Salmonella enterica serovar Typhimurium ST313 sublineage 2.2 has emerged in Malawi with a characteristic gene expression signature and a fitness advantage.

Invasive non-typhoidal Salmonella (iNTS) disease is a serious bloodstream infection that targets immune-compromised individuals, and causes significant mortality in sub-Saharan Africa. Salmonella enterica serovar Typhimurium ST313 causes the majority of iNTS in Malawi. We performed an intensive comparative genomic analysis of 608 S. Typhimurium ST313 isolates dating between 1996 and 2018 from Blantyre, Malawi. We discovered that following the arrival of the well-characterized S. Typhimurium ST313 lineage 2 in 1999, two multidrug-resistant variants emerged in Malawi in 2006 and 2008, designated sublineages 2.2 and 2.3, respectively. The majority of S. Typhimurium isolates from human bloodstream infections in Malawi now belong to sublineages 2.2 or 2.3. To understand the emergence of the prevalent ST313 sublineage 2.2, we studied two representative strains, D23580 (lineage 2) and D37712 (sublineage 2.2). The chromosome of ST313 lineage 2 and sublineage 2.2 only differed by 29 SNPs/small indels and a 3 kb deletion of a Gifsy-2 prophage region including the sseI pseudogene. Lineage 2 and sublineage 2.2 had distinctive plasmid profiles. The transcriptome was investigated in 15 infection-relevant in vitro conditions and within macrophages. During growth in physiological conditions that do not usually trigger S. Typhimurium SPI2 gene expression, the SPI2 genes of D37712 were transcriptionally active. We identified down-regulation of flagellar genes in D37712 compared with D23580. Following phenotypic confirmation of transcriptomic differences, we discovered that sublineage 2.2 had increased fitness compared with lineage 2 during mixed growth in minimal media. We speculate that this competitive advantage is contributing to the emergence of sublineage 2.2 in Malawi.

The Enterics for Global Health (EFGH) Shigella Surveillance Study in Malawi.

Background: Malawi is among 7 countries participating in the Enterics for Global Health (EFGH) Shigella surveillance study, which aims to determine the incidence of medically attended diarrhea attributed to Shigella, a leading bacterial cause of diarrhea in children in low-resource settings. Methods: We describe the EFGH study site in the densely populated informal settlement of Ndirande Township, Blantyre, Malawi. We explore the site's geographical location, demographic characteristics, and the healthcare-seeking behavior of its population, particularly for childhood diarrhea. We also describe the management of childhood diarrhea at the health facility, and the associated challenges to attaining optimum adherence to local and national guidelines at the site. Conclusions: Our overarching aim is to improve global health through understanding and mitigating the impact of diarrhea attributed to Shigella.

Evaluating the relationship between ciprofloxacin prescription and non-susceptibility in Salmonella Typhi in Blantyre, Malawi: an observational study.

BACKGROUND: Ciprofloxacin is the first-line drug for treating typhoid fever in many countries in Africa with a high disease burden, but the emergence of non-susceptibility poses a challenge to public health programmes. Through enhanced surveillance as part of vaccine evaluation, we investigated the occurrence and potential determinants of ciprofloxacin non-susceptibility in Blantyre, Malawi. METHODS: We conducted systematic surveillance of typhoid fever cases and antibiotic prescription in two health centres in Blantyre, Malawi, between Oct 1, 2016, and Oct 31, 2019, as part of the STRATAA and TyVAC studies. In addition, blood cultures were taken from eligible patients presenting at Queen Elizabeth Central Hospital, Blantyre, as part of routine diagnosis. Inclusion criteria were measured or reported fever, or clinical suspicion of sepsis. Microbiologically, we identified Salmonella enterica serotype Typhi (S Typhi) isolates with a ciprofloxacin non-susceptible phenotype from blood cultures, and used whole-genome sequencing to identify drug-resistance mutations and phylogenetic relationships. We constructed generalised linear regression models to investigate associations between the number of ciprofloxacin prescriptions given per month to study participants and the proportion of S Typhi isolates with quinolone resistance-determining region (QRDR) mutations in the following month. FINDINGS: From 46 989 blood cultures from Queen Elizabeth Central Hospital, 502 S Typhi isolates were obtained, 30 (6%) of which had either decreased ciprofloxacin susceptibility, or ciprofloxacin resistance. From 11 295 blood cultures from STRATAA and TyVAC studies, 241 microbiologically confirmed cases of typhoid fever were identified, and 198 isolates from 195 participants sequenced (mean age 12·8 years [SD 10·2], 53% female, 47% male). Between Oct 1, 2016, and Aug 31, 2019, of 177 typhoid fever cases confirmed by whole-genome sequencing, four (2%) were caused by S Typhi with QRDR mutations, compared with six (33%) of 18 cases between Sept 1 and Oct 31, 2019. This increase was associated with a preceding spike in ciprofloxacin prescriptions. Every additional prescription of ciprofloxacin given to study participants in the preceding month was associated with a 4·2% increase (95% CI 1·8-7·0) in the relative risk of isolating S Typhi with a QRDR mutation (p=0·0008). Phylogenetic analysis showed that S Typhi isolates with QRDR mutations from September and October, 2019, belonged to two distinct subclades encoding two different QRDR mutations, and were closely related (4-10 single-nucleotide polymorphisms) to susceptible S Typhi endemic to Blantyre. INTERPRETATION: We postulate a causal relationship between increased ciprofloxacin prescriptions and an increase in fluoroquinolone non-susceptibility in S Typhi. Decreasing ciprofloxacin use by improving typhoid diagnostics, and reducing typhoid fever cases through the use of an efficacious vaccine, could help to limit the emergence of resistance. FUNDING: Wellcome Trust, Bill & Melinda Gates Foundation, and National Institute for Health and Care Research (UK).

Clofazimine pharmacokinetics in HIV-infected adults with diarrhea: Implications of diarrheal disease on absorption of orally administered therapeutics.

Oral drug absorption kinetics are usually established in populations with a properly functioning gastrointestinal tract. However, many diseases and therapeutics can alter gastrointestinal physiology and cause diarrhea. The extent of diarrhea-associated impact on drug pharmacokinetics has not been quantitatively described. To address this knowledge gap, we used a population pharmacokinetic modeling approach with data collected in a phase IIa study of matched human immunodeficiency virus (HIV)-infected adults with/without cryptosporidiosis and diarrhea to examine diarrhea-associated impact on oral clofazimine pharmacokinetics. A population pharmacokinetic model was developed with 428 plasma samples from 23 HIV-infected adults with/without Cryptosporidium infection using nonlinear mixed-effects modeling. Covariates describing cryptosporidiosis-associated diarrhea severity (e.g., number of diarrhea episodes, diarrhea grade) or HIV infection (e.g., viral load, CD4+ T cell count) were evaluated. A two-compartment model with lag time and first-order absorption and elimination best fit the data. Maximum diarrhea grade over the study duration was found to be associated with a more than sixfold reduction in clofazimine bioavailability. Apparent clofazimine clearance, intercompartmental clearance, central volume of distribution, and peripheral volume of distribution were 3.71 L/h, 18.2 L/h (interindividual variability [IIV] 45.0%), 473 L (IIV 3.46%), and 3434 L, respectively. The absorption rate constant was 0.625 h-1 (IIV 149%) and absorption lag time was 1.83 h. In conclusion, the maximum diarrhea grade observed for the duration of oral clofazimine administration was associated with a significant reduction in clofazimine bioavailability. Our results highlight the importance of studying disease impacts on oral therapeutic pharmacokinetics to inform dose optimization and maximize the chance of treatment success.

Protocol for the challenge non-typhoidal Salmonella (CHANTS) study: a first-in-human, in-patient, double-blind, randomised, safety and dose-escalation controlled human infection model in the UK.

INTRODUCTION: Invasive non-typhoidal Salmonella (iNTS) serovars are a major cause of community-acquired bloodstream infections in sub-Saharan Africa (SSA). In this setting, Salmonella enterica serovar Typhimurium accounts for two-thirds of infections and is associated with an estimated case fatality rate of 15%-20%. Several iNTS vaccine candidates are in early-stage assessment which-if found effective-would provide a valuable public health tool to reduce iNTS disease burden. The CHANTS study aims to develop a first-in-human Salmonella Typhimurium controlled human infection model, which can act as a platform for future vaccine evaluation, in addition to providing novel insights into iNTS disease pathogenesis. METHODS AND ANALYSIS: This double-blind, safety and dose-escalation study will randomise 40-80 healthy UK participants aged 18-50 to receive oral challenge with one of two strains of S. Typhimurium belonging to the ST19 (strain 4/74) or ST313 (strain D23580) lineages. 4/74 is a global strain often associated with diarrhoeal illness predominantly in high-income settings, while D23580 is an archetypal strain representing invasive disease-causing isolates found in SSA. The primary objective is to determine the minimum infectious dose (colony-forming unit) required for 60%-75% of participants to develop clinical or microbiological features of systemic salmonellosis. Secondary endpoints are to describe and compare the clinical, microbiological and immunological responses following challenge. Dose escalation or de-escalation will be undertaken by continual-reassessment methodology and limited within prespecified safety thresholds. Exploratory objectives are to describe mechanisms of iNTS virulence, identify putative immune correlates of protection and describe host-pathogen interactions in response to infection. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the NHS Health Research Authority (London-Fulham Research Ethics Committee 21/PR/0051; IRAS Project ID 301659). The study findings will be disseminated in international peer-reviewed journals and presented at national/international stakeholder meetings. Study outcome summaries will be provided to both funders and participants. TRIAL REGISTRATION NUMBER: NCT05870150.

Efficacy of typhoid conjugate vaccine: final analysis of a 4-year, phase 3, randomised controlled trial in Malawian children.

BACKGROUND: Randomised controlled trials of typhoid conjugate vaccines among children in Africa and Asia have shown high short-term efficacy. Data on the durability of protection beyond 2 years are sparse. We present the final analysis of a randomised controlled trial in Malawi, encompassing more than 4 years of follow-up, with the aim of investigating vaccine efficacy over time and by age group. METHODS: In this phase 3, double-blind, randomised controlled efficacy trial in Blantyre, Malawi, healthy children aged 9 months to 12 years were randomly assigned (1:1) by an unmasked statistician to receive a single dose of Vi polysaccharide conjugated to tetanus toxoid vaccine (Vi-TT) or meningococcal capsular group A conjugate (MenA) vaccine. Children had to have no previous history of typhoid vaccination and reside in the study areas for inclusion and were recruited from government schools and health centres. Participants, their parents or guardians, and the study team were masked to vaccine allocation. Nurses administering vaccines were unmasked. We did surveillance for febrile illness from vaccination until follow-up completion. The primary outcome was first occurrence of blood culture-confirmed typhoid fever. Eligible children who were randomly assigned and vaccinated were included in the intention-to-treat analyses. This trial is registered at ClinicalTrials.gov, NCT03299426. FINDINGS: Between Feb 21, 2018, and Sept 27, 2018, 28 130 children were vaccinated; 14 069 were assigned to receive Vi-TT and 14 061 to receive MenA. After a median follow-up of 4·3 years (IQR 4·2-4·5), 24 (39·7 cases per 100 000 person-years) children in the Vi-TT group and 110 (182·7 cases per 100 000 person-years) children in the MenA group were diagnosed with a first episode of blood culture-confirmed typhoid fever. In the intention-to-treat population, efficacy of Vi-TT was 78·3% (95% CI 66·3-86·1), and 163 (129-222) children needed to be vaccinated to prevent one case. Efficacies by age group were 70·6% (6·4-93·0) for children aged 9 months to 2 years; 79·6% (45·8-93·9) for children aged 2-4 years; and 79·3% (63·5-89·0) for children aged 5-12 years. INTERPRETATION: A single dose of Vi-TT is durably efficacious for at least 4 years among children aged 9 months to 12 years and shows efficacy in all age groups, including children younger than 2 years. These results support current WHO recommendations in typhoid-endemic areas for mass campaigns among children aged 9 months to 15 years, followed by routine introduction in the first 2 years of life. FUNDING: Bill & Melinda Gates Foundation.

Typhoid fever.

Typhoid fever is an invasive bacterial disease associated with bloodstream infection that causes a high burden of disease in Africa and Asia. Typhoid primarily affects individuals ranging from infants through to young adults. The causative organism, Salmonella enterica subsp. enterica serovar Typhi is transmitted via the faecal-oral route, crossing the intestinal epithelium and disseminating to systemic and intracellular sites, causing an undifferentiated febrile illness. Blood culture remains the practical reference standard for diagnosis of typhoid fever, where culture testing is available, but novel diagnostic modalities are an important priority under investigation. Since 2017, remarkable progress has been made in defining the global burden of both typhoid fever and antimicrobial resistance; in understanding disease pathogenesis and immunological protection through the use of controlled human infection; and in advancing effective vaccination programmes through strategic multipartner collaboration and targeted clinical trials in multiple high-incidence priority settings. This Primer thus offers a timely update of progress and perspective on future priorities for the global scientific community.

Global diversity and antimicrobial resistance of typhoid fever pathogens: Insights from a meta-analysis of 13,000 Salmonella Typhi genomes.

Background: The Global Typhoid Genomics Consortium was established to bring together the typhoid research community to aggregate and analyse Salmonella enterica serovar Typhi (Typhi) genomic data to inform public health action. This analysis, which marks 22 years since the publication of the first Typhi genome, represents the largest Typhi genome sequence collection to date (n=13,000). Methods: This is a meta-analysis of global genotype and antimicrobial resistance (AMR) determinants extracted from previously sequenced genome data and analysed using consistent methods implemented in open analysis platforms GenoTyphi and Pathogenwatch. Results: Compared with previous global snapshots, the data highlight that genotype 4.3.1 (H58) has not spread beyond Asia and Eastern/Southern Africa; in other regions, distinct genotypes dominate and have independently evolved AMR. Data gaps remain in many parts of the world, and we show the potential of travel-associated sequences to provide informal 'sentinel' surveillance for such locations. The data indicate that ciprofloxacin non-susceptibility (>1 resistance determinant) is widespread across geographies and genotypes, with high-level ciprofloxacin resistance (≥3 determinants) reaching 20% prevalence in South Asia. Extensively drug-resistant (XDR) typhoid has become dominant in Pakistan (70% in 2020) but has not yet become established elsewhere. Ceftriaxone resistance has emerged in eight non-XDR genotypes, including a ciprofloxacin-resistant lineage (4.3.1.2.1) in India. Azithromycin resistance mutations were detected at low prevalence in South Asia, including in two common ciprofloxacin-resistant genotypes. Conclusions: The consortium's aim is to encourage continued data sharing and collaboration to monitor the emergence and global spread of AMR Typhi, and to inform decision-making around the introduction of typhoid conjugate vaccines (TCVs) and other prevention and control strategies. Funding: No specific funding was awarded for this meta-analysis. Coordinators were supported by fellowships from the European Union (ZAD received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 845681), the Wellcome Trust (SB, Wellcome Trust Senior Fellowship), and the National Health and Medical Research Council (DJI is supported by an NHMRC Investigator Grant [GNT1195210]).

Salmonella Typhi (Typhi) is a type of bacteria that causes typhoid fever. More than 110,000 people die from this disease each year, predominantly in areas of sub-Saharan Africa and South Asia with limited access to safe water and sanitation. Clinicians use antibiotics to treat typhoid fever, but scientists worry that the spread of antimicrobial-resistant Typhi could render the drugs ineffective, leading to increased typhoid fever mortality. The World Health Organization has prequalified two vaccines that are highly effective in preventing typhoid fever and may also help limit the emergence and spread of resistant Typhi. In low resource settings, public health officials must make difficult trade-off decisions about which new vaccines to introduce into already crowded immunization schedules. Understanding the local burden of antimicrobial-resistant Typhi and how it is spreading could help inform their actions. The Global Typhoid Genomics Consortium analyzed 13,000 Typhi genomes from 110 countries to provide a global overview of genetic diversity and antimicrobial-resistant patterns. The analysis showed great genetic diversity of the different strains between countries and regions. For example, the H58 Typhi variant, which is often drug-resistant, has spread rapidly through Asia and Eastern and Southern Africa, but is less common in other regions. However, distinct strains of other drug-resistant Typhi have emerged in other parts of the world. Resistance to the antibiotic ciprofloxacin was widespread and accounted for over 85% of cases in South Africa. Around 70% of Typhi from Pakistan were extensively drug-resistant in 2020, but these hard-to-treat variants have not yet become established elsewhere. Variants that are resistant to both ciprofloxacin and ceftriaxone have been identified, and azithromycin resistance has also appeared in several different variants across South Asia. The Consortium's analyses provide valuable insights into the global distribution and transmission patterns of drug-resistant Typhi. Limited genetic data were available fromseveral regions, but data from travel-associated cases helped fill some regional gaps. These findings may help serve as a starting point for collective sharing and analyses of genetic data to inform local public health action. Funders need to provide ongoing supportto help fill global surveillance data gaps.

Nontyphoidal Salmonella Invasive Disease: Challenges and Solutions.

Nontyphoidal Salmonella are a leading cause of community-onset bacteremia and other serious infections in sub-Saharan African countries where large studies indicate that they are an uncommon cause of moderate-to-severe diarrhea. Approximately 535 000 nontyphoidal Salmonella invasive disease illnesses and 77 500 deaths were estimated to occur in 2017; 422 000 (78.9%) illnesses and 66 500 (85.9%) deaths in countries in sub-Saharan Africa. Lineages of Salmonella enterica serovar Typhimurium sequence type (ST) 313 and lineages of Salmonella enterica serovar Enteritidis ST11 dominate as causes of invasive disease. A major reservoir for these specific strains outside of humans has not been identified to date. Human fecal shedding of such strains is common in areas where nontyphoidal Salmonella invasive disease incidence is high. The case-fatality ratio of nontyphoidal Salmonella invasive disease is approximately 15%. Early diagnosis and treatment are needed to avert fatal outcomes. Antimicrobial resistance, including multiple drug resistance, decreased fluoroquinolone susceptibility, and resistance to third-generation cephalosporins, is increasing in prevalence and is likely to further compromise patient outcomes. Naturally acquired immunity against invasive disease develops in children aged >3 years in endemic areas, likely mediated in part by the sequential acquisition of T-cell immunity, followed by antigen-specific immunoglobulin G antibodies. Vaccines in preclinical or clinical development include live-attenuated S. enterica serovar Typhimurium, nontyphoidal S. enterica core and O-polysaccharide glycoconjugates, multiple antigen-presenting system complexes, and generalized modules for membrane antigens vaccines. The latter are in phase I trials in Europe and Africa. Both vaccine use, and other effective, evidence-based nonvaccine interventions, are needed to prevent and control nontyphoidal Salmonella invasive disease.

Bacterial shedding and serologic responses following an outbreak of Salmonella Typhi in an endemic cohort.

BACKGROUND: Salmonella enterica serovar Typhi (Salmonella Typhi) is the cause of typhoid fever. Salmonella Typhi may be transmitted through shedding in the stool, which can continue after recovery from acute illness. Shedding is detected by culturing stool, which is challenging to co-ordinate at scale. We hypothesised that sero-surveillance would direct us to those shedding Salmonella Typhi in stool following a typhoid outbreak. METHODS: In 2016 a typhoid outbreak affected one in four residents of a Nursing School in Malosa, Malawi. The Department of Health asked for assistance to identify nursing students that might spread the outbreak to other health facilities. We measured IgG antibody titres against Vi capsular polysaccharide (anti-Vi IgG) and IgM / IgG antibodies against H:d flagellin (anti-H:d) three and six months after the outbreak. We selected participants in the highest and lowest deciles for anti-Vi IgG titre (measured at visit one) and obtained stool for Salmonella culture and PCR. All participants reported whether they had experienced fever persisting for three days or more during the outbreak (in keeping with the WHO definitions of 'suspected typhoid'). We tested for salmonellae in the Nursing School environment. RESULTS: We obtained 320 paired serum samples from 407 residents. We cultured stool from 25 residents with high anti-Vi IgG titres and 24 residents with low titres. We did not recover Salmonella Typhi from stool; four stool samples yielded non-typhoidal salmonellae; one sample produced a positive PCR amplification for a Salmonella Typhi target. Median anti-Vi and anti-H:d IgG titres fell among participants who reported persistent fever. There was a smaller fall in anti-H:d IgG titres among participants who did not report persistent fever. Non-typhoidal salmonellae were identified in water sampled at source and from a kitchen tap. CONCLUSION: High titres of anti-Vi IgG did not identify culture-confirmed shedding of Salmonella Typhi. There was a clear serologic signal of recent typhoid exposure in the cohort, represented by waning IgG antibody titres over time. The presence of non-typhoidal salmonellae in drinking water indicates sub-optimal sanitation. Developing methods to detect and treat shedding remains an important priority to complement typhoid conjugate vaccination in efforts to achieve typhoid elimination.

Cost-effectiveness analysis of typhoid conjugate vaccines in an outbreak setting: a modeling study.

BACKGROUND: Several prolonged typhoid fever epidemics have been reported since 2010 throughout eastern and southern Africa, including Malawi, caused by multidrug-resistant Salmonella Typhi. The World Health Organization recommends the use of typhoid conjugate vaccines (TCVs) in outbreak settings; however, current data are limited on how and when TCVs might be introduced in response to outbreaks. METHODOLOGY: We developed a stochastic model of typhoid transmission fitted to data from Queen Elizabeth Central Hospital in Blantyre, Malawi from January 1996 to February 2015. We used the model to evaluate the cost-effectiveness of vaccination strategies over a 10-year time horizon in three scenarios: (1) when an outbreak is likely to occur; (2) when an outbreak is unlikely to occur within the next ten years; and (3) when an outbreak has already occurred and is unlikely to occur again. We considered three vaccination strategies compared to the status quo of no vaccination: (a) preventative routine vaccination at 9 months of age; (b) preventative routine vaccination plus a catch-up campaign to 15 years of age; and (c) reactive vaccination with a catch-up campaign to age 15 (for Scenario 1). We also explored variations in outbreak definitions, delays in implementation of reactive vaccination, and the timing of preventive vaccination relative to the outbreak. RESULTS: Assuming an outbreak occurs within 10 years, we estimated that the various vaccination strategies would prevent a median of 15-60% of disability-adjusted life-years (DALYs). Reactive vaccination was the preferred strategy for WTP values of $0-300 per DALY averted. For WTP values > $300, introduction of preventative routine TCV immunization with a catch-up campaign was the preferred strategy. Routine vaccination with a catch-up campaign was cost-effective for WTP values above $890 per DALY averted if no outbreak occurs and > $140 per DALY averted if implemented after the outbreak has already occurred. CONCLUSIONS: Countries for which the spread of antimicrobial resistance is likely to lead to outbreaks of typhoid fever should consider TCV introduction. Reactive vaccination can be a cost-effective strategy, but only if delays in vaccine deployment are minimal; otherwise, introduction of preventive routine immunization with a catch-up campaign is the preferred strategy.

What is the Source of Infections Causing Invasive Nontyphoidal Salmonella Disease?

Invasive nontyphoidal Salmonella (iNTS) disease is a clinical condition distinct from Salmonella gastroenteritis. With an overall case-fatality rate of 14.5%, iNTS remains a major cause of morbidity and mortality, particularly in sub-Saharan Africa. However, the sources of infections that lead to cases of iNTS remain unclear. Broadly, there are 2 hypotheses as to the source of infections: (i) transmission from a zoonotic reservoir, similar to other nontyphoidal salmonelloses; or (ii) person-to-person transmission. Here we review several recent studies that have asked, "What is the source of infections causing invasive nontyphoidal Salmonella disease?" Two studies reported isolates in the stool of household members of iNTS cases that were very closely related (<3 single-nucleotide polymorphisms) to the iNTS case isolates; this is consistent with the hypothesis of person-to-person transmission, but infection from a common source (eg, a foodstuff) cannot be excluded. On the other hand, thorough investigations of the domestic environment of iNTS cases and the food pathway found only a single iNTS-associated Salmonella Enteritidis isolate. Therefore, we recommend that future studies test the hypothesis that iNTS is transmitted between people within the domestic environment. Further studies of food and water pathways are also warranted.

Typhoid conjugate vaccine effectiveness in Malawi: evaluation of a test-negative design using randomised, controlled clinical trial data.

BACKGROUND: Typhoid conjugate vaccines are being introduced in low-income and middle-income countries to prevent typhoid illness in children. Vaccine effectiveness studies assess vaccine performance after introduction. The test-negative design is a commonly used method to estimate vaccine effectiveness that has not been applied to typhoid vaccines because of concerns over blood culture insensitivity. The overall aim of the study was to evaluate the appropriateness of using a test-negative design to assess typhoid Vi polysaccharide-tetanus toxoid conjugate vaccine (Vi-TT) effectiveness using a gold standard randomised controlled trial database. METHODS: Using blood culture data from a randomised controlled trial of Vi-TT in Malawi, we simulated a test-negative design to derive vaccine effectiveness estimates using three different approaches and compared these to randomised trial efficacy results. In the randomised trial, 27 882 children aged 9 months to 12 years were randomly assigned (1:1) to receive a single dose of Vi-TT or meningococcal capsular group A conjugate vaccine between Feb 21 and Sept 27, 2018, and were followed up for blood culture-confirmed typhoid fever until Sept 30, 2021. FINDINGS: For all three test-negative design approaches, vaccine effectiveness estimates (test-negative design A, 80·3% [95% CI 66·2 to 88·5] vs test-negative design B, 80·5% [66·5 to 88·6] vs test-negative design C, 80·4% [66·9 to 88·4]) were almost identical to the randomised trial results (80·4% [95% CI 66·4 to 88·5]). Receipt of Vi-TT did not affect the risk of non-typhoid fever (vaccine efficacy against non-typhoid fever -0·4% [95% CI -4·9 to 3·9] vs -1% [-5·6 to 3·3] vs -2·5% [-6·4 to 1·3] for test-negative design A, test-negative design B, and test-negative design C, respectively). INTERPRETATION: This study validates the test-negative design core assumption for typhoid vaccine effectiveness estimation and shows the accuracy and precision of the estimates compared with the randomised controlled trial. These results show that the test-negative design is suitable for assessing typhoid conjugate vaccine effectiveness in post-introduction studies using blood culture surveillance. FUNDING: Bill & Melinda Gates Foundation.

Case-control investigation of invasive Salmonella disease in Malawi reveals no evidence of environmental or animal transmission of invasive strains, and supports human to human transmission.

BACKGROUND: Invasive Salmonella infections cause significant morbidity and mortality in Sub-Saharan Africa. However, the routes of transmission are uncertain. We conducted a case-control study of index-case and geographically-matched control households in Blantyre, Malawi, sampling Salmonella isolates from index cases, healthy people, animals, and the household environment. METHODOLOGY: Sixty index cases of human invasive Salmonella infection were recruited (March 2015-Oct 2016). Twenty-eight invasive Non-Typhoidal Salmonella (iNTS) disease and 32 typhoid patients consented to household sampling. Each index-case household was geographically matched to a control household. Extensive microbiological sampling included stool sampling from healthy household members, stool or rectal swabs from household-associated animals and boot-sock sampling of the household environment. FINDINGS: 1203 samples from 120 households, yielded 43 non-Typhoidal Salmonella (NTS) isolates from 25 households (overall sample positivity 3.6%). In the 28 iNTS patients, disease was caused by 3 STs of Salmonella Typhimurium, mainly ST313. In contrast, the isolates from households spanned 15 sequence types (STs). Two S. Typhimurium isolates from index cases closely matched isolates from their respective asymptomatic household members (2 and 3 SNP differences respectively). Despite the recovery of a diverse range of NTS, there was no overlap between the STs causing iNTS disease with any environmental or animal isolates. CONCLUSIONS: The finding of NTS strains from index cases that matched household members, coupled with lack of related animal or environmental isolates, supports a hypothesis of human to human transmission of iNTS infections in the household. The breadth of NTS strains found in animals and the household environment demonstrated the robustness of NTS sampling and culture methodology, and suggests a diverse ecology of Salmonella in this setting. Healthy typhoid (S. Typhi) carrier state was not detected. The lack of S. Typhi isolates from the household environment suggests that further methodological development is needed to culture S. Typhi from the environment.

Estimating the economic burden of typhoid in children and adults in Blantyre, Malawi: A costing cohort study.

BACKGROUND: Typhoid causes preventable death and disease. The World Health Organization recommends Typhoid Conjugate Vaccine for endemic countries, but introduction decisions depend on cost-effectiveness. We estimated household and healthcare economic burdens of typhoid in Blantyre, Malawi. METHODS: In a prospective cohort of culture-confirmed typhoid cases at two primary- and a referral-level health facility, we collected direct medical, non-medical costs (2020 U.S. dollars) to healthcare provider, plus indirect costs to households. RESULTS: From July 2019-March 2020, of 109 cases, 63 (58%) were <15 years old, 44 (40%) were inpatients. Mean hospitalization length was 7.7 days (SD 4.1). For inpatients, mean total household and provider costs were $93.85 (95%CI: 68.87-118.84) and $296.52 (95%CI: 225.79-367.25), respectively. For outpatients, these costs were $19.05 (95%CI: 4.38-33.71) and $39.65 (95%CI: 33.93-45.39), respectively. Household costs were due mainly to direct non-medical and indirect costs, medical care was free. Catastrophic illness cost, defined as cost >40% of non-food monthly household expenditure, occurred in 48 (44%) households. CONCLUSIONS: Typhoid can be economically catastrophic for families, despite accessible free medical care. Typhoid is costly for government healthcare provision. These data make an economic case for TCV introduction in Malawi and the region and will be used to derive vaccine cost-effectiveness.

Quality of antibody responses by adults and young children to 13-valent pneumococcal conjugate vaccination and Streptococcus pneumoniae colonisation.

Childhood pneumococcal conjugate vaccine (PCV) protects against invasive pneumococcal disease caused by vaccine-serotype (VT) Streptococcus pneumoniae by generating opsonophagocytic anti-capsular antibodies, but how vaccination protects against and reduces VT carriage is less well understood. Using serological samples from PCV-vaccinated Malawian individuals and a UK human challenge model, we explored whether antibody quality (IgG subclass, opsonophagocytic killing, and avidity) is associated with protection from carriage. Following experimental challenge of adults with S. pneumoniae serotype 6B, 3/21 PCV13-vaccinees were colonised with pneumococcus compared to 12/24 hepatitis A-vaccinated controls; PCV13-vaccination induced serotype-specific IgG, IgG1, and IgG2, and strong opsonophagocytic responses. However, there was no clear relationship between antibody quality and protection from carriage or carriage intensity after vaccination. Similarly, among PCV13-vaccinated Malawian infants there was no relationship between serotype-specific antibody titre or quality and carriage through exposure to circulating serotypes. Although opsonophagocytic responses were low in infants, antibody titre and avidity to circulating serotypes 19F and 6A were maintained or increased with age. These data suggest a complex relationship between antibody-mediated immunity and pneumococcal carriage, and that PCV13-driven antibody quality may mature with age and exposure.

Safety and immunogenicity of a typhoid conjugate vaccine among children aged 9 months to 12 years in Malawi: a nested substudy of a double-blind, randomised controlled trial.

BACKGROUND: Typhoid fever is a substantial public health problem in Africa, yet there are few clinical trials of typhoid conjugate vaccine (TCV). We assessed immunogenicity and safety of Typbar TCV in Malawi. METHODS: This substudy was nested within a phase 3, double-blind, parallel design, randomised controlled trial of TCV in children from Ndirande Health Centre in Ndirande township, Blantyre, Malawi. To be eligible, participants had to be aged between 9 months and 12 years with no known immunosuppression or chronic health conditions, including HIV or severe malnutrition; eligible participants were enrolled into three strata of approximately 200 children (9-11 months, 1-5 years, and 6-12 years), randomly assigned (1:1) to receive TCV or control (meningococcal serogroup A conjugate vaccine [MCV-A]) intramuscularly. Serum was collected before vaccination and at 28 days and 730-1035 days after vaccination to measure anti-Vi antibodies by ELISA. Because of COVID-19, day 730 visits were extended up to 1035 days. This nested substudy evaluated reactogenicity, safety, and immunogenicity by age stratum. Safety outcomes, analysed in the intention-to-treat population, included solicited adverse events within 7 days of vaccination (assessed on 3 separate days) and unsolicited adverse events within 28 days of vaccination. This trial is registered with ClinicalTrials.gov, NCT03299426. FINDINGS: Between Feb 22 and Sept 6, 2018, 664 participants were screened, and 631 participants were enrolled and randomly assigned (320 to the TCV group and 311 to the MCV-A group). 305 participants in the TCV group and 297 participants in the MCV-A group were vaccinated. Among TCV recipients, anti-Vi IgG geometric mean titres increased more than 500 times from 4·2 ELISA units (EU)/mL (95% CI 4·0-4·4) at baseline to 2383·7 EU/mL (2087·2-2722·3) at day 28, then decreased to 48·0 EU/mL (39·9-57·8) at day 730-1035, remaining more than 11 times higher than baseline. Among MCV-A recipients, anti-Vi IgG titres remained unchanged: 4·3 EU/mL (4·0-4·5) at baseline, 4·4 EU/mL (4·0-4·7) on day 28, and 4·6 EU/mL (4·2-5·0) on day 730-1035. TCV and MCV-A recipients had similar solicited local (eight [3%] of 304, 95% CI 1·3-5·1 and three [1%] of 293, 0·4-3·0) and systemic (27 [9%] of 304, 6·2-12·6 and 27 [9%] of 293, 6·4-13·1) reactogenicity. Related unsolicited adverse events occurred similarly in TCV and MCV-A recipients in eight (3%) of 304 (1·3-5·1) and eight (3%) of 293 (1·4-5·3). INTERPRETATION: This study provides evidence of TCV safety, tolerability, and immunogenicity up to 730-1035 days in Malawian children aged 9 months to 12 years. FUNDING: Bill & Melinda Gates Foundation.

Differential localization and limited cytotoxic potential of duodenal CD8+ T cells.

The duodenum is a major site of HIV persistence during suppressive antiretroviral therapy despite harboring abundant tissue-resident memory (Trm) CD8+ T cells. The role of duodenal Trm CD8+ T cells in viral control is still not well defined. We examined the spatial localization, phenotype, and function of CD8+ T cells in the human duodenal tissue from people living with HIV (PLHIV) and healthy controls. We found that Trm (CD69+CD103hi) cells were the predominant CD8+ T cell population in the duodenum. Immunofluorescence imaging of the duodenal tissue revealed that CD103+CD8+ T cells were localized in the intraepithelial region, while CD103-CD8+ T cells and CD4+ T cells were mostly localized in the lamina propria (LP). Furthermore, HIV-specific CD8+ T cells were enriched in the CD69+CD103-/lo population. However, the duodenal HIV-specific CD8+ Trm cells rarely expressed canonical molecules for potent cytolytic function (perforin and granzyme B) but were more polyfunctional than those from peripheral blood. Taken together, our results show that duodenal CD8+ Trm cells possess limited perforin-mediated cytolytic potential and are spatially separated from HIV-susceptible LP CD4+ T cells. This could contribute to HIV persistence in the duodenum and provides critical information for the design of cure therapies.

A clinical and molecular epidemiological survey of hepatitis C in Blantyre, Malawi, suggests a historic mechanism of transmission.

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. There are no previous representative community HCV prevalence studies from Southern Africa, and limited genotypic data. Epidemiological data are required to inform an effective public health response. We conducted a household census-based random sampling serological survey, and a prospective hospital-based study of patients with cirrhosis and hepatocellular carcinoma (HCC) in Blantyre, Malawi. We tested participants with an HCV antigen/antibody ELISA (Monolisa, Bio-Rad), confirmed with PCR (GeneXpert, Cepheid) and used line immunoassay (Inno-LIA, Fujiribio) for RNA-negative participants. We did target-enrichment whole-genome HCV sequencing (NextSeq, Illumina). Among 96,386 censused individuals, we randomly selected 1661 people aged ≥16 years. Population-standardized HCV RNA prevalence was 0.2% (95% CI 0.1-0.5). Among 236 patients with cirrhosis and HCC, HCV RNA prevalence was 1.9% and 5.0%, respectively. Mapping showed that HCV RNA+ patients were from peri-urban areas surrounding Blantyre. Community and hospital HCV RNA+ participants were older than comparator HCV RNA-negative populations (median 53 vs 30 years for community, p = 0.01 and 68 vs 40 years for cirrhosis/HCC, p < 0.001). Endemic HCV genotypes (n = 10) were 4v (50%), 4r (30%) and 4w (10%). In this first census-based community serological study in Southern Africa, HCV was uncommon in the general population, was centred on peri-urban regions and was attributable for <5% of liver disease. HCV infection was observed only among older people, suggesting a historic mechanism of transmission. Genotype 4r, which has been associated with treatment failure with ledipasvir and daclatasvir, is endemic.