-lactamase were performed on all pathogens. RESULTS: Five hundred and five pathogenic Gram-negative bloodstream isolates were isolated during the study period and available for further characterization. This included 423 Enterobacteriaceae. Phenotypically, 61 (12.1%) isolates exhibited ESBL activity, and genotypically, 47 (9.3%) yielded a PCR amplicon for at least one of the screened ESBL genes. Among specific Gram-negative isolates, 40 (45.5%) of 88 Klebsiella spp., 7 (5.7%) of 122 Escherichia coli, 6 (16.2%) of 37 Acinetobacter spp., and 2 (1.3%) of 159 of nontyphoidal Salmonella (NTS) showed phenotypic ESBL activity. CONCLUSIONS: Our findings confirm the presence of ESBL production among pathogens causing bloodstream infections in sub-Saharan Africa. With few alternatives for managing ESBL-producing pathogens in the African setting, measures to control the development and proliferation of AMR organisms are urgently needed.

The phylogeography and incidence of multi-drug resistant typhoid fever in sub-Saharan Africa.

There is paucity of data regarding the geographical distribution, incidence, and phylogenetics of multi-drug resistant (MDR) Salmonella Typhi in sub-Saharan Africa. Here we present a phylogenetic reconstruction of whole genome sequenced 249 contemporaneous S. Typhi isolated between 2008-2015 in 11 sub-Saharan African countries, in context of the 2,057 global S. Typhi genomic framework. Despite the broad genetic diversity, the majority of organisms (225/249; 90%) belong to only three genotypes, 4.3.1 (H58) (99/249; 40%), 3.1.1 (97/249; 39%), and 2.3.2 (29/249; 12%). Genotypes 4.3.1 and 3.1.1 are confined within East and West Africa, respectively. MDR phenotype is found in over 50% of organisms restricted within these dominant genotypes. High incidences of MDR S. Typhi are calculated in locations with a high burden of typhoid, specifically in children aged <15 years. Antimicrobial stewardship, MDR surveillance, and the introduction of typhoid conjugate vaccines will be critical for the control of MDR typhoid in Africa.

Molecular Characterization of Diarrheagenic Escherichia Coli in Children Less Than 5 Years of Age with Diarrhea in Ouagadougou, Burkina Faso.

Diarrheagenic Escherichia coli (DEC) is important bacteria of children's endemic and epidemic diarrhea worldwide. The aim of this study was to determine the prevalence of DEC isolated from stool samples collected from children with acute diarrhea living in Ouagadougou, Burkina Faso. From August 2013 to October 2015, stool samples were collected from 315 children under 5 years of age suffering from diarrhea in the "Centre Médical avec Antenne Chirurgicale (CMA)" Paul VI and the CMA of Schiphra. E. coli were isolated and identified by standard microbiological methods, and the 16-plex PCR method was used to further characterize them. Four hundred and nineteen (419) E. coli strains were characterized, of which 31 (7.4%) DEC pathotypes were identified and classified in five E. coli pathotypes: 15 enteroaggregative E. coli (EAEC) (48.4%), 8 enteropathogenic E. coli (EPEC) (25.8%) with 4 typical EPEC and 4 atypical EPEC, 4 enteroinvasive E. coli (EIEC) (12.9%), 3 enterohemorrhagic E. coli (EHEC) 9.67%, and 1 enterotoxigenic E. coli (ETEC) 3.2%. The use of multiplex PCR as a routine in clinical laboratory for the detection of DEC would be a useful mean for a rapid management of an acute diarrhea in children.

Incidence of invasive salmonella disease in sub-Saharan Africa: a multicentre population-based surveillance study.

BACKGROUND: Available incidence data for invasive salmonella disease in sub-Saharan Africa are scarce. Standardised, multicountry data are required to better understand the nature and burden of disease in Africa. We aimed to measure the adjusted incidence estimates of typhoid fever and invasive non-typhoidal salmonella (iNTS) disease in sub-Saharan Africa, and the antimicrobial susceptibility profiles of the causative agents. METHODS: We established a systematic, standardised surveillance of blood culture-based febrile illness in 13 African sentinel sites with previous reports of typhoid fever: Burkina Faso (two sites), Ethiopia, Ghana, Guinea-Bissau, Kenya, Madagascar (two sites), Senegal, South Africa, Sudan, and Tanzania (two sites). We used census data and health-care records to define study catchment areas and populations. Eligible participants were either inpatients or outpatients who resided within the catchment area and presented with tympanic (≥38·0°C) or axillary temperature (≥37·5°C). Inpatients with a reported history of fever for 72 h or longer were excluded. We also implemented a health-care utilisation survey in a sample of households randomly selected from each study area to investigate health-seeking behaviour in cases of self-reported fever lasting less than 3 days. Typhoid fever and iNTS disease incidences were corrected for health-care-seeking behaviour and recruitment. FINDINGS: Between March 1, 2010, and Jan 31, 2014, 135 Salmonella enterica serotype Typhi (S Typhi) and 94 iNTS isolates were cultured from the blood of 13 431 febrile patients. Salmonella spp accounted for 33% or more of all bacterial pathogens at nine sites. The adjusted incidence rate (AIR) of S Typhi per 100 000 person-years of observation ranged from 0 (95% CI 0-0) in Sudan to 383 (274-535) at one site in Burkina Faso; the AIR of iNTS ranged from 0 in Sudan, Ethiopia, Madagascar (Isotry site), and South Africa to 237 (178-316) at the second site in Burkina Faso. The AIR of iNTS and typhoid fever in individuals younger than 15 years old was typically higher than in those aged 15 years or older. Multidrug-resistant S Typhi was isolated in Ghana, Kenya, and Tanzania (both sites combined), and multidrug-resistant iNTS was isolated in Burkina Faso (both sites combined), Ghana, Kenya, and Guinea-Bissau. INTERPRETATION: Typhoid fever and iNTS disease are major causes of invasive bacterial febrile illness in the sampled locations, most commonly affecting children in both low and high population density settings. The development of iNTS vaccines and the introduction of S Typhi conjugate vaccines should be considered for high-incidence settings, such as those identified in this study. FUNDING: Bill & Melinda Gates Foundation.

The Relationship Between Invasive Nontyphoidal Salmonella Disease, Other Bacterial Bloodstream Infections, and Malaria in Sub-Saharan Africa.

BACKGROUND: Country-specific studies in Africa have indicated that Plasmodium falciparum is associated with invasive nontyphoidal Salmonella (iNTS) disease. We conducted a multicenter study in 13 sites in Burkina Faso, Ethiopia, Ghana, Guinea-Bissau, Kenya, Madagascar, Senegal, South Africa, Sudan, and Tanzania to investigate the relationship between the occurrence of iNTS disease, other systemic bacterial infections, and malaria. METHODS: Febrile patients received a blood culture and a malaria test. Isolated bacteria underwent antimicrobial susceptibility testing, and the association between iNTS disease and malaria was assessed. RESULTS: A positive correlation between frequency proportions of malaria and iNTS was observed (P = .01; r = 0.70). Areas with higher burden of malaria exhibited higher odds of iNTS disease compared to other bacterial infections (odds ratio [OR], 4.89; 95% CI, 1.61-14.90; P = .005) than areas with lower malaria burden. Malaria parasite positivity was associated with iNTS disease (OR, 2.44; P = .031) and gram-positive bacteremias, particularly Staphylococcus aureus, exhibited a high proportion of coinfection with Plasmodium malaria. Salmonella Typhimurium and Salmonella Enteritidis were the predominant NTS serovars (53/73; 73%). Both moderate (OR, 6.05; P = .0001) and severe (OR, 14.62; P < .0001) anemia were associated with iNTS disease. CONCLUSIONS: A positive correlation between iNTS disease and malaria endemicity, and the association between Plasmodium parasite positivity and iNTS disease across sub-Saharan Africa, indicates the necessity to consider iNTS as a major cause of febrile illness in malaria-holoendemic areas. Prevention of iNTS disease through iNTS vaccines for areas of high malaria endemicity, targeting high-risk groups for Plasmodium parasitic infection, should be considered.

Diagnosing Salmonella enterica Serovar Typhi Infections by Polymerase Chain Reaction Using EDTA Blood Samples of Febrile Patients From Burkina Faso.

BACKGROUND: Globally, there are an estimated 22 million cases of Salmonella enterica serovar Typhi infection each year. However, this figure is likely to be an underestimate due to the low sensitivity of blood culture in S. Typhi diagnosis. The aim of this study was to diagnose S. Typhi by conventional polymerase chain reaction (PCR) using patient's blood preserved with ethylenediamine tetraacetic acid (EDTA). METHODS: From April 2012 to September 2013, typhoid fever surveillance was conducted in Polesgo and Nioko, 2 dry slum areas in Ouagadougou, Burkina Faso. Blood culture was performed for febrile patients using an automated blood culture system. Additional blood was collected in EDTA tubes from those patients and preserved at -80°C. DNA was extracted from EDTA blood and PCR was performed to identify presence of S. Typhi. Randomly selected PCR products were further sequenced to identify S. Typhi-specific amplicons. RESULTS: Of 1674 patients, S. Typhi was isolated from 18 (1.1%) individuals by blood culture. EDTA blood was collected from 1578 patients, of which 298 EDTA samples were tested by PCR. Salmonella Typhi-specific DNA was identified in 44 (14.8%) samples. The sensitivity of S. Typhi-specific PCR from EDTA blood was 89% (74%-100%) among the blood culture-positive cases. Sixteen S. Typhi-positive PCR products were sequenced, and 13 retrieved the sequence of a S. Typhi-specific amplicon. CONCLUSIONS: These findings suggest that blood culture-based diagnoses of S. Typhi underestimate the burden of typhoid fever in Burkina Faso. PCR could be considered as an alternative method for the identification and diagnosis of S. Typhi in blood samples.

A Multicountry Molecular Analysis of Salmonella enterica Serovar Typhi With Reduced Susceptibility to Ciprofloxacin in Sub-Saharan Africa.

BACKGROUND: Salmonella enterica serovar Typhi is a predominant cause of bloodstream infections in sub-Saharan Africa (SSA). Increasing numbers of S. Typhi with resistance to ciprofloxacin have been reported from different parts of the world. However, data from SSA are limited. In this study, we aimed to measure the ciprofloxacin susceptibility of S. Typhi isolated from patients with febrile illness in SSA. METHODS: Febrile patients from 9 sites within 6 countries in SSA with a body temperature of ≥38.0°C were enrolled in this study. Blood samples were obtained for bacterial culture, and Salmonella isolates were identified biochemically and confirmed by multiplex polymerase chain reaction (PCR). Antimicrobial susceptibility of all Salmonella isolates was performed by disk diffusion test, and minimum inhibitory concentrations (MICs) against ciprofloxacin were measured by Etest. All Salmonella isolates with reduced susceptibility to ciprofloxacin (MIC > 0.06 µg/mL) were screened for mutations in quinolone resistance-determining regions in target genes, and the presence of plasmid-mediated quinolone resistance (PMQR) genes was assessed by PCR. RESULTS: A total of 8161 blood cultures were performed, and 100 (1.2%) S. Typhi, 2 (<0.1%) Salmonella enterica serovar Paratyphi A, and 27 (0.3%) nontyphoid Salmonella (NTS) were isolated. Multidrug-resistant S. Typhi were isolated in Kenya (79% [n = 38]) and Tanzania (89% [n = 8]) only. Reduced ciprofloxacin-susceptible (22% [n = 11]) S. Typhi were isolated only in Kenya. Among those 11 isolates, all had a Glu133Gly mutation in the gyrA gene combined with either a gyrA (Ser83Phe) or gyrB mutation (Ser464Phe). One Salmonella Paratyphi A isolate with reduced susceptibility to ciprofloxacin was found in Senegal, with 1 mutation in gyrA (Ser83Phe) and a second mutation in parC (Ser57Phe). Mutations in the parE gene and PMQR genes were not detected in any isolate. CONCLUSIONS: Salmonella Typhi with reduced susceptibility to ciprofloxacin was not distributed homogenously throughout SSA. Its prevalence was very high in Kenya, and was not observed in other study countries. Continuous monitoring of antimicrobial susceptibility is required to follow the potential spread of antimicrobial-resistant isolates throughout SSA.

Validation and Identification of Invasive Salmonella Serotypes in Sub-Saharan Africa by Multiplex Polymerase Chain Reaction.

Salmonella enterica serovar Typhi and nontyphoidal Salmonella (NTS) cause the majority of bloodstream infections in sub-Saharan Africa; however, serotyping is rarely performed. We validated a multiplex polymerase chain reaction (PCR) assay with the White-Kauffmann-Le Minor (WKLM) scheme of serotyping using 110 Salmonella isolates from blood cultures of febrile children in Ghana and applied the method in other Typhoid Fever Surveillance in Africa Program study sites. In Ghana, 47 (43%) S. Typhi, 36 (33%) Salmonella enterica serovar Typhimurium, 14 (13%) Salmonella enterica serovar Dublin, and 13 (12%) Salmonella enterica serovar Enteritidis were identified by both multiplex PCR and the WKLM scheme separately. Using the validated multiplex PCR assay, we identified 42 (66%) S. Typhi, 14 (22%) S. Typhimurium, 2 (3%) S. Dublin, 2 (3%) S. Enteritidis, and 4 (6%) other Salmonella species from the febrile patients in Burkina Faso, Guinea-Bissau, Madagascar, Senegal, and Tanzania. Application of this multiplex PCR assay in sub-Saharan Africa could advance the knowledge of serotype distribution of Salmonella.