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. 2025 Oct 16:10:581.
doi: 10.12688/wellcomeopenres.24663.1. eCollection 2025.

Transmission dynamics for invasive Non-Typhoidal S almonella serovars (TiNTS): protocol for a household study of transmission and immune response to non-typhoidal Salmonella in Malawi

Peter I Johnston  1   2 , Kenneth Chizani  2   3 , Esmeda Chirwa  1   2 , Helen Dale  1   2 , Pyianka Patel  2 , Niza Silungwe  2   3 , Chifundo Mkwangwanya  2   3 , Tamando Kachala  2   3 , Chipiliro Mhango  2   3 , Grace Nyirenda  2   3 , Yohane Diness  2 , Star Mpesi  2 , Richard Wachepa  2 , Florence Shumba  2 , Felistas Mwakiseghile  2 , Vincent Rashid  2 , Theresa Misiri  2 , Philip M Ashton  1   2   4 , Angeziwa Chunga  2   3 , Derek Cocker  1 , Edward Cunningham-Oakes  5   6 , Chris Jewell  7 , Nicholas Feasey  2   8 , Melita A Gordon #  1   2   9 , Tonney Nyirenda #  2   3
Affiliations

Transmission dynamics for invasive Non-Typhoidal S almonella serovars (TiNTS): protocol for a household study of transmission and immune response to non-typhoidal Salmonella in Malawi

Peter I Johnston et al. Wellcome Open Res. .

Abstract

Background: Invasive non-typhoidal Salmonella (iNTS) disease is a leading cause of community-onset bloodstream infection in Africa, driving high morbidity in young children. The World Health Organization has published preferred product characteristics for an iNTS vaccine, but lack of transmission data is an impediment to vaccine licensure. Enteric NTS (eNTS) is the asymptomatic carriage of NTS in stool that precedes invasive disease. We do not know how long eNTS shedding lasts, how often infection spreads in endemic settings, or how an eNTS episode shapes immunity against later invasion. These gaps make it difficult to define trial sites, select cohorts, refine target product profiles, and build reliable models of vaccine impact. Here we describe TiNTS, a prospective household study in Blantyre, Malawi, which will measure real-time eNTS incidence, transmission, and antibody responses to close these evidence gaps and accelerate rational vaccine deployment.

Methods: We will recruit all members of at least 60 households in Ndirande, Blantyre, Malawi. Stool samples will be collected every other day for at least four weeks and tested for NTS using culture and pan- Salmonella PCR on growth media. Environmental samples collected at enrolment will be tested using the same methods. Symptoms and exposure risks will be recorded throughout.We will collect blood samples at enrolment, after four weeks, and four weeks after the first eNTS episode in each household. We will measure serum IgG responses to Salmonella Typhimurium and Enteritidis LPS antigens. We will extend follow-up if participants continue shedding or if the first household case occurs with fewer than 14 days of follow-up remaining.All culture-positive isolates and PCR-positive broths will undergo Illumina sequencing to enable genome and metagenome reconstruction for transmission inference.

Conclusions: TiNTS will define the burden, transmission patterns, and immune response to eNTS. Findings will inform vaccine modelling, trial design, and targeted introduction strategies.

Keywords: Environmental reservoirs; Non-typhoidal Salmonella (NTS); Urban informal settlement; antimicrobial resistance (AMR); genomics; longitudinal cohort study; metagenomics; transmission.

Plain language summary

Non-typhoidal salmonellae (NTS) are bacteria that are spread by the faecal-oral route. Once in someone’s gut, they can do one of three things: 1) cause no symptoms (termed “enteric NTS”, or “eNTS”) 2) cause diarrhoea (termed “diarrhoeal NTS”, or “dNTS” 3) escape from the gut and cause infection in the bloodstream (termed “invasive NTS” or “iNTS”). Many who develop iNTS disease will die. In Africa, iNTS disease is common. This is because NTS bacteria have adapted to escape immune defences in the gut, and because illnesses that dampen the body’s defence against the bacteria, such as malaria, malnutrition, anaemia, and HIV, are frequent. In order to prevent iNTS disease, we need to understand how the bacteria that cause it are circulating in communities. How long are individuals infectious? How many people can they spread infection to? What factors predispose to eNTS infection? It is also important to understand the degree to which eNTS can protect against iNTS disease by stimulating natural immunity. Our study will track entire households over at least four weeks, in a setting where NTS bacteria are known to be circulating in the community, and where iNTS disease is seen in local hospitals. We will see how often members of the household experience eNTS infection, and whether they pass it on to their household contacts. We will find out how long individuals are shedding the bacteria in their stool (the period for which they are infectious). We will look for NTS bacteria in the water supply and household environment. We will find out what effect an episode of eNTS has on a participants’ immune response to NTS bacteria. This information will help to estimate whether vaccines and other interventions might reduce the burden of iNTS disease.

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Conflict of interest statement

No competing interests were disclosed.

Figures

Figure 1.
Figure 1.. Map of Ndirande, showing the randomly generated co-ordinates from which households will be selected for the study.
The Nasolo river is shown with a 100 metre buffer zone surrounding it. Grey borders represent enumeration areas (EA) from the 2018 Malawi census, and the density of households within each of these is represented by colour as detailed in the legend. The location of Ndirande Health Centre is shown as a red circle.
Figure 2.
Figure 2.. Simulation-based estimation of expected events and statistical power for within-household Salmonella transmission.
Panel A shows SIS model dynamics over 28 days in a 5-person household with one initial infection. Panel B plots the expected number of detected events in 60 households across a range of daily introduction hazards. Panel C shows the effect of increasing the number of contacts sampled per household on expected detected events, assuming a fixed hazard of 0.0115. Panel D displays statistical power to detect ≥25 onward transmission events, plotted against increasing household sample size, with a 90% power threshold indicated (red dashed line). Results are shown for both PCR and culture-based detection methods, incorporating sensitivity estimates from Chirambo et al. (2021) .
Figure 3.
Figure 3.. Overview of TiNTS study visits.
Households are enrolled at day zero and followed through day 28. When Salmonella is first cultured from a participant's stool, an additional visit is scheduled for 28 days after the positive stool sample date (illustrated by the arrow from day 8). If the first positive culture occurs with fewer than 14 days remaining in the routine follow-up, visits are extended to ensure 14 days of monitoring after the positive result (illustrated by the downward arrow from day 18). Participants who remain stool culture-positive at the end of household follow-up undergo weekly stool sampling until two consecutive samples are negative (arrows at right). Blood draws for antibody measurements are indicated by vacutainer tubes.
Figure 4.
Figure 4.. Overview of laboratory procedures applied to stool samples.
The diagram outlines selective enrichment and culture steps for Salmonella detection, PCR screening, archiving of isolates, and the downstream selection of samples for whole-genome sequencing (single colonies) or low-diversity metagenomic sequencing (PCR-positive enriched media).
See this image and copyright information in PMC

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