Detection of Salmonella Typhi nucleic acid by RT-PCR and anti-HlyE, -CdtB, -PilL, and -Vi IgM by ELISA at sites in Ghana, Madagascar and Ethiopia.

BACKGROUND: We aimed to assess the prevalence of Salmonella Typhi through DNA and IgM-antibody detection methods as a prelude to extended surveillance activities at sites in Ghana, Madagascar, and Ethiopia. METHODS: We performed species-specific real-time polymerase reaction (RT-PCR) to identify bacterial nucleic acid, and enzyme-linked immunosorbent assay (ELISA) for detecting HlyE/STY1498-, CdtB/STY1886-, pilL/STY4539- and Vi-antigens in blood and biopsy specimens of febrile and non-febrile subjects. We generated antigen-specific ELISA proxy cut-offs by change-point analyses, and utilized cumulative sum as detection method coupled with 1000 repetitive bootstrap analyses. We computed prevalence rates in addition to odds ratios to assess correlations between ELISA outcomes and participant characteristics. RESULTS: Definitive positive RT-PCR results were obtained from samples of febrile subjects originating from Adama Zuria/Ethiopia (1.9%, 2/104), Wolayita Sodo/Ethiopia (1.0%, 1/100), Diego/Madagascar (1.0%, 1/100), and Kintampo/Ghana (1.0%, 1/100), and from samples of non-febrile subjects from Wolayita Sodo/Ethiopia (1%, 2/201). While IgM antibodies against all antigens were identified across all sites, prevalence rates were highest at all Ethiopian sites, albeit in non-febrile populations. Significant correlations in febrile subjects aged < 15 years versus ≥ 15 years were detected for Vi (Odds Ratio (OR): 8.00, p = 0.034) in Adama Zuria/Ethiopia, STY1498 (OR: 3.21, p = 0.008), STY1886 (OR: 2.31, p = 0.054) and STY4539 (OR: 2.82, p = 0.022) in Diego/Madagascar, and STY1498 (OR: 2.45, p = 0.034) in Kintampo/Ghana. We found statistical significance in non-febrile male versus female subjects for STY1498 (OR: 1.96, p = 0.020) in Adama Zuria/Ethiopia, Vi (OR: 2.84, p = 0.048) in Diego/Madagascar, and STY4539 (OR: 0.46, p = 0.009) in Kintampo/Ghana. CONCLUSIONS: Findings indicate non-discriminatory stages of acute infections, though with site-specific differences. Immune responses among non-febrile, presumably healthy participants may mask recall and/or reporting bias leading to misclassification, or asymptomatic, subclinical infection signs induced by suppression of inflammatory responses. As most Ethiopian participants were ≥ 15 years of age and not at high-risk, the true S. Typhi burden was likely missed. Change-point analyses for generating ELISA proxy cut-offs appeared robust, though misclassification is possible. Our findings provided important information that may be useful to assess sites prior to implementing surveillance for febrile illness including Salmonella disease.

A retrospective review of vaccine wastage and associated risk factors in the Littoral region of Cameroon during 2016-2017.

BACKGROUND: Immunization is an effective preventive health intervention. In Cameroon, the Expanded Program on Immunization (EPI) aims to vaccinate children under 5 years of age for free, but vaccination coverage has consistently remained below the national target. Vaccines are distributed based on the target population size, factoring in wastage norms. However, the vaccine wastage rate (VWR) may differ among various settings. Our study aimed to assess vaccine wastage for different site settings, seasonality, and vaccine types in comparison to vaccination coverage in order to provide comprehensive insights on vaccine wastage. METHODS: A retrospective data collection and analysis were conducted on immunization and vaccine wastage data in the Littoral Region of Cameroon during 2016 and 2017. Health districts were classified as urban or rural, seasonality was categorized as rainy or dry season, and vaccine types were grouped into liquid, lyophilized, oral, and injectable vaccines. VWRs and vaccination coverage rates (VCRs) were calculated, and the vaccine waste factor was investigated. RESULTS: The VWR of Bacillus Calmette-Guérin (BCG; 32.19%) was the highest, followed by measles and rubella (MR; 19.05%) and yellow fever (YF; 18.34%) among all EPI vaccines in the Littoral Region of Cameroon during 2016 and 2017. Single-dose vaccine vials exhibited lower VWRs than multi-dose vials. Dry season was associated with higher VWRs for most vaccines, although more lyophilized vaccines (BCG, MR, YF vaccines) were wasted in rainy season in 2016. The VWR was persistently higher in rural than urban health districts. The months of February and November saw a decrease in VCRs. The study found an overall negative correlation between VCR and VWR. CONCLUSIONS: Multiple factors may cause wastage of EPI vaccines in Cameroon. Vaccination area characteristics, seasonality, types of vaccines such as multi- or single-dose, lyophilized or injectable vaccines are related to VWRs in Littoral Region. Further research on vaccine wastage and vaccination coverage across Cameroon is needed to better understand the socio-behavioral aspect of vaccine in-take that may affect the level of vaccination and vaccine wastage. Public health system strengthening is warranted to adapt more real-time monitoring of the VWR and VCR for each vaccine in the government's immunization programs.

Spatial and Temporal Patterns of Typhoid and Paratyphoid Fever Outbreaks: A Worldwide Review, 1990-2018.

BACKGROUND: Analyses of the global spatial and temporal distribution of enteric fever outbreaks worldwide are important factors to consider in estimating the disease burden of enteric fever disease burden. METHODS: We conducted a global literature review of enteric fever outbreak data by systematically using multiple databases from 1 January 1990 to 31 December 2018 and classified them by time, place, diagnostic methods, and drug susceptibility, to illustrate outbreak characteristics including spatial and temporal patterns. RESULTS: There were 180 940 cases in 303 identified outbreaks caused by infection with Salmonella enterica serovar Typhi (S. Typhi) and Salmonella enterica serovar Paratyphi A or B (S. Paratyphi). The size of outbreak ranged from 1 to 42 564. Fifty-one percent of outbreaks occurred in Asia, 15% in Africa, 14% in Oceania, and the rest in other regions. Forty-six percent of outbreaks specified confirmation by blood culture, and 82 outbreaks reported drug susceptibility, of which 54% had multidrug-resistant pathogens. Paratyphoid outbreaks were less common compared to typhoid (22 vs 281) and more prevalent in Asia than Africa. Risk factors were multifactorial, with contaminated water being the main factor. CONCLUSIONS: Enteric fever outbreak burden remains high in endemic low- and middle-income countries and, despite its limitations, outbreak data provide valuable contemporary evidence in prioritizing resources, public health policies, and actions. This review highlights geographical locations where urgent attention is needed for enteric fever control and calls for global action to prevent and contain outbreaks.

A Multicenter Cost-of-Illness and Long-term Socioeconomic Follow-up Study in the Severe Typhoid Fever in Africa Program: Study Protocol.

BACKGROUND: There are limited data on typhoid fever cost of illness (COI) and economic impact from Africa. Health economic data are essential for measuring the cost-effectiveness of vaccination or other disease control interventions. Here, we describe the protocol and methods for conducting the health economic studies under the Severe Typhoid Fever in Africa (SETA) program. METHODS: The SETA health economic studies will rely on the platform for SETA typhoid surveillance in 4 African countries-Burkina Faso, Ethiopia, Ghana, and Madagascar. A COI and long-term socioeconomic study (LT-SES) will be its components. The COI will be assessed among blood culture-positive typhoid fever cases, blood culture-negative clinically suspected cases (clinical cases), and typhoid fever cases with pathognomonic gastrointestinal perforations (special cases). Repeated surveys using pretested questionnaires will be used to measure out-of-pocket expenses, quality of life, and the long-term socioeconomic impact. The cost of resources consumed for diagnosis and treatment will be collected at health facilities. RESULTS: Results from these studies will be published in peer-reviewed journals and presented at scientific conferences to make the data available to the wider health economics and public health research communities. CONCLUSIONS: The health economic data will be analyzed to estimate the average cost per case, the quality of life at different stages of illness, financial stress due to illness, and the burden on the family due to caregiving during illness. The data generated are expected to be used in economic analysis and policy making on typhoid control interventions in sub-Saharan Africa.

Acute Febrile Illness Among Children in Butajira, South-Central Ethiopia During the Typhoid Fever Surveillance in Africa Program.

BACKGROUND: Clearly differentiating causes of fever is challenging where diagnostic capacities are limited, resulting in poor patient management. We investigated acute febrile illness in children aged ≤15 years enrolled at healthcare facilities in Butajira, Ethiopia, during January 2012 to January 2014 for the Typhoid Fever Surveillance in Africa Program. METHODS: Blood culture, malaria microscopy, and blood analyses followed by microbiological, biochemical, and antimicrobial susceptibility testing of isolates were performed. We applied a retrospectively developed scheme to classify children as malaria or acute respiratory, gastrointestinal or urinary tract infection, or other febrile infections and syndromes. Incidence rates per 100 000 population derived from the classification scheme and multivariate logistic regression to determine fever predictors were performed. RESULTS: We rarely observed stunting (4/513, 0.8%), underweight (1/513, 0.2%), wasting (1/513, 0.2%), and hospitalization (21/513, 4.1%) among 513 children with mild transient fever and a mean disease severity score of 12 (95% confidence interval [CI], 11-13). Blood cultures yielded 1.6% (8/513) growth of pathogenic agents; microscopy detected 13.5% (69/513) malaria with 20 611/µL blood (95% CI, 15 352-25 870) mean parasite density. Incidences were generally higher in children aged ≤5 years than >5 to ≤15 years; annual incidences in young children were 301.3 (95% CI, 269.2-337.2) for malaria and 1860.1 (95% CI, 1778.0-1946.0) for acute respiratory and 379.9 (95% CI, 343.6-420.0) for gastrointestinal tract infections. CONCLUSIONS: We could not detect the etiological agents in all febrile children. Our findings may prompt further investigations and the reconsideration of policies and frameworks for the management of acute febrile illness.

The Typhoid Fever Surveillance in Africa Program: Geospatial Sampling Frames for Household-based Studies: Lessons Learned From a Multicountry Surveillance Network in Senegal, South Africa, and Sudan.

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.

The Monitoring and Evaluation of a Multicountry Surveillance Study, the Severe Typhoid Fever in Africa Program.

BACKGROUND: There is limited information on the best practices for monitoring multicountry epidemiological studies. Here, we describe the monitoring and evaluation procedures created for the multicountry Severe Typhoid Fever in Africa (SETA) study. METHODS: Elements from the US Food and Drug Administration (FDA) and European Centre for Disease Prevention and Control (ECDC) recommendations on monitoring clinical trials and data quality, respectively were applied in the development of the SETA monitoring plan. The SETA core activities as well as the key data and activities required for the delivery of SETA outcomes were identified. With this information, a list of key monitorable indicators was developed using on-site and centralized monitoring methods, and a dedicated monitoring team was formed. The core activities were monitored on-site in each country at least twice per year and the SETA databases were monitored centrally as a collaborative effort between the International Vaccine Institute and study sites. Monthly reports were generated for key indicators and used to guide risk-based monitoring specific for each country. RESULTS: Preliminary results show that monitoring activities have increased compliance with protocol and standard operating procedures. A reduction in blood culture contamination following monitoring field visits in two of the SETA countries are preliminary results of the impact of monitoring activities. CONCLUSIONS: Current monitoring recommendations applicable to clinical trials and routine surveillance systems can be adapted for monitoring epidemiological studies. Continued monitoring efforts ensure that the procedures are harmonized across sites. Flexibility, ongoing feedback, and team participation yield sustainable solutions.

The Severe Typhoid Fever in Africa Program: Study Design and Methodology to Assess Disease Severity, Host Immunity, and Carriage Associated With Invasive Salmonellosis.

BACKGROUND: Invasive salmonellosis is a common community-acquired bacteremia in persons residing in sub-Saharan Africa. However, there is a paucity of data on severe typhoid fever and its associated acute and chronic host immune response and carriage. The Severe Typhoid Fever in Africa (SETA) program, a multicountry surveillance study, aimed to address these research gaps and contribute to the control and prevention of invasive salmonellosis. METHODS: A prospective healthcare facility-based surveillance with active screening of enteric fever and clinically suspected severe typhoid fever with complications was performed using a standardized protocol across the study sites in Burkina Faso, the Democratic Republic of Congo (DRC), Ethiopia, Ghana, Madagascar, and Nigeria. Defined inclusion criteria were used for screening of eligible patients for enrollment into the study. Enrolled patients with confirmed invasive salmonellosis by blood culture or patients with clinically suspected severe typhoid fever with perforation were eligible for clinical follow-up. Asymptomatic neighborhood controls and immediate household contacts of each case were enrolled as a comparison group to assess the level of Salmonella-specific antibodies and shedding patterns. Healthcare utilization surveys were performed to permit adjustment of incidence estimations. Postmortem questionnaires were conducted in medically underserved areas to assess death attributed to invasive Salmonella infections in selected sites. RESULTS: Research data generated through SETA aimed to address scientific knowledge gaps concerning the severe typhoid fever and mortality, long-term host immune responses, and bacterial shedding and carriage associated with natural infection by invasive salmonellae. CONCLUSIONS: SETA supports public health policy on typhoid immunization strategy in Africa.

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.

Determining the Best Immunization Strategy for Protecting African Children Against Invasive Salmonella Disease.

Background: The World Health Organization recently prequalified a typhoid conjugate vaccine (TCV), recommending its use in persons ≥6 months to 45 years residing in typhoid fever (TF)-endemic areas. We now need to consider how TCVs can have the greatest impact in the most vulnerable populations. Methods: The Typhoid Fever Surveillance in Africa Program (TSAP) was a blood culture-based surveillance of febrile patients from defined populations presenting at healthcare facilities in 10 African countries. TF and invasive non-typhoidal Salmonella (iNTS) disease incidences were estimated for 0-10 year-olds in one-year age increments. Results: Salmonella Typhi and iNTS were the most frequently isolated pathogens; 135 and 94 cases were identified, respectively. Analysis from three countries was excluded (incomplete person-years of observation (PYO) data). Thirty-seven of 123 TF cases (30.1%) and 71/90 iNTS disease cases (78.9%) occurred in children aged <5 years. No TF and 8/90 iNTS infections (8.9%) were observed in infants aged <9 months. The TF incidences (/100 000 PYO) for children aged <1 year and 1 to <2 years were 5 and 39, respectively; the highest incidence was 304 per 100 000 PYO in 4 to <5 year-olds. The iNTS disease incidence in the defined age groups ranged between 81 and 233 per 100 000 PYO, highest in 1 to <2 year-olds. TF and iNTS disease incidences were higher in West Africa. Conclusions: High burden of TF detected in young children strengthens the need for TCV introduction. Given the concurrent iNTS disease burden, development of a trivalent vaccine against S. Typhi, S. Typhimurium, and S. Enteritidis may be timely in this region.

A Qualitative Study Investigating Experiences, Perceptions, and Healthcare System Performance in Relation to the Surveillance of Typhoid Fever in Madagascar.

BACKGROUND: The burden of typhoid fever (TF) in sub-Saharan Africa is largely unknown but is increasingly thought to be high, given that water and sanitary conditions remain unimproved in many countries. To address this gap in information, the Typhoid Fever Surveillance in Africa Program (TSAP) founded a surveillance system for TF in 10 African countries. This study was a component of the TSAP surveillance project in Madagascar. METHODS: The study entailed a qualitative assessment of patients' experiences and perceptions of services for febrile symptoms at the studies' rural and urban sentinel public health clinics. The study examined influences on the use of these facilities, alternative sources of care, and providers' descriptions of medical consultations and challenges in providing services. Data were collected through semistructured and open-ended individual interviews and a focus group with patients, caregivers, and medical personnel. RESULTS: Thirty-three patients and 12 healthcare providers participated in the data collection across the 2 healthcare facilities. The quality of services, cost, and travel distance were key factors that enabled access to and use of these clinics. Divergent healthcare-seeking patterns were related to variability in the care utilized, socioeconomic status, and potential distance from the facilities : These factors influenced delivery of care, patient access, and the health facilities' capacity to identify cases of febrile illness such as TF. CONCLUSIONS: This approach provided an in-depth investigation and understanding of healthcare-seeking behavior at the study facilities, and factors that facilitated or acted as barriers to their use. Our findings demonstrate the relevance of these public health clinics as sites for the surveillance of TF in their role as central healthcare sources for families and communities within these rural and urban areas of Madagascar.

The Emergence of Reduced Ciprofloxacin Susceptibility in Salmonella enterica Causing Bloodstream Infections in Rural Ghana.

BACKGROUND: Salmonella ranks among the leading causes of bloodstream infections in sub-Saharan Africa. Multidrug resistant typhoidal and nontyphoidal Salmonella (NTS) isolates have been previously identified in this region. However, resistance to ciprofloxacin has rarely been reported in West Africa. This study aims to assess susceptibility against ciprofloxacin in Salmonella causing invasive bloodstream infections among children in rural Ghana. METHODS: From May 2007 until May 2012, children attending a rural district hospital in central Ghana were eligible for recruitment. Salmonella enterica isolated from blood cultures were assessed for ciprofloxacin susceptibility by Etest (susceptible minimum inhibitory concentration [MIC] ≤ 0.06 µg/mL). The gyrA, gyrB, parC, and parE genes were sequenced to identify mutations associated with changes in susceptibility to fluoroquinolones. RESULTS: Two hundred eighty-five Salmonella enterica isolates from 5211 blood cultures were most commonly identified as Salmonella enterica serovar Typhimurium (n = 129 [45%]), Salmonella enterica serovar Typhi (n = 89 [31%]), Salmonella enterica serovar Dublin (n = 20 [7%]), and Salmonella enterica serovar Enteritidis (n = 19 [7%]). All S. Typhi and S. Dublin were susceptible to ciprofloxacin. Reduced susceptibility (MIC >0.06 µg/mL) was found in 53% (10/19) of S. Enteritidis and in 2% (3/129) of S. Typhimurium isolates. Sequencing detected a single gyrB mutation (Glu466Asp) and a single gyrA mutation (Ser83Tyr) in all 3 S. Typhimurium isolates, while 9 of 10 S. Enteritidis harbored single gyrA mutations (Asp87Gly, Asp87Asn, or Asp87Tyr). No mutations were found in the parC and parE genes. CONCLUSIONS: Ciprofloxacin susceptibility in invasive NTS in rural Ghana is highly dependent on serotype. Although reduced ciprofloxacin susceptibility is low in S. Typhimurium, more than half of all S. Enteritidis isolates are affected. Healthcare practitioners in Ghana should be aware of potential treatment failure in patients with invasive S. Enteritidis infections.

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.

Prevalence of Salmonella Excretion in Stool: A Community Survey in 2 Sites, Guinea-Bissau and Senegal.

BACKGROUND: Chronic and convalescent carriers play an important role in the transmission and endemicity of many communicable diseases. A high incidence of Salmonella enterica serovar Typhi and invasive nontyphoidal Salmonella (NTS) infection has been reported in parts of sub-Saharan Africa, yet the prevalence of Salmonella excretion in the general population is unknown. METHODS: Stool specimens were collected from a random sample of households in 2 populations in West Africa: Bissau, Guinea-Bissau, and Dakar, Senegal. Stool was cultured to detect presence of Salmonella, and antimicrobial susceptibility testing was performed on the isolated organisms. RESULTS: Stool was cultured from 1077 and 1359 individuals from Guinea-Bissau and Senegal, respectively. Salmonella Typhi was not isolated from stool samples at either site. Prevalence of NTS in stool samples was 24.1 (95% confidence interval [CI], 16.5-35.1; n = 26/1077) per 1000 population in Guinea-Bissau and 10.3 (95% CI, 6.1-17.2; n = 14/1359) per 1000 population in Senegal. CONCLUSIONS: Evidence of NTS excretion in stool in both study populations indicates a possible NTS transmission route in these settings.

Variations of Invasive Salmonella Infections by Population Size in Asante Akim North Municipal, Ghana.

BACKGROUND: The Typhoid Fever Surveillance in Africa Program (TSAP) estimated adjusted incidence rates (IRs) for Salmonella enterica serovar Typhi and invasive nontyphoidal S. enterica serovars (iNTS) of >100 cases per 100 000 person-years of observation (PYO) for children aged <15 years in Asante Akim North Municipal (AAN), Ghana, between March 2010 and May 2012. We analyzed how much these rates differed between rural and urban settings. METHODS: Children recruited at the Agogo Presbyterian Hospital and meeting TSAP inclusion criteria were included in the analysis. Towns with >32 000 inhabitants were considered urban; towns with populations <5200 were considered rural. Adjusted IRs for Salmonella bloodstream infections were estimated for both settings. Setting-specific age-standardized incidence rates for children aged <15 years were derived and used to calculate age-standardized rate ratios (SRRs) to evaluate differences between settings. RESULTS: Eighty-eight percent (2651/3000) of recruited patients met inclusion criteria and were analyzed. IRs of Salmonella bloodstream infections in children <15 years old were >100 per 100 000 PYO in both settings. Among rural children, the Salmonella Typhi and iNTS rates were 2 times (SRR, 2.2; 95% confidence interval [CI], 1.3-3.5) and almost 3 times (SRR, 2.8; 95% CI, 1.9-4.3) higher, respectively, than rates in urban children. CONCLUSIONS: IRs of Salmonella bloodstream infections in children <15 years old in AAN, Ghana, differed by setting, with 2 to nearly 3 times higher rates in the less populated setting. Variations in the distribution of the disease should be considered to implement future studies and intervention strategies.

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 program's 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.

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.

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.