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Diarrhoea is an important cause of morbidity and mortality in children from low-income and middle-income countries (LMICs), despite advances in the management of this condition. Understanding of the causes of diarrhoea in children in LMICs has advanced owing to large multinational studies and big data analytics computing the disease burden, identifying the important variables that have contributed to reducing this burden. The advent of the mobile phone has further enabled the management of childhood diarrhoea by providing both clinical support to health-care workers (such as diagnosis and management) and communicating preventive measures to carers (such as breastfeeding and vaccination reminders) in some settings. There are still challenges in addressing the burden of diarrhoeal diseases, such as incomplete patient information, underrepresented geographical areas, concerns about patient confidentiality, unequal partnerships between study investigators, and the reactive approach to outbreaks. A transparent approach to promote the inclusion of researchers in LMICs could address partnership imbalances. A big data umbrella encompassing cloud-based centralised databases to analyse interlinked human, animal, agricultural, social, and climate data would provide an informative solution to the development of appropriate management protocols in LMICs.

Treatment outcomes among multidrug-resistant tuberculosis (MDR-TB) patients receiving ethambutol, cycloserine, or terizidone as part of a standardized regimen were compared, determining occurrence of serious adverse drug events (SADEs). Newly diagnosed adult MDR-TB patients were enrolled between 2000 and 2004, receiving a standardized multidrug regimen for 18 to 24 months, including ethambutol, cycloserine, or terizidone. Cycloserine and terizidone were recorded individually. SADEs and factors associated with culture conversion and unfavorable treatment outcomes (default, death, treatment failure) were determined. Of 858 patients, 435 (51%) received ethambutol, 278 (32%) received cycloserine, and 145 (17%) received terizidone. Demographic and baseline clinical data were comparable. Successful treatment occurred in 56%, significantly more in patients receiving cycloserine (60%) and terizidone (62%) than in those receiving ethambutol (52% [P = 0.03]). Defaults rates were 30% in ethambutol patients versus 15% and 11% for cycloserine and terizidone patients, respectively. Terizidone was associated with fewer unfavorable outcomes (adjusted odds ratio [AOR], 0.4; P = 0.008; 95% confidence interval [CI], 0.2 to 0.8). Patients receiving cycloserine were more likely to achieve culture conversion than those receiving ethambutol or terizidone (AOR, 2.2; P = 0.02; 95% CI, 1.12 to 4.38). Failure to convert increased the odds of unfavorable outcomes (AOR, 23.7; P < 0.001; 95% CI, 13 to 44). SADEs were reported in two patients receiving ethambutol, seven patients receiving cycloserine, and three receiving terizidone (P = 0.05). Ethambutol was associated with high culture conversion and default rates. Cycloserine achieved higher culture conversion rates than terizidone. Fewer patients on terizidone experienced SADEs, with lower default rates. The differences that we observed between cycloserine and terizidone require further elucidation.

Objectives Shigella sonnei is a globally important diarrhoeal pathogen tracked through the surveillance network PulseNet Latin America and Caribbean (PNLA&C), which participates in PulseNet International. PNLA&C laboratories use common molecular techniques to track pathogens causing foodborne illness. We aimed to demonstrate the possibility and advantages of transitioning to whole genome sequencing (WGS) for surveillance within existing networks across a continent where S. sonnei is endemic. Methods We applied WGS to representative archive isolates of S. sonnei (n = 323) from laboratories in nine PNLA&C countries to generate a regional phylogenomic reference for S. sonnei and put this in the global context. We used this reference to contextualise 16 S. sonnei from three Argentinian outbreaks, using locally generated sequence data. Assembled genome sequences were used to predict antimicrobial resistance (AMR) phenotypes and identify AMR determinants. Results S. sonnei isolates clustered in five Latin American sublineages in the global phylogeny, with many (46%, 149 of 323) belonging to previously undescribed sublineages. Predicted multidrug resistance was common (77%, 249 of 323), and clinically relevant differences in AMR were found among sublineages. The regional overview showed that Argentinian outbreak isolates belonged to distinct sublineages and had different epidemiologic origins. Conclusions Latin America contains novel genetic diversity of S. sonnei that is relevant on a global scale and commonly exhibits multidrug resistance. Retrospective passive surveillance with WGS has utility for informing treatment, identifying regionally epidemic sublineages and providing a framework for interpretation of prospective, locally sequenced outbreaks., Graphical abstract Image 1

Background Robust household sampling, commonly applied for population-based investigations, requires sampling frames or household lists to minimize selection bias. We have applied Google Earth Pro satellite imagery to constitute structure-based sampling frames at sites in Pikine, Senegal; Pietermaritzburg, South Africa; and Wad-Medani, Sudan. Here we present our experiences in using this approach and findings from assessing its applicability by determining positional accuracy. Methods Printouts of satellite imagery combined with Global Positioning System receivers were used to locate and to verify the locations of sample structures (simple random selection; weighted-stratified sampling). Positional accuracy was assessed by study site and administrative subareas by calculating normalized distances (meters) between coordinates taken from the sampling frame and on the ground using receivers. A higher accuracy in conjunction with smaller distances was assumed. Kruskal-Wallis and Dunn multiple pairwise comparisons were performed to evaluate positional accuracy by setting and by individual surveyor in Pietermaritzburg. Results The median normalized distances and interquartile ranges were 0.05 and 0.03–0.08 in Pikine, 0.09 and 0.05–0.19 in Pietermaritzburg, and 0.05 and 0.00–0.10 in Wad-Medani, respectively. Root mean square errors were 0.08 in Pikine, 0.42 in Pietermaritzburg, and 0.17 in Wad-Medani. Kruskal-Wallis and Dunn comparisons indicated significant differences by low- and high-density setting and interviewers who performed the presented approach with high accuracy compared to interviewers with poor accuracy. Conclusions The geospatial approach presented minimizes systematic errors and increases robustness and representativeness of a sample. However, the findings imply that this approach may not be applicable at all sites and settings; its success also depends on skills of surveyors working with aerial data. Methodological modifications are required, especially for resource-challenged sites that may be affected by constraints in data availability and area size.

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 program’s estimates of salmonellosis and other conditions associated with fever.

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.

BackgroundTuberculosis (TB) is amongst the top five causes of death in women of childbearing age (15-≤44 years). Little is known about treatment of pregnant women with drug-resistant TB (DR-TB). Treatment for pregnant women remains challenging and more complex in DR-TB/HIV co-infection, where an evidence-based guide to clinical practice is limited. The study reviewed treatment and pregnancy outcomes and birth outcomes of their new-born in a cohort of pregnant women with DR-TB from three MDR-TB hospitals during 2010 and 2018.Design/methodsData were extracted from: TB register and patient clinic notes using a standardized case record form. Information on DR-TB treatment, pregnancy and Adverse Drug Events (ADEs) of twenty-six pregnant women treated with individualized second-line TB medications were captured. The frequency of favourable and adverse outcomes regarding disease and pregnancy were evaluated.ResultsThe mean age was 29 years (SD ±5.1), with the minimum and maximum age of 21 and 40 years, respectively. Eleven (42.3%) were previously treated with first-line TB drugs, 11 (42.3%) never treated before and 4 (15.4%) were previously treated for DR-TB. Of the 26 women, 15 (57.7%) had at least one ADE, but most had more than one ADE. Seventeen women were successfully treated, and 22 live births recorded. Live birth outcome was significantly associated with trimester of initiation of DR-TB treatment (p = 0.036). The proportion of live births for the pregnancy trimester when DR-TB treatment was initiated, were 60.0%, 90.9% and 100.0%, for first, second and third trimester, respectively.ConclusionDR-TB treatment should be delayed until after the first trimester. Routine pharmacovigilance surveillance integrated antenatal and delivery services with an integrated record of DR-TB treatment during pregnancy is recommended. Prospective studies using standardised case record forms for DR-TB treatment for pregnant women could provide more insight on the effect of DR-TB treatment on the birth outcome.

BACKGROUND: Nontyphoidal salmonellosis continues to pose a global threat to human health, primarily by causing food-borne illnesses, and food-producing animals are the principal reservoirs of many pathogenic serovars. To identify key control points and generate information that may enable future estimation of the transmission routes between the environment, animals, and humans, we examined data on Salmonella isolates in South Africa. METHODS: Samples were obtained from livestock and poultry on farms, meat at abattoirs, raw materials at feed mills, animal feed, and environmental sources (eg, poultry houses, abattoirs, feed mills, water) from 2012 to 2014 in compliance with each establishment's protocols conforming to International Organization for Standardization (ISO) (ISO/TS 17728, ISO 18593:2004 and ISO 17604:2003) standards. Isolation and serotyping of Salmonella were performed according to the scope of accreditation of the respective laboratories conforming to ISO/IEC 17025:2005 standard techniques. RESULTS: Salmonella was isolated from 9031 of 180 298 (5.0%) samples, and these isolates were distributed among 188 different serovars. Salmonella Enteritidis was the most frequent isolate, with 1944 of 180 298 (21.5%) originating from poultry on farms, poultry meat, and poultry houses, followed by Salmonella Havana, with 677 of 180 298 (7.5%), mostly from environmental samples. Serovars that are uncommonly associated with human disease (Salmonella Idikan, Salmonella Salford, and Salmonella Brancaster) were isolated at higher frequencies than Salmonella Typhimurium, a common cause of human illness. Environmental samples accounted for 3869 of 9031 (42.8%) samples positive for Salmonella. CONCLUSIONS: We describe the frequent isolation of Salmonella of a wide variety of serovars, from an array of animal feeds, food animals, and food animal environment. As prevention of human salmonellosis requires the effective control of Salmonella in food animals, these data can be used to facilitate Salmonella control in food animals and thereby prevent human infections.

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 (

This disease is associated with approximately 3.4 million illnesses and 681,316 deaths, particularly in Africa., Nontyphoidal Salmonella is a major cause of bloodstream infections worldwide, and HIV-infected persons and malaria-infected and malnourished children are at increased risk for the disease. We conducted a systematic literature review to obtain age group–specific, population-based invasive nontyphoidal Salmonella (iNTS) incidence data. Data were categorized by HIV and malaria prevalence and then extrapolated by using 2010 population data. The case-fatality ratio (CFR) was determined by expert opinion consensus. We estimated that 3.4 (range 2.1–6.5) million cases of iNTS disease occur annually (overall incidence 49 cases [range 30–94] per 100,000 population). Africa, where infants, young children, and young adults are most affected, had the highest incidence (227 cases [range 152–341] per 100,000 population) and number of cases (1.9 [range 1.3–2.9] million cases). An iNTS CFR of 20% yielded 681,316 (range 415,164–1,301,520) deaths annually. iNTS disease is a major cause of illness and death globally, particularly in Africa. Improved understanding of the epidemiology of iNTS is needed.

Introduction: Salmonella is well recognized as an aetiological agent of gastrointestinal and diarrhoeal disease. Salmonella enterica serotype Enteritidis (Salmonella Enteritidis) is one of the commonest serotypes associated with foodborne illness. In South Africa, we compared Salmonella Enteritidis strains isolated from humans with gastroenteritis and strains isolated from captive wild animals, between June 2011 and July 2012. Methodology: Bacteria were phenotypically characterized using standard microbiological techniques. Genotypic relatedness of isolates was investigated by pulsed-field gel electrophoresis (PFGE) analysis. Results: a diversity of 27 PFGE patterns amongst 196 human non-invasive isolates was shown; two PFGE patterns predominated and accounted for 74% of all human isolates. Human isolates showed a 12% prevalence rate for nalidixic acid resistance. Animal isolates from 5 different sources were investigated. With the exception of an isolate from a ground hornbill, all animal isolates (jaguar, crocodile, lion and poultry) showed PFGE pattern matches to a human isolate. Animal isolates showed susceptibility to all antimicrobial agents tested, with the exception of nalidixic acid resistance in isolates from the lion and poultry source. Conclusions: Our data showed similarities between Salmonella Enteritidis strains isolated from humans and captive wild animals, suggesting a probable common source for strains from humans and animals.

Introduction: Guillain-Barré Syndrome (GBS) is an autoimmune disease characterized by acute or subacute symmetrical ascending motor weakness, areflexia, and mild-to-moderate sensory abnormalities. Campylobacter jejuni is reported to be the most common bacterium associated with GBS cases. Despite the eradication of polio, the number of reported GBS cases remains considerably high in South Africa with the causative agents not being well described. Methodology: The aim of the study was to investigate the proportion of Campylobacter spp. detected in stool specimens from patients with symptoms of acute flaccid paralysis (AFP). Stool specimens from patients presenting with AFP, that were negative for polio and non-polio enteroviruses (NPENT), were processed and screened for the presence of Campylobacter spp. using quantitative PCR (qPCR). Results: Of the 512 stool specimens screened between October 2014 to December 2015, 12% (62/512) were positive for Campylobacter spp. Of these 62 Campylobacter infections: 77.4% (48/62) was C. jejuni; 19.4% (12/62) was Campylobacter coli; 3.2% (2/62) was mixed infections of C. jejuni and C. coli. Conclusions: True association of the disease with Campylobacter spp. will enable the proportion of Campylobacter-induced GBS to be better described in South Africa; this can only be done through systematic studies that include bacterial culture and serology together with molecular methodologies.

Background Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) has become a significant pathogen in South Africa, and the need for improved molecular surveillance of this pathogen has become important. Over the years, multi-locus variable-number tandem-repeats analysis (MLVA) has become a valuable molecular subtyping technique for Salmonella, particularly for highly homogenic serotypes such as Salmonella Enteritidis. This study describes the use of MLVA in the molecular epidemiological investigation of outbreak isolates in South Africa. Methods Between the years 2013 and 2015, the Centre for Enteric Diseases (CED) received 39 Salmonella Enteritidis isolates from seven foodborne illness outbreaks, which occurred in six provinces. MLVA was performed on all isolates. Results Three MLVA profiles (MLVA profiles 21, 22 and 28) were identified among the 39 isolates. MLVA profile 28 accounted for 77% (30/39) of the isolates. Isolates from a single outbreak were grouped into a single MLVA profile. A minimum spanning tree (MST) created from the MLVA data showed a close relationship between MLVA profiles 21, 22 and 28, with a single VNTR locus difference between them. Conclusions MLVA has proven to be a reliable method for the molecular epidemiological investigation of Salmonella Enteritidis outbreaks in South Africa. These foodborne outbreaks emphasize the importance of the One Health approach as an essential component for combating the spread of zoonotic pathogens such as Salmonella Enteritidis.

Background Workers in clinical microbiology laboratories are exposed to a variety of pathogenic microorganisms. Salmonella species is among the most commonly reported bacterial causes of laboratory-acquired infections. We report on three cases of laboratory-acquired Salmonella enterica serotype Typhi (Salmonella Typhi) infection which occurred over the period 2012 to 2016 in South Africa. Methods Laboratory investigation included phenotypic and genotypic characterization of isolates. Phenotypic analysis included standard microbiological identification techniques, serotyping and antimicrobial susceptibility testing. Genotypic analysis included the molecular subtyping methodologies of pulsed-field gel electrophoresis analysis, multilocus sequence typing and whole-genome sequencing (WGS); with WGS data analysis including phylogenetic analysis based upon comparison of single nucleotide polymorphism profiles of isolates. Results All cases of laboratory-acquired infection were most likely the result of lapses in good laboratory practice and laboratory safety. The following critical issues were highlighted. There was misdiagnosis and misreporting of Salmonella Typhi as nontyphoidal Salmonella by a diagnostic laboratory, with associated public health implications. We highlight issues concerning the importance of accurate fluoroquinolone susceptibility testing and interpretation of results according to updated guidelines. We describe potential shortcomings of a single disk susceptibility screening test for fluoroquinolone susceptibility and suggest that confirmatory minimum inhibitory concentration testing should always be performed in cases of invasive Salmonella infections. These antimicrobial susceptibility testing issues resulted in inappropriate ciprofloxacin therapy which may have been responsible for failure in clearance of pathogen from patients. Salmonella Typhi capsular polysaccharide vaccine was not protective in one case, possibly secondarily to a faulty vaccine. Conclusions Molecular subtyping of isolates proved effective to investigate the genetic relatedness of isolates. Molecular subtyping data interpreted together with epidemiological data allowed us to pinpoint the most likely sources for our cases of laboratory-acquired infection.

SUMMARYPublic health interest in norovirus (NoV) has increased in recent years following improved diagnostics, global burden estimates and the development of NoV vaccine candidates. This study aimed to describe the detection rate, clinical characteristics and environmental features associated with NoV detection in hospitalized children P = 0·011) with none-to-mild dehydration (adjusted OR 0·5; 95% CI 0·3–0·7) compared with NoV-negative children. Amongst children testing NoV positive, HIV-infected children were more likely to have prolonged hospitalization and increased mortality compared with HIV-uninfected children. Continued surveillance will be important to consider the epidemic trends and estimate the burden and risk of NoV infection in SA.

Background A total of 720 Vibrio cholerae O1 strains were recovered for investigation from an outbreak of cholera in South Africa between November 2008 and April 2009. Methods Strains were characterized by serotype testing. Antimicrobial susceptibility testing. Genetic diversity of 248 strains was investigated using pulsed-field gel electrophoresis (PFGE) analysis. Extended characterization was performed on 90 strains. Molecular analysis included: polymerase chain reaction (PCR) identification of ctxA and tcpA genes, sequencing the ctxAB gene, and investigation of molecular mechanisms conferring antimicrobial resistance. Results The majority of strains were characterized as serotype Ogawa. Strains showed multidrug resistance. Approximately 1.0% of strains displayed extended-spectrum β-lactamase (ESBL) activity. Strains showed very similar PFGE patterns. Ninety strains selected for extended characterization showed the following results: Strains possessed the cholera toxin (CT) and all were PCR positive for the tcpA-El Tor variant. Sequencing of the ctxB gene matched the B-1 allele. Strains harbored the SXT element. Strains that displayed ESBL activity possessed a 140-kilobase-pair plasmid that produced the TEM-63 β-lactamase. Nalidixic acid-resistant strains harbored mutations in GyrA (Ser83-Ile) and ParC (Ser85-Leu). Conclusions These data highlight the rapid development of antimicrobial resistance and spread of V. cholerae O1 El Tor variants expressing the classical CT within South Africa.

Isolation of Vibrio cholerae O1 is necessary for cholera outbreak confirmation. Rapid diagnostic testing of fecal specimens, based on lipopolysaccharide detection of V. cholerae O1 or O139, may assist in early outbreak detection and surveillance. Cary-Blair transport medium is recommended for specimen transport. Filter paper, although used in epidemics, needs evaluation against rectal swab specimens. Fecal specimens are subcultured onto selective and nonselective media, including 5% blood agar and TCBS agar, for detection of V. cholerae O1 or O139. Suspicious, oxidase-positive isolates are serotyped in monovalent antisera. Antimicrobial-susceptibility testing is performed to detect resistance. Molecular characterization supports phenotypic identification and outbreak investigations. The presence of genes encoding cholera toxin, lipopolysaccharide, and El Tor biotype traits can be confirmed. Standardized pulsed-field gel electrophoresis analysis facilitates strain comparison. Quality management ensures reliability of results through validation and verification of functional laboratory equipment; quality control of testing procedures, laboratory reagents, and consumables; and participation in proficiency-testing schemes.

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Background Typhoid fever remains an important disease in Africa, associated with outbreaks and the emerging multidrug resistant Salmonella enterica serotype Typhi (Salmonella Typhi) haplotype, H58. This study describes the incidence of, and factors associated with mortality due to, typhoid fever in South Africa, where HIV prevalence is high. Methods and Findings Nationwide active laboratory-based surveillance for culture-confirmed typhoid fever was undertaken from 2003–2013. At selected institutions, additional clinical data from patients were collected including age, sex, HIV status, disease severity and outcome. HIV prevalence among typhoid fever patients was compared to national HIV seroprevalence estimates. The national reference laboratory tested Salmonella Typhi isolates for antimicrobial susceptibility and haplotype. Unadjusted and adjusted logistic regression analyses were conducted determining factors associated with typhoid fever mortality. We identified 855 typhoid fever cases: annual incidence ranged from 0.11 to 0.39 per 100,000 population. Additional clinical data were available for 369 (46.8%) cases presenting to the selected sites. Among typhoid fever patients with known HIV status, 19.3% (29/150) were HIV-infected. In adult females, HIV prevalence in typhoid fever patients was 43.2% (19/44) versus 15.7% national HIV seroprevalence (P < .001); in adult males, 16.3% (7/43) versus 12.3% national HIV seroprevalence (P = .2). H58 represented 11.9% (22/185) of Salmonella Typhi isolates tested. Increased mortality was associated with HIV infection (AOR 10.7; 95% CI 2.3–50.3) and disease severity (AOR 9.8; 95% CI 1.6–60.0) on multivariate analysis. Conclusions Typhoid fever incidence in South Africa was largely unchanged from 2003–2013. Typhoid fever mortality was associated disease severity. HIV infection may be a contributing factor. Interventions mandate improved health care access, including to HIV management programmes as well as patient education. Further studies are necessary to clarify relationships between HIV infection and typhoid fever in adults.

Background The burden of typhoid in sub-Saharan African (SSA) countries has been difficult to estimate, in part, due to suboptimal laboratory diagnostics. However, surveillance blood cultures at two sites in Nigeria have identified typhoid associated with Salmonella enterica serovar Typhi (S. Typhi) as an important cause of bacteremia in children. Methods A total of 128 S. Typhi isolates from these studies in Nigeria were whole-genome sequenced, and the resulting data was used to place these Nigerian isolates into a worldwide context based on their phylogeny and carriage of molecular determinants of antibiotic resistance. Results Several distinct S. Typhi genotypes were identified in Nigeria that were related to other clusters of S. Typhi isolates from north, west and central regions of Africa. The rapidly expanding S. Typhi clade 4.3.1 (H58) previously associated with multiple antimicrobial resistances in Asia and in east, central and southern Africa, was not detected in this study. However, antimicrobial resistance was common amongst the Nigerian isolates and was associated with several plasmids, including the IncHI1 plasmid commonly associated with S. Typhi. Conclusions These data indicate that typhoid in Nigeria was established through multiple independent introductions into the country, with evidence of regional spread. MDR typhoid appears to be evolving independently of the haplotype H58 found in other typhoid endemic countries. This study highlights an urgent need for routine surveillance to monitor the epidemiology of typhoid and evolution of antimicrobial resistance within the bacterial population as a means to facilitate public health interventions to reduce the substantial morbidity and mortality of typhoid., Author Summary Typhoid fever, a serious bloodstream infection caused by the bacterium Salmonella Typhi, is a major cause of disease and death around the world. There have been limited data on the epidemiology of typhoid in many countries in sub-Saharan African, including Nigeria. Recent evidence, however, showed that typhoid was an important cause of bacteraemia in children residing in two regions of Nigeria. Here, we analyzed the whole genome sequences of 128 S. Typhi isolates from two studies in order to elucidate the population structure and characterize the genetic components of antimicrobial resistance. We found that the multiple S. Typhi genotypes identified were closely related to other S. Typhi from neighboring regions of Africa and that multidrug resistance (MDR) was common among these isolates, and in many cases was associated with the IncHI1 plasmid known to cause MDR typhoid. These results provide evidence that typhoid was established in Nigeria as a result of several independent introductions into the country and that there has been extensive exchange of S. Typhi in and around the region of West Africa. This study emphasizes the importance of surveillance to improve our understanding of the epidemiology of typhoid, which is needed to underpin public health measures to reduce the spread of disease and facilitate patient management.

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. 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. 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. 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.

Although sapovirus (SaV) has been detected in 2.2-12.7% of gastroenteritis cases globally, there are limited data on SaV epidemiology.Describe the epidemiology, clinical characteristics and factors associated with SaV gastroenteritis in hospitalised children5 years of age in South Africa.Between 2009 and 2013 during prospective diarrhoeal surveillance, stool specimens were collected from four sites and screened for SaVs and associated enteric pathogens using ELISA, microscopy, conventional and real-time PCR. Epidemiological and clinical data were compared in patients with or without SaV. Odds ratios were assessed by bivariate and stepwise multivariable logistic regression analysis.Sapoviruses were detected in 7.7% (238/3103) of children admitted to hospital and 11.4% (9/79) of deaths. Sapovirus was detected more commonly in children 19-24 months compared to6months (aOR=2.3; p=0.018) and in males (aOR=2.0; p=0.001). Additional factors associated with SaV detection included residing with≥7 inhabitants compared to ≤3 (aOR=2.2; p=0.011) and concomitant norovirus infections (aOR=3.0; p=0.003). HIV-infected children with SaV were more likely to have bloody stools (aOR=16.8; p0.001), low birth weight (2.5kg; aOR=5.8; p=0.007) and live in environments without flush toilets (aOR=8.1; p=0.003) compared to HIV-uninfected children.Sapoviruses, which are perceived to cause mild diarrhoea, were detected in hospitalised children and diarrhoeal deaths in South Africa. Determinants increasing the odds of SaV included overcrowding and concomitant infections while HIV-infected children with SaV displayed bloody stools, low birth weight and reduced access to proper sanitation. Mitigation strategies against SaV infections include improved sanitation.

SUMMARYIn 2005, over 600 clinically diagnosed typhoid fever cases occurred in South Africa, where an outbreak had been previously described in 1993. Case-control and molecular investigations, including Salmonella enterica serovar Typhi (S. Typhi) isolates from that area from 1993, 2005 and later, were undertaken. Controls were significantly older than cases (P=0·003), possibly due to immunity from previous infection, and a significantly larger proportion had attended a gathering (P=0·035). Exposure to commercial food outlets and person-to-person transmission was not significant. Pulsed-field gel electrophoresis and multi-locus tandem repeat analysis revealed common clusters of S. Typhi strains identified in 1993 and 2005 as well as in 2007 and 2009. This outbreak probably occurred in a non-immune population due to faecally contaminated water. S. Typhi strains appeared to be related to strains from 1993; failure to address unsafe water may lead to further outbreaks in the area if the current population immunity wanes or is lost.

To determine the prevalence of invasive nontyphoid salmonellosis and typhoid fever in Malawi and South Africa, we compared case frequency and patient age distribution. Invasive nontyphoid salmonellosis showed a clear bimodal age distribution; the infection developed in women at a younger age than in men. Case frequency for typhoid fever was lower than for salmonellosis.

BACKGROUND: Non-typhoidal Salmonella are frequently food-borne zoonotic pathogens that may cause invasive disease in HIV-positive individuals. METHODOLOGY: Invasive isolates (n = 652) of Salmonella Typhimurium from human patients in Gauteng Province of South Africa were investigated for the years 2006 and 2007. Bacteria were identified using standard microbiological techniques and serotyping was performed using commercially available antisera. Susceptibility testing to antimicrobial agents was determined by the E-test. Genotypic relatedness of isolates was investigated by pulsed-field gel electrophoresis analysis of digested genomic DNA. RESULTS: Forty-five clusters of isolates were identified, of which four (clusters 3, 5, 6 and 11) were major clusters. Most isolates originated from hospital H2 and most were isolated from patients in the age range of 15 to 64 years. Ninety-three percent (213/230) of patients with a known HIV status were HIV-positive. Most isolates showed resistance to multiple antibiotics. The most commonly expressed antibiotic resistance profiles were ACSSuNa (14%; 75/555) and ACSSuTNa (13%; 72/555). Some evidence was found for nosocomial acquisition of isolates. Of the isolates from the major clusters 3, 5, 6, and 11, 33% (8/24), 6% (7/117), 4% (1/26) and 6% (3/52) were of nosocomial origin, respectively. CONCLUSIONS: In South Africa, Salmonella Typhimurium remains a major opportunistic infection of predominantly HIV-positive patients. Clonally diverse strains that are resistant to multiple antibiotics may circulate in patients aged between 15 and 64 years over prolonged periods within the hospital environment, adding to the health care burden.

The clinical and microbiological characteristics of nontyphoidal Salmonella (NTS) meningitis in South Africa, where human immunodeficiency virus (HIV) prevalence is high (approximately 15% in persons ≥15 years of age), were reviewed.From 2003 through 2013, 278 cases were identified through national laboratory-based surveillance. Clinical information (age, sex, outcome, Glasgow Coma Scale [GCS], and HIV status) was ascertained at selected sites. Isolates were serotyped; susceptibility testing and multilocus sequence typing on Salmonella enterica serovar Typhimurium isolates was performed. Multivariable logistic regression was used to determine factors associated with mortality outcome, using Stata software, version 13.Where age was ascertained, 139 of 256 (54.3%) patients were15 years. Males represented 151 of 267 (56.6%). Mortality outcome was recorded for 112 of 146 (76.7%) enhanced surveillance patients; 53 of 112 (47.3%) died. Death was associated with GCS ≤13 (adjusted odds ratio [OR], 18.7; 95% confidence interval [CI], 3.0-118.5; P = .002) on multivariable analysis. Where data were available, all 45 patients aged15 years were HIV infected, compared with 24 of 46 (52.2%) patients aged5 years. Neonates were less likely to be HIV infected than infants aged 2-12 months (OR, 4.8; 95% CI, 1.1-21.1; P = .039).Salmonella Typhimurium represented 106 of 238 (44.5%) serotyped isolates: 65 of 95 (68.4%) were ST313 vs ST19, respectively, and significantly associated with HIV-infected patients (P = .03) and multidrug resistance (OR, 6.6; 95% CI, 2.5-17.2; P.001).NTS meningitis in South Africa is highly associated with HIV in adults, with neonates (irrespective of HIV status), and with Salmonella Typhimurium ST313. GCS is the best predictor of mortality: early diagnosis and treatment are critical. Focused prevention requires further studies to understand the sources and transmission routes.

© 2015 Elsevier Ltd. Background: Shigellosis is an acute, severe bacterial colitis that, in high-income countries, is typically associated with travel to high-risk regions (Africa, Asia, and Latin America). Since the 1970s, shigellosis has also been reported as a sexually transmitted infection in men who have sex with men (MSM), in whom transmission is an important component of shigellosis epidemiology in high-income nations. We aimed to use sophisticated subtyping and international sampling to determine factors driving shigellosis emergence in MSM linked to an outbreak in the UK. Methods: We did a large-scale, cross-sectional genomic epidemiological study of shigellosis cases collected from 29 countries between December, 1995, and June 8, 2014. Focusing on an ongoing epidemic in the UK, we collected and whole-genome sequenced clinical isolates of Shigella flexneri serotype 3a from high-risk and low-risk regions, including cases associated with travel and sex between men. We examined relationships between geographical, demographic, and clinical patient data with the isolate antimicrobial susceptibility, genetic data, and inferred evolutionary relationships. Findings: We obtained 331 clinical isolates of S flexneri serotype 3a, including 275 from low-risk regions (44 from individuals who travelled to high-risk regions), 52 from high-risk regions, and four outgroup samples (ie, closely related, but genetically distinct isolates used to determine the root of the phylogenetic tree). We identified a recently emerged lineage of S flexneri 3a that has spread intercontinentally in less than 20 years throughout regions traditionally at low risk for shigellosis via sexual transmission in MSM. The lineage had acquired multiple antimicrobial resistance determinants, and prevailing sublineages were strongly associated with resistance to the macrolide azithromycin. Eight (4%) of 206 isolates from the MSM-associated lineage were obtained from patients who had previously provided an isolate; these serial isolations indicated atypical infection patterns (eg, reinfection). Interpretation: We identified transmission-facilitating behaviours and atypical course(s) of infection as precipitating factors in shigellosis-affected MSM. The intercontinental spread of antimicrobial-resistant shigella through established transmission routes emphasises the need for new approaches to tackle the public health challenge of sexually transmitted infections in MSM. Funding: Wellcome Trust (grant number 098051).

Salmonella spp. producing extended-spectrum beta-lactamases (ESBLs) have been reported in many countries, but there is no information on their prevalence in Africa. ESBL-producing Salmonella enterica serotype Isangi and S. enterica serotype Typhimurium strains have been noted in South Africa since 2001. A total of 160 consecutive isolates of Salmonella spp. were collected from 13 hospitals located in different cities in South Africa over a 5-month period from December 2002 to April 2003. All strains were screened for production of ESBLs by the double disk diffusion test and for AmpC production by assessing resistance to cefoxitin. bla SHV , bla TEM , bla CTX-M , and bla CMY-2 were sought from all ESBL-positive and cefoxitin-resistant isolates. A total of 15.6% (25 of 160) isolates produced SHV or TEM ESBLs, and 1.9% (3 of 160) produced CMY-2. Nine S. enterica serotype Typhimurium, eight S. enterica serotype Isangi, and three S. enterica serotype Muenchen strains produced either TEM-63 or a derivative of TEM-63 designated TEM-131. Both TEM-63 and TEM-131 have an isoelectric point of 5.6, and their sequences have the following amino acid substitutions compared to the TEM-1 sequence: Leu21Phe, Glu104Lys, Arg164Ser, and Met182Thr. Additionally, TEM-131 has an Ala237Thr substitution. ESBL-producing Salmonella spp. have become a significant public health problem in South Africa with particular implications for the treatment of serious nontyphoidal Salmonella infections in children, for whom extended-spectrum cephalosporins were the preferred treatment.

Background HIV-infected persons are at increased risk of opportunistic infections, including invasive nontyphoidal Salmonella (iNTS) infections; antiretroviral therapy (ART) reduces this risk. We explored changing iNTS incidence associated with increasing ART availability in South Africa. Methods Laboratory-based surveillance for iNTS was conducted in Gauteng Province, South Africa, with verification using the National Health Laboratory Service’s Central Data Warehouse (CDW), between 2003 and 2013. Isolates were serotyped at the Centre for Enteric Diseases. CDW data on patient numbers obtaining HIV viral load measurements provided estimates of numbers of HIV-infected patients receiving ART. A Poisson regression model was used to measure the changing incidence of iNTS infection from 2003 to 2013. The correlation between the incidence of iNTS and ART use from 2004 to 2013 was determined using Pearson’s correlation coefficient. Results From 2003–2013, the incidence of iNTS per 100,000 population per year decreased from 5.0 to 2.2 (p < .001). From 2004 to 2013, the incidence per 100,000 population of HIV viral load testing increased from 75.2 to 3,620.3 (p < .001). The most common serotypes causing invasive disease were Salmonella enterica serovar Typhimurium (Salmonella Typhimurium), and Salmonella Enteritidis: 2,469 (55.4%) and 1,156 (25.9%) of 4,459 isolates serotyped, respectively. A strong negative correlation was observed between decreasing iNTS incidence and increasing ART use from 2004 to 2013 (r = -0.94, p < .001). Similarly, decreasing incidence of invasive Salmonella Typhimurium infection correlated with increasing ART use (r = -0.93, p < .001). Incidence of invasive Salmonella Enteritidis infection increased, however (r = 0.95, p < .001). Between 2003 and 2004, fewer adult men than women presented with iNTS (male-to-female rate ratio 0.73 and 0.89, respectively). This was reversed from 2005 through 2013 (ranging from 1.07 in 2005 to 1.44 in 2013). Adult men accessed ART less (male-to-female rate ratio ranging from 0.61 [2004] to 0.67 [2013]). Conclusions The incidence of iNTS infections including Salmonella Typhimurium decreased significantly in Gauteng Province in association with increased ART utilization. Adult men accessed ART programs less than women, translating into increasing iNTS incidence in this group. Monitoring iNTS incidence may assist in monitoring the ART program. Increasing incidence of invasive Salmonella Enteritidis infections needs further elucidation.

The characteristics of Vibrio cholerae O1 biotype El Tor, serotype Ogawa isolates from outbreaks of cholera in 1998 amongst migrant workers in the South African provinces of Gauteng and Mpumalanga, on the border of Mozambique, are reported. The isolates seem to have originated from the same clone since they are of two closely related BglI ribotypes. These ribotypes had a high similarity to ribotypes of V. cholerae O1 recently found in three South-east Asian countries. Isolates were resistant to furazolidone, streptomycin, sulfamethoxazole, trimethoprim and tetracycline. Only two isolates contained plasmids of 54 and 63 kb in size. PCR and DNA sequencing revealed that the chromosomally located resistance determinants present included an aadA2 gene cassette contained in a class 1 integron; the SXT element, which is a transposon-like element containing resistance genes; and the tetA gene. A co-transfer of chromosomal closely located genes encoding the SXT element and tetA was shown by mating experiments, PCR and pulsed-field gel electrophoresis analyses. Our study shows for the first time that multiple-resistant V. cholerae O1 isolates containing class 1 integrons and the SXT element were responsible for cholera outbreaks in Southern Africa. Studies are needed to determine the spread of this multiple-resistant O1 strain and further genetic details of the association of the SXT element, tetA and class 1 integrons, including their means of transfer.

Background Drug resistance of Salmonella enterica serovar Typhi (Salmonella Typhi) to first-line antibiotics is emerging in Central Africa. Although increased use of fluoroquinolones is associated with spread of resistance, Salmonella Typhi with decreased ciprofloxacin susceptibility (DCS) has rarely been reported in Central Africa. Methodology/Principal Findings As part of a microbiological surveillance study in the Democratic Republic of the Congo (DR Congo), Salmonella Typhi isolates from bloodstream infections were collected prospectively between 2007 and 2011. The genetic relationship of the Salmonella Typhi isolates was assessed by pulsed-field gel electrophoresis (PFGE). The antimicrobial resistance profile of the isolates was determined and mutations associated with DCS were studied. In total, 201 Salmonella Typhi isolates were collected. More than half of the Salmonella Typhi isolates originated from children and young adults aged 5–19. Thirty different PFGE profiles were identified, with 72% of the isolates showing a single profile. Multidrug resistance, DCS and azithromycin resistance were 30.3%, 15.4% and 1.0%, respectively. DCS was associated with point mutations in the gyrA gene at codons 83 and 87. Conclusions/Significance Our study describes the first report of widespread multidrug resistance and DCS among Salmonella Typhi isolates from DR Congo. Our findings highlight the need for increased microbiological diagnosis and surveillance in DR Congo, being a prerequisite for rational use of antimicrobials and the development of standard treatment guidelines., Author Summary Typhoid fever, caused by infection with Salmonella enterica serovar Typhi (Salmonella Typhi), is an important health problem in sub-Saharan Africa. Multidrug resistance of Salmonella Typhi to the first line antibiotics is spreading and treatment of typhoid fever increasingly relies on fluoroquinolone antibiotics such as ciprofloxacin. Increased use of fluoroquinolones is however associated with spread of resistance as well. In sub-Saharan Africa, microbiological cultures to detect invasive bacterial diseases are frequently absent and the extent of the problem is poorly known. In the present study, 201 Salmonella Typhi isolates were collected between 2007 and 2011 in DR Congo, mainly from children and young adults. For the first time, widespread Salmonella Typhi multidrug resistance (30.3%) and decreased ciprofloxacin susceptibility (15.4%) is described in Central Africa. Decreased ciprofloxacin susceptibility was associated with point mutations in the quinolone resistance determining region of the gyrA gene. Resistance to azithromycin, an alternative for treatment of uncomplicated typhoid fever in the case of decreased ciprofloxacin susceptibility, was still rare (1.0%). Our findings demonstrate emergence of multidrug resistance and fluoroquinolone decreased susceptibility in DR Congo, and highlight the need for increased microbiological diagnosis and surveillance, being a prerequisite for rational use of antimicrobials and the development of standard treatment guidelines.

The publication of studies using next generation sequencing to analyse large numbers of bacterial isolates from global epidemics is transforming microbiology, epidemiology and public health. The emergence of multidrug resistant Salmonella Typhimurium ST313 is one example. While the epidemiology in Africa appears to be human-to-human spread and the association with invasive disease almost absolute, more needs to be done to exclude the possibility of animal reservoirs and to transfer the ability to track all Salmonella infections to the laboratories in the front line. In this mini-review we summarise what is currently known about non-typhoidal Salmonella in sub-Saharan Africa and discuss some of the issues which remain.

To determine the origin of >4,000 suspected diarrheagenic Escherichia coli strains isolated during 2004–2011 in South Africa, we identified 7 isolates as serotype O104; 5 as enteroaggregative E. coli O104:H4, and 2 as enteropathogenic E. coli O104:non-H4. Pulsed-field gel electrophoresis showed that these isolates were unrelated to the 2011 E. coli O104:H4 outbreak strain from Germany.

Background: Shigellosis is a global human health problem. The disease is most prevalent in developing countries with poor access to safe potable water and sanitation. Shigella boydii is of particular epidemiological importance in developing nations such as African and Asian countries. In the present study, we report on the analysis of a temporal cluster of 29 S. boydii serotype 2 strains, isolated in the Mpumalanga Province of South Africa (SA) over the period of November to December 2007. Methodology: Bacteria were identified as S. boydii using standard microbiological identification techniques and serotyped using commercially available antisera. Susceptibility testing to antimicrobial agents was determined by the Etest. Genotypic relatedness of strains was investigated by pulsed-field gel electrophoresis (PFGE) analysis of digested genomic DNA. Results: The cluster of 29 isolates revealed comparable antimicrobial susceptibility profiles, while dendrogram analysis of PFGE patterns showed that the cluster of isolates grouped together and could clearly be differentiated from a random selection of unrelated S. boydii serotype 2 strains. Our data has strongly suggested that this cluster of isolates may share a common ancestry. However, this cannot be substantiated by epidemiological data because a detailed epidemiological investigation was not conducted. Conclusions: We have documented the first cluster of S. boydii infection in SA. Due to the lack of adequate epidemiological investigation, we cannot emphatically state that an outbreak had occurred. However, we do hypothesis that this was an outbreak for which a waterborne source cannot be excluded. This study has highlighted the urgent need for timely and appropriate systems of epidemiological investigation of all suspected outbreaks of disease in developing countries.

SUMMARYWe report on the first recorded outbreak of cholera in Namibia. From December 2006 to February 2007, more than 250 cases of cholera were reported from the Omusati and Kunene provinces of Namibia. However, only nine bacterial isolates were obtainable for analysis. Isolates were all identified as Vibrio cholerae O1 serotype Inaba biotype El Tor. All isolates were susceptible to ampicillin, augmentin, chloramphenicol, nalidixic acid, ciprofloxacin, tetracycline, kanamycin, imipenem, ceftriaxone and ceftazidime; and they all showed resistance to trimethoprim, sulfamethoxazole and streptomycin. Pulsed-field gel electrophoresis analysis of bacteria incorporating either SfiI or NotI digestion revealed an identical fingerprint pattern for all isolates. These data together with results indicating identical antimicrobial susceptibility profiles for all isolates determined that the outbreak was caused by a single strain of V. cholerae.

Since May 2000, extended-spectrum beta-lactamase-producing (ESBL) Salmonella Isangi were isolated from pediatric patients at a tertiary hospital. A total of 41 patients with positive cultures were reviewed, and the majority presented with gastroenteritis, fever, or both. One ESBL phenotype was noted in all isolates, and clonality was confirmed by pulsed-field gel electrophoresis. This is the first report of Salmonella sp. ESBL resistance in our hospital.

To the Editor: Salmonella enterica serotype Typhi, the causal pathogen for typhoid, is a major public health hazard in many parts of the world, with an estimated 21.6 million cases of typhoid and 217,000 deaths occurring each year (1). Most isolates in South Africa are susceptible to quinolones, and fluoroquinolones remain the treatment of choice (2). The disease is primarily water or foodborne, but person-to-person spread is well recognized (3). Travelers to disease-endemic regions may be at risk for typhoid, which may result in the importation of strains of S. Typhi with unfamiliar or unusual resistance patterns (4). Such infections present a challenge to local clinicians on optimal patient management. S. Typhi was isolated from the blood culture of a woman 65 years of age from Cape Town; she had been in contact with a traveler to Bangladesh. The patient was treated first with ciprofloxacin, but this medication was changed to high-dose ceftriaxone combined with doxycycline for 8 days; she recovered well. Contact tracing indicated no family members had typhoid fever or carried the organism. The person who had traveled to Bangladesh was unavailable to provide further history or a stool specimen. No other potential source of infection could be elucidated: the patient lived in an urban area with safe water sources and shared meals with her family. The isolate was referred to the Enteric Diseases Reference Unit for confirmation of identification, serotyping (Kauffman-White scheme), and antimicrobial drug susceptibility testing using the Etest (bioMerieux, Marcy l’Etoile, France) and agar dilution methods, according to criteria of the Clinical and Laboratory Standards Institute (Wayne, PA, USA) (www.clsi.org). The isolate was resistant to ampicillin, chloramphenicol, sulfamethoxazole, nalidixic acid, and ciprofloxacin, but susceptible to ceftriaxone and tetracycline. Pulsed-field gel electrophoresis (PFGE) analysis was performed on the isolate, following the standard PulseNet protocol (5). The PFGE pattern was compared with a database of S. Typhi PFGE patterns from South Africa by using BioNumerics version 6.01 software (Applied Maths, Sint-Martens-Latem, Belgium). The PFGE pattern of this isolate was 100% identical to pattern JPPX01.0026 in the Global PulseNet Salmonella Typhi Database. This pattern, which has been reported to PulseNet from India, Kenya, Tanzania, and Taiwan, is the most common pattern in the database (www.pulsenetinternational.org/projects/styphidatabase.asp); it is rarely seen in South Africa (Figure). Figure Dendrogram of pulsed-field gel electrophoresis patterns representative of the 15 largest clusters of Salmonella enterica serotype Typhi isolates identified in South Africa during 2005–2009. The pattern of isolate 372460 is indicated. Scale bar ... PCR was used to isolate the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, and parE (6). Genes were sequenced by using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and an Applied Biosystems 3130 genetic analyzer. The QRDR DNA sequences were compared with those of S. Typhi strain Ty2 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AE014613","term_id":"29140506","term_text":"AE014613"}}AE014613). PCR also used to confirm the presence of qnrA, qnrB, and qnrS genes (6). Analysis for mutations in the QRDR of gyrA, gyrB, parC, and par found a single amino-acid mutation (Ser83 to Tyr) in gyrA. No amino acid mutations were identified in gyrB, parC, and parE. PCR for detection of qnr genes confirmed the presence of a qnrS gene, which was identified as the qnrS1 variant by nucleotide sequence analysis. The efflux of quinolones from bacterial cells was investigated in the following manner. For nalidixic acid and ciprofloxacin, agar dilution MIC testing was performed in the absence and presence of 40 µg/mL of the efflux pump inhibitor, Phe-Arg-β-naphthylamide (6). In the presence of efflux pump inhibitor, the MIC to ciprofloxacin decreased from 4 µg/mL to 1 µg/mL, and the MIC to nalidixic acid decreased from >512 µg/mL to 32 µg/mL, establishing the involvement of an efflux pump in conferring quinolone resistance. The mutation in gyrA (Ser83 to Tyr) confers reduced susceptibility to ciprofloxacin to a maximum MIC ≈0.5 µg/mL (7) and the QnrS1 protein confers reduced susceptibility to ciprofloxacin to a maximum MIC ≈0.5 µg/mL (8). We showed that a single amino acid mutation in gyrA (Ser83 to Tyr) with the QnrS1 protein and active efflux, conferred ciprofloxacin resistance, at least to an MIC level of 4 µg/mL. Previously, Smith et al. reported quinolone resistance in South African isolates of S. Typhi mediated by mutations in gyrA and parC in combination with active efflux (6). We report qnrS1 from S. Typhi, confirming the role of plasmid-mediated fluoroquinolone resistance in S. Typhi (9) and a fluoroquinolone- resistant strain in South Africa. Fluoroquinolone resistance is well recognized in Bangladesh; other researchers have described multidrug-resistant S. Typhi isolates imported from that country (10). Molecular epidemiology supports the conclusion that this strain likely originated in Bangladesh (L. Theobald, pers.comm.). In conclusion, fluoroquinolone-resistant typhoid fever is a reality in South Africa in patients who have a history of travel or contact with travelers. Blood cultures are mandatory to guide antimicrobial drug management. Plasmid-mediated fluoroquinolone resistance has implications for cotransference of resistance to the major antimicrobial agents used to treat typhoid fever and for the potential for rapid spread of fluoroquinolone resistance through S. Typhi strains in South Africa. The presence of fluoroquinolone-resistant typhoid fever could force a change in current treatment guidelines for this disease.

Between 10 and 11 years after children were vaccinated with Vi capsular polysaccharide of Salmonella typhi or meningococcal A + C control vaccine in a double blind randomized trial, we traced 83 subjects, aged 16-20 years. A blood sample was taken for determination of Vi antibody titres in both groups by radioimmunoassay. TO and TH titres were also done to assess if the participants had had recent exposure to typhoid fever. Fifty-eight percent of subjects in both groups had protective levels of Vi antibody against Salmonella typhi (a titre greater than 1 microgram ml-1). There was no significant difference in the levels of Vi antibodies in the cases versus the controls (p = 0.5). Two of the children who had received meningococcal A + C vaccine had recently had typhoid fever. Our data show that adolescents in typhoid endemic areas have high levels of Vi antibodies regardless of previous vaccination status, suggesting that Vi antibodies are acquired in adolescence by a large percentage of the population in this area. Moreover, Vi vaccination has led to ongoing antibody production in greater than 50% of Vi vaccinated children in an endemic area for a period of 10 years. Ongoing antigenic exposure may have contributed to these antibody levels.