Developing biomarker assays to accelerate tuberculosis drug development: defining target product profiles.

Drug development for tuberculosis is hindered by the methodological limitations in the definitions of patient outcomes, particularly the slow organism growth and difficulty in obtaining suitable and representative samples throughout the treatment. We developed target product profiles for biomarker assays suitable for early-phase and late-phase clinical drug trials by consulting subject-matter experts on the desirable performance and operational characteristics of such assays for monitoring of tuberculosis treatment in drug trials. Minimal and optimal criteria were defined for scope, intended use, pricing, performance, and operational characteristics of the biomarkers. Early-stage trial assays should accurately quantify the number of viable bacilli, whereas late-stage trial assays should match the number, predict relapse-free cure, and replace culture conversion endpoints. The operational criteria reflect the infrastructure and resources available for drug trials. The effective tools should define the sterilising activity of the drug and lower the probability of treatment failure or relapse in people with tuberculosis. The target product profiles outlined in this Review should guide and de-risk the development of biomarker-based assays suitable for phase 2 and 3 clinical drug trials.

Incidence of typhoid fever in Burkina Faso, Democratic Republic of the Congo, Ethiopia, Ghana, Madagascar, and Nigeria (the Severe Typhoid in Africa programme): a population-based study.

BACKGROUND: Typhoid Fever remains a major cause of morbidity and mortality in low-income settings. The Severe Typhoid in Africa programme was designed to address regional gaps in typhoid burden data and identify populations eligible for interventions using novel typhoid conjugate vaccines. METHODS: A hybrid design, hospital-based prospective surveillance with population-based health-care utilisation surveys, was implemented in six countries in sub-Saharan Africa. Patients presenting with fever (≥37·5°C axillary or ≥38·0°C tympanic) or reporting fever for three consecutive days within the previous 7 days were invited to participate. Typhoid fever was ascertained by culture of blood collected upon enrolment. Disease incidence at the population level was estimated using a Bayesian mixture model. FINDINGS: 27 866 (33·8%) of 82 491 participants who met inclusion criteria were recruited. Blood cultures were performed for 27 544 (98·8%) of enrolled participants. Clinically significant organisms were detected in 2136 (7·7%) of these cultures, and 346 (16·2%) Salmonella enterica serovar Typhi were isolated. The overall adjusted incidence per 100 000 person-years of observation was highest in Kavuaya and Nkandu 1, Democratic Republic of the Congo (315, 95% credible interval 254-390). Overall, 46 (16·4%) of 280 tested isolates showed ciprofloxacin non-susceptibility. INTERPRETATION: High disease incidence (ie, >100 per 100 000 person-years of observation) recorded in four countries, the prevalence of typhoid hospitalisations and complicated disease, and the threat of resistant typhoid strains strengthen the need for rapid dispatch and implementation of effective typhoid conjugate vaccines along with measures designed to improve clean water, sanitation, and hygiene practices. FUNDING: The Bill & Melinda Gates Foundation.

Early risk assessment in paediatric and adult household contacts of confirmed tuberculosis cases by novel diagnostic tests (ERASE-TB): protocol for a prospective, non-interventional, longitudinal, multicountry cohort study.

INTRODUCTION: The WHO End-TB Strategy calls for the development of novel diagnostics to detect tuberculosis (TB) earlier and more accurately. Better diagnostics, together with tools to predict disease progression, are critical for achieving WHO End-TB targets. The Early Risk Assessment in TB Contacts by new diagnoStic tEsts (ERASE-TB) study aims to evaluate novel diagnostics and testing algorithms for early TB diagnosis and accurate prediction of disease progression among household contacts (HHCs) exposed to confirmed index cases in Mozambique, Tanzania and Zimbabwe. METHODS AND ANALYSIS: A total of 2100 HHCs (aged ≥10 years) of adults with microbiologically-confirmed pulmonary TB will be recruited and followed up at 6-month intervals for 18-24 months. At each time point, a WHO symptom screen and digital chest radiograph (dCXR) will be performed, and blood and urine samples will be collected. Individuals screening positive (WHO symptom screen or dCXR) will be requested to provide sputum for Xpert MTB/Rif Ultra. At baseline, HHCs will also be screened for HIV, diabetes (HbA1c), chronic lung disease (spirometry), hypertension and anaemia. Study outcomes will be coprevalent TB (diagnosed at enrolment), incident TB (diagnosed during follow-up) or no TB at completion of follow-up. Novel diagnostics will be validated using fresh and biobanked samples with a nested case-control design. Cases are defined as HHCs diagnosed with TB (for early diagnosis) or with incident TB (for prediction of progression) and will be matched by age, sex and country to HHCs who remain healthy (controls). Statistical analyses will include assessment of diagnostic accuracy by constructing receiver operating curves and calculation of sensitivity and specificity. ETHICS AND DISSEMINATION: ERASE-TB has been approved by regulatory and ethical committees in each African country and by each partner organisation. Consent, with additional assent for participants <18 years, is voluntary. Attestation by impartial witnesses is sought in case of illiteracy. Confidentiality of participants is being maintained throughout. Study findings will be presented at scientific conferences and published in peer-reviewed international journals. TRIAL REGISTRATION NUMBER: NCT04781257.Cite Now.

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.

Detection of Pathogens of Acute Febrile Illness Using Polymerase Chain Reaction from Dried Blood Spots.

Quantitative polymerase chain reaction (qPCR) of dried blood spots (DBS) for pathogen detection is a potentially convenient method for infectious disease diagnosis. This study tested 115 DBS samples paired with whole blood specimens of children and adolescent from Burkina Faso, Sudan, and Madagascar by qPCR for a wide range of pathogens, including protozoans, helminths, fungi, bacteria, and viruses. Plasmodium spp. was consistently detected from DBS but yielded a mean cycle threshold (Ct) 5.7 ± 1.6 higher than that from whole blood samples. A DBS qPCR Ct cutoff of 27 yielded 94.1% sensitivity and 95.1% specificity against the whole blood qPCR cutoff of 21 that has been previously suggested for malaria diagnosis. For other pathogens investigated, DBS testing yielded a sensitivity of only 8.5% but a specificity of 98.6% compared with whole blood qPCR. In sum, direct PCR of DBS had reasonable performance for Plasmodium but requires further investigation for the other pathogens assessed in this study.

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

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

Natural Intra- and Interclade Human Hybrid Schistosomes in Africa with Considerations on Prevention through Vaccination.

Causal agents of schistosomiasis are dioecious, digenean schistosomes affecting mankind in 76 countries. Preventive measures are manifold but need to be complemented by vaccination for long-term protection; vaccine candidates in advanced pre-clinical/clinical stages include Sm14, Sm-TSP-2/Sm-TSP-2Al®, Smp80/SchistoShield®, and Sh28GST/Bilhvax®. Natural and anthropogenic changes impact on breaking species isolation barriers favoring introgressive hybridization, i.e., allelic exchange among gene pools of sympatric, interbreeding species leading to instant large genetic diversity. Phylogenetic distance matters, thus the less species differ phylogenetically the more likely they hybridize. PubMed and Embase databases were searched for publications limited to hybridale confirmation by mitochondrial cytochrome c oxidase (COX) and/or nuclear ribosomal internal transcribed spacer (ITS). Human schistosomal hybrids are predominantly reported from West Africa with clustering in the Senegal River Basin, and scattering to Europe, Central and Eastern Africa. Noteworthy is the dominance of Schistosoma haematobium interbreeding with human and veterinary species leading due to hybrid vigor to extinction and homogenization as seen for S. guineensis in Cameroon and S. haematobium in Niger, respectively. Heterosis seems to advantage S. haematobium/S. bovis interbreeds with dominant S. haematobium-ITS/S. bovis-COX1 profile to spread from West to East Africa and reoccur in France. S. haematobium/S. mansoni interactions seen among Senegalese and Côte d'Ivoirian children are unexpected due to their high phylogenetic distance. Detecting pure S. bovis and S. bovis/S. curassoni crosses capable of infecting humans observed in Corsica and Côte d'Ivoire, and Niger, respectively, is worrisome. Taken together, species hybridization urges control and preventive measures targeting human and veterinary sectors in line with the One-Health concept to be complemented by vaccination protecting against transmission, infection, and disease recurrence. Functional and structural diversity of naturally occurring human schistosomal hybrids may impact current vaccine candidates requiring further research including natural history studies in endemic areas targeted for clinical trials.

Pathogens That Cause Acute Febrile Illness Among Children and Adolescents in Burkina Faso, Madagascar, and Sudan.

BACKGROUND: The etiology and optimal clinical management of acute febrile illness (AFI) is poorly understood. METHODS: Blood samples taken from study participants with acute fever (≥37.5°C) or a history of fever and recruited into the previous Typhoid Fever Surveillance in Africa (TSAP) study were evaluated using a polymerase chain reaction (PCR)-based TaqMan-Array Card designed to detect a panel of bacterial, viral, and parasitic pathogens. Clinical metadata were also assessed. RESULTS: A total of 615 blood samples available for analysis originated from Burkina Faso (n = 53), Madagascar (n = 364), and Sudan (n = 198) and were taken from participants ranging in age from 0-19 years. Through the TaqMan-Array Card, at least 1 pathogen was detected in 62% (33 of 53), 24% (86 of 364), and 60% (118 of 198) of specimens from Burkina Faso, Madagascar, and Sudan, respectively. The leading identified pathogen overall was Plasmodium spp., accounting for 47% (25 of 53), 2.2% (8 of 364), and 45% (90 of 198) of AFI at the respective sites. In Madagascar, dengue virus was the most prevalent pathogen (10.2%). Overall, 69% (357 of 516) of patients with clinical diagnoses of malaria, respiratory infection, or gastrointestinal infection were prescribed a World Health Organization guideline-recommended empiric antibiotic, whereas only 45% (106 of 237) of patients with pathogens detected were treated with an antibiotic exerting likely activity. CONCLUSIONS: A PCR approach for identifying multiple bacterial, viral, and parasitic pathogens in whole blood unveiled a diversity of previously undetected pathogens in AFI cases and carries implications for the appropriate management of this common syndrome.

How Can the Typhoid Fever Surveillance in Africa and the Severe Typhoid Fever in Africa Programs Contribute to the Introduction of Typhoid Conjugate Vaccines?

BACKGROUND: The World Health Organization now recommends the use of typhoid conjugate vaccines (TCVs) in typhoid-endemic countries, and Gavi, the Vaccine Alliance, added TCVs into the portfolio of subsidized vaccines. Data from the Severe Typhoid Fever in Africa (SETA) program were used to contribute to TCV introduction decision-making processes, exemplified for Ghana and Madagascar. METHODS: Data collected from both countries were evaluated, and barriers to and benefits of introduction scenarios are discussed. No standardized methodological framework was applied. RESULTS: The Ghanaian healthcare system differs from its Malagasy counterpart: Ghana features a functioning insurance system, antimicrobials are available nationwide, and several sites in Ghana deploy blood culture-based typhoid diagnosis. A higher incidence of antimicrobial-resistant Salmonella Typhi is reported in Ghana, which has not been identified as an issue in Madagascar. The Malagasy people have a low expectation of provided healthcare and experience frequent unavailability of medicines, resulting in limited healthcare-seeking behavior and extended consequences of untreated disease. CONCLUSIONS: For Ghana, high typhoid fever incidence coupled with spatiotemporal heterogeneity was observed. A phased TCV introduction through an initial mass campaign in high-risk areas followed by inclusion into routine national immunizations prior to expansion to other areas of the country can be considered. For Madagascar, a national mass campaign followed by routine introduction would be the introduction scenario of choice as it would protect the population, reduce transmission, and prevent an often-deadly disease in a setting characterized by lack of access to healthcare infrastructure. New, easy-to-use diagnostic tools, potentially including environmental surveillance, should be explored and improved to facilitate identification of high-risk areas.

Bacteremia Among Febrile Patients Attending Selected Healthcare Facilities in Ibadan, Nigeria.

BACKGROUND: The relative contribution of bacterial infections to febrile disease is poorly understood in many African countries due to diagnostic limitations. This study screened pediatric and adult patients attending 4 healthcare facilities in Ibadan, Nigeria, for bacteremia and malaria parasitemia. METHODS: Febrile patients underwent clinical diagnosis, malaria parasite testing, and blood culture. Bacteria from positive blood cultures were isolated and speciated using biochemical and serological methods, and Salmonella subtyping was performed by polymerase chain reaction. Antimicrobial susceptibility was tested by disk diffusion. RESULTS: A total of 682 patients were recruited between 16 June and 16 October 2017; 467 (68.5%) were <18 years of age. Bacterial pathogens were cultured from the blood of 117 (17.2%) patients, with Staphylococcus aureus (69 [59.0%]) and Salmonella enterica (34 [29.1%]) being the most common species recovered. Twenty-seven (79.4%) of the Salmonella isolates were serovar Typhi and the other 7 belonged to nontyphoidal Salmonella serovarieties. Thirty-four individuals were found to be coinfected with Plasmodium falciparum and bacteria. Five (14.7%) of these coinfections were with Salmonella, all in children aged <5 years. Antimicrobial susceptibility testing revealed that most of the Salmonella and Staphylococcus isolates were multidrug resistant. CONCLUSIONS: The study demonstrates that bacteria were commonly recovered from febrile patients with or without malaria in this location. Focused and extended epidemiological studies are needed for the introduction of typhoid conjugate vaccines that have the potential to prevent a major cause of severe community-acquired febrile diseases in our locality.

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.

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.

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 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.

Occurrence of Typhoid Fever Complications and Their Relation to Duration of Illness Preceding Hospitalization: A Systematic Literature Review and Meta-analysis.

BACKGROUND: Complications from typhoid fever disease have been estimated to occur in 10%-15% of hospitalized patients, with evidence of a higher risk in children and when delaying the implementation of effective antimicrobial treatment. We estimated the prevalence of complications in hospitalized patients with culture-confirmed typhoid fever and the effects of delaying the implementation of effective antimicrobial treatment and age on the prevalence and risk of complications. METHODS: A systematic review and meta-analysis were performed using studies in the PubMed database. We rated risk of bias and conducted random-effects meta-analyses. Days of disease at hospitalization (DDA) was used as a surrogate for delaying the implementation of effective antimicrobial treatment. Analyses were stratified by DDA (DDA <10 versus ≥10 mean/median days of disease) and by age (children versus adults). Differences in risk were assessed using odds ratios (ORs) and 95% confidence intervals (CIs). Heterogeneity and publication bias were evaluated with the I2 value and funnel plot analysis, respectively. RESULTS: The pooled prevalence of complications estimated among hospitalized typhoid fever patients was 27% (95% CI, 21%-32%; I2 = 90.9%, P < .0001). Patients with a DDA ≥ 10 days presented higher prevalence (36% [95% CI, 29%-43%]) and three times greater risk of severe disease (OR, 3.00 [95% CI, 2.14-4.17]; P < .0001) than patients arriving earlier (16% [95% CI, 13%- 18%]). Difference in prevalence and risk by age groups were not significant. CONCLUSIONS: This meta-analysis identified a higher overall prevalence of complications than previously reported and a strong association between duration of symptoms prior to hospitalization and risk of serious complications.

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.

Ralstonia mannitolilytica sepsis: a case report.

BACKGROUND: Ralstonia mannitolilytica is an emerging opportunistic pathogen that is associated with severe disease, including septic shock, meningitis, and renal transplant infections. Reports on this pathogen are limited, however, especially on the African continent. CASE PRESENTATION: A 2-year-old Akan child was presented to a hospital in the northeastern part of Ghana with a 1-week history of fever and chills. We identified Ralstonia mannitolilytica in her blood culture using both conventional and 16S ribosomal deoxyribonucleic acid (rDNA) techniques. The patient's condition improved clinically upon treatment with cefuroxime. CONCLUSION: Our report highlights the potential of Ralstonia mannitolilytica to cause sepsis and thus emphasizes the need for improved laboratory diagnosis and evidence for use of appropriate antibiotics in rural settings of Africa, where presumptive treatment using antimicrobial agents is rife.

Emergence of phylogenetically diverse and fluoroquinolone resistant Salmonella Enteritidis as a cause of invasive nontyphoidal Salmonella disease in Ghana.

BACKGROUND: Salmonella enterica serovar Enteritidis is a cause of both poultry- and egg-associated enterocolitis globally and bloodstream-invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa (sSA). Distinct, multi-drug resistant genotypes associated with iNTS disease in sSA have recently been described, often requiring treatment with fluoroquinolone antibiotics. In industrialised countries, antimicrobial use in poultry production has led to frequent fluoroquinolone resistance amongst globally prevalent enterocolitis-associated lineages. METHODOLOGY/PRINCIPAL FINDINGS: Twenty seven S. Enteritidis isolates from patients with iNTS disease and two poultry isolates, collected between 2007 and 2015 in the Ashanti region of Ghana, were whole-genome sequenced. These isolates, notable for a high rate of diminished ciprofloxacin susceptibility (DCS), were placed in the phyletic context of 1,067 sequences from the Public Health England (PHE) S. Enteritidis genome database to understand whether DCS was associated with African or globally-circulating clades of S. Enteritidis. Analysis showed four of the major S. Enteritidis clades were represented, two global and two African. All thirteen DCS isolates, containing a single gyrA mutation at codon 87, belonged to a global PT4-like clade responsible for epidemics of poultry-associated enterocolitis. Apart from two DCS isolates, which clustered with PHE isolates associated with travel to Spain and Brazil, the remaining DCS isolates, including one poultry isolate, belonged to two monophyletic clusters in which gyrA 87 mutations appear to have developed within the region. CONCLUSIONS/SIGNIFICANCE: Extensive phylogenetic diversity is evident amongst iNTS disease-associated S. Enteritidis in Ghana. Antimicrobial resistance profiles differed by clade, highlighting the challenges of devising empirical sepsis guidelines. The detection of fluoroquinolone resistance in phyletically-related poultry and human isolates is of major concern and surveillance and control measures within the region's burgeoning poultry industry are required to protect a human population at high risk of iNTS disease.

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

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