Amy Gassama Sow

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

Summary 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.

Carbapenem Resistance and Acinetobacter baumannii in Senegal: The Paradigm of a Common Phenomenon in Natural Reservoirs

Incidence of carbapenem-resistant Acinetobacter baumannii is rising in several parts of the world. In Africa, data concerning this species and its resistance to carbapenems are limited. The objective of the present study was to identify the presence of A. baumannii carbapenem-resistant encoding genes in natural reservoirs in Senegal, where antibiotic pressure is believed to be low. From October 2010 to January 2011, 354 human head lice, 717 human fecal samples and 118 animal fecal samples were screened for the presence of A. baumannii by real time PCR targeting bla OXA51-like gene. For all samples positive for A. baumannii, the carbapenemase-hydrolysing oxacillinases blaOXA23-like and blaOXA24-like were searched for and sequenced, and the isolates harbouring an oxacillinase were genotyped using PCR amplification and sequencing of recA gene. The presence of A. baumannii was detected in 4.0% of the head lice, in 5.4% of the human stool samples and in 5.1% of the animal stool samples tested. No bla OXA24 gene was detected but six fecal samples and three lice were positive for bla OXA23-like gene. The bla OXA23-like gene isolated in lice was likely a new oxacillinase sequence. Finally, the A. baumannii detected in stools were all of recA genotype 3 and those detected in lice, of recA genotype 4. This study shows for the first time a reservoir of bla OXA23-like-positive gene in human head lice and stool samples in Senegal.

The Typhoid Fever Surveillance in Africa Program (TSAP): Clinical, Diagnostic, and Epidemiological Methodologies.

BACKGROUND New immunization programs are dependent on data from surveillance networks and disease burden estimates to prioritize target areas and risk groups. Data regarding invasive Salmonella disease in sub-Saharan Africa are currently limited, thus hindering the implementation of preventive measures. The Typhoid Fever Surveillance in Africa Program (TSAP) was established by the International Vaccine Institute to obtain comparable incidence data on typhoid fever and invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa through standardized surveillance in multiple countries. METHODS Standardized procedures were developed and deployed across sites for study site selection, patient enrolment, laboratory procedures, quality control and quality assurance, assessment of healthcare utilization and incidence calculations. RESULTS Passive surveillance for bloodstream infections among febrile patients was initiated at thirteen sentinel sites in ten countries (Burkina Faso, Ethiopia, Ghana, Guinea-Bissau, Kenya, Madagascar, Senegal, South Africa, Sudan, and Tanzania). Each TSAP site conducted case detection using these standardized methods to isolate and identify aerobic bacteria from the bloodstream of febrile patients. Healthcare utilization surveys were conducted to adjust population denominators in incidence calculations for differing healthcare utilization patterns and improve comparability of incidence rates across sites. CONCLUSIONS By providing standardized data on the incidence of typhoid fever and iNTS disease in sub-Saharan Africa, TSAP will provide vital input for targeted typhoid fever prevention programs.

Global phylogeography and evolutionary history of Shigella dysenteriae type 1.

Together with plague, smallpox and typhus, epidemics of dysentery have been a major scourge of human populations for centuries1. A previous genomic study concluded that Shigella dysenteriae type 1 (Sd1), the epidemic dysentery bacillus, emerged and spread worldwide after the First World War, with no clear pattern of transmission2. This is not consistent with the massive cyclic dysentery epidemics reported in Europe during the eighteenth and nineteenth centuries1,3,4 and the first isolation of Sd1 in Japan in 18975. Here, we report a whole-genome analysis of 331 Sd1 isolates from around the world, collected between 1915 and 2011, providing us with unprecedented insight into the historical spread of this pathogen. We show here that Sd1 has existed since at least the eighteenth century and that it swept the globe at the end of the nineteenth century, diversifying into distinct lineages associated with the First World War, Second World War and various conflicts or natural disasters across Africa, Asia and Central America. We also provide a unique historical perspective on the evolution of antibiotic resistance over a 100-year period, beginning decades before the antibiotic era, and identify a prevalent multiple antibiotic-resistant lineage in South Asia that was transmitted in several waves to Africa, where it caused severe outbreaks of disease.

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.

Description of an unusual class 2 integron in Shigella sonnei isolates in Senegal (sub-Saharan Africa)

Sir, Integrons are genetic elements that acquire gene cassettes by integrase-catalysed site-specific recombination. Integrons contain various combinations of gene cassettes encoding antibiotic resistance determinants and are widespread among Gram-negative bacteria. Five classes of integrons have been described to date, based on the integrase coding sequence. Classes 1 and 2 are the most frequent. Class 2 was found in transposon Tn7 and its derivatives (Tn1825, Tn1826 and Tn4132), and its 30 segment contains five tns genes involved in transposon movements. The intI2 integrase gene of class 2 integrons contains a premature stop codon at position 179, rendering the integrase non-functional. Therefore, class 2 integrons usually contain the same array of four gene cassettes, three antibiotic resistance gene cassettes (dfrA1, sat and aadA1, conferring resistance to trimethoprim, streptothricin and spectinomycin/streptomycin, respectively) and an orfX of unknown function. However, variations of the cassette contents have been described, resulting from Intl1 integrase-catalysed co-integrate formation between a class 1 and a class 2 integron, or from an RecA-dependent homologous recombination between two copies of the same cassette found in both classes of integron. Indeed, the dfrA1 and aadA1 cassettes, initially described in class 2 integrons, have also been found in class 1 integrons. Class 1 and 2 integrons have frequently been found in Shigella strains, and class 2 integrons predominate in Shigella sonnei and Shigella flexneri. – 5 We have previously described integron structures in S. sonnei strains isolated in Senegal; they included a class 2 integron of 4 kb found in two epidemiologically related isolates. Here, we describe the organization of this class 2 integron. The following primers were used for PCR mapping of the class 2 integron: hep74 in attI2, ORFX1 (CTCGTACTTG CGATGGCATC) in ORFX, and int2CS2 and int7S, both located in tnsE. Amplification was performed with the Expand Long Template PCR System (Roche Molecular Biochemicals, Roche Diagnostics GmbH, Germany). With primers hep74 and ORFX1, agarose gel electrophoresis identified a 4074 bp DNA fragment (data not shown) in both isolates, instead of the expected 3095 bp product. No PCR product was obtained with primers int2CS2 and int7S, suggesting a different arrangement in the 30 region of this class 2 integron. The entire class 2 integron was amplified with primer int2S in intI2 and with primer ORFX3 located in Tn7, downstream of the ORFX cassette. A PCR product of 4174 bp was purified with the QIAquick kit (Qiagen SA, Courtaboeuf, France), cloned with the pGEM-T vector system (Promega, Madison, WI, USA), transformed into XL1-Blue competent cells (Stratagene, Garden Grove, CA, USA) and sequenced. Sequence analysis of the 4174 bp insert of the recombinant plasmid showed that this integron harboured the four gene cassettes usually found in class 2 integrons, namely dfrA1, sat1, aadA1 and orfX (accession no. EU732664). However, the sat1 cassette was interrupted at position 186 by a complete copy of the insertion sequence IS911, a member of the IS3 family. IS911 was initially isolated from Shigella dysenteriae, but has since been detected (10–20 copies per cell) in the four Shigella species. As usually described for IS911, its insertion generated a 3 bp duplication (TAT) in the target sat1 sequence. To our knowledge, two ISs have previously been described in class 2 integrons, but not within the array of gene cassettes. Dubois et al. described a class 2 integron with an IS630 element in a strain of S. flexneri, and Biskri and Mazel described a class 2 integron with an IS1 in a strain of Escherichia coli. Both were located between intI2 and the first gene cassette. To determine whether the resistance determinants carried by the integron were transferable, we performed a conjugation experiment on MH agar plates from S. sonnei to an E. coli strain resistant to nalidixic acid. We used a selective medium containing 50 mg/L nalidixic acid plus 100 mg/L trimethoprim. No transconjugants were obtained, suggesting the chromosomal location of this Tn7-containing class 2 integron as previously described in Shigella isolates. Pan et al. described a class 2 integron on transposon Tn7 inserted into the Tn7-specific target attTn7 of the S. sonnei chromosome downstream of the glmUS operon. Thus, to confirm the chromosomal insertion of Tn7, we successfully performed a PCR with primers Tn7R-F and glmS-R as described by Pan et al. Another feature of this atypical class 2 integron is the deletion of the tns genes, described in Tn7 and its derivatives. A class 2 integron lacking the tns genes has previously been described in Acinetobacter baumannii, but the 30 segment of the class 2 integron contained the ORFs found in the 30 segment of class 1 integrons (qacED1, sul1 and ORF5). The PCR method previously used to detect such a chimeric integron with primers aadAL and ORFR was unsuccessful, suggesting that the class 2 integron that we describe here is not a hybrid class 2/class 1 integron. In conclusion, we describe an unusual class 2 integron in a S. sonnei strain isolated in Africa. This new class 2 integron is characterized by the presence of one complete copy of the IS911 element inserted within the sat1 gene cassette. Journal of Antimicrobial Chemotherapy 62, 843–851 doi:10.1093/jac/dkn264 Advance Access publication 18 June 2008

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.

Utilization of Healthcare in the Typhoid Fever Surveillance in Africa Program

BACKGROUND Assessing healthcare utilization is important to identify weaknesses of healthcare systems, to outline action points for preventive measures and interventions, and to more accurately estimate the disease burden in a population. METHODS A healthcare utilization survey was developed for the Typhoid Fever Surveillance in Africa Program (TSAP) to adjust incidences of salmonellosis determined through passive, healthcare facility-based surveillance. This cross-sectional survey was conducted at 11 sites in 9 sub-Saharan African countries. Demographic data and healthcare-seeking behavior were assessed at selected households. Overall and age-stratified percentages of each study population that sought healthcare at a TSAP healthcare facility and elsewhere were determined. RESULTS Overall, 88% (1007/1145) and 81% (1811/2238) of the population in Polesgo and Nioko 2, Burkina Faso, respectively, and 63% (1636/2590) in Butajira, Ethiopia, sought healthcare for fever at any TSAP healthcare facility. A far smaller proportion-namely, 20%-45% of the population in Bissau, Guinea-Bissau (1743/3885), Pikine, Senegal (1473/4659), Wad-Medani, Sudan (861/3169), and Pietermaritzburg, South Africa (667/2819); 18% (483/2622) and 9% (197/2293) in Imerintsiatosika and Isotry, Madagascar, respectively; and 4% (127/3089) in Moshi, Tanzania-sought healthcare at a TSAP healthcare facility. Patients with fever preferred to visit pharmacies in Imerintsiatosika and Isotry, and favored self-management of fever in Moshi. Age-dependent differences in healthcare utilization were also observed within and across sites. CONCLUSIONS Healthcare utilization for fever varied greatly across sites, and revealed that not all studied populations were under optimal surveillance. This demonstrates the importance of assessing healthcare utilization. Survey data were pivotal for the adjustment of the programs estimates of salmonellosis and other conditions associated with fever.

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.

Bloodstream Infections and Frequency of Pretreatment Associated With Age and Hospitalization Status in Sub-Saharan Africa.

BACKGROUND The clinical diagnosis of bacterial bloodstream infections (BSIs) in sub-Saharan Africa is routinely confused with malaria due to overlapping symptoms. The Typhoid Surveillance in Africa Program (TSAP) recruited febrile inpatients and outpatients of all ages using identical study procedures and enrollment criteria, thus providing an opportunity to assess disease etiology and pretreatment patterns among children and adults. METHODS Inpatients and outpatients of all ages with tympanic or axillary temperatures of ≥38.0 or ≥37.5°C, respectively, and inpatients only reporting fever within the previous 72 hours were eligible for recruitment. All recruited patients had one blood sample drawn and cultured for microorganisms. Data from 11 TSAP surveillance sites in nine different countries were used in the analysis. Bivariate analysis was used to compare frequencies of pretreatment and BSIs in febrile children (<15 years old) and adults (≥15 years old) in each country. Pooled Cochran Mantel-Haenszel odds ratios (ORs) were calculated for overall trends. RESULTS There was no significant difference in the odds of a culture-proven BSI between children and adults among inpatients or outpatients. Among both inpatients and outpatients, children had significantly higher odds of having a contaminated blood culture compared with adults. Using country-pooled data, child outpatients had 66% higher odds of having Salmonella Typhi in their bloodstream than adults (OR, 1.66; 95% confidence interval [CI], 1.01-2.73). Overall, inpatient children had 59% higher odds of pretreatment with analgesics in comparison to inpatient adults (OR, 1.59; 95% CI, 1.28-1.97). CONCLUSIONS The proportion of patients with culture-proven BSIs in children compared with adults was similar across the TSAP study population; however, outpatient children were more likely to have Salmonella Typhi infections than outpatient adults. This finding points to the importance of including outpatient facilities in surveillance efforts, particularly for the surveillance of typhoid fever. Strategies to reduce contamination among pediatric blood cultures are needed across the continent to prevent the misdiagnosis of BSI cases in children.