Blood culture-PCR to optimise typhoid fever diagnosis after controlled human infection identifies frequent asymptomatic cases and evidence of primary bacteraemia

- Culture in ox-bile/tryptone soy broth selectively enriches for bile-tolerant Salmonella Typhi while lysing human cells.
- PCR sensitivity for detecting typhoid in clinical blood is limited by very low level bacteraemia during clinical illness.
- PCR amplification of S. Typhi fliC-d in pre-cultured blood can accurately identify typhoid infection in challenge study participants.
- Daily culture-PCR of blood collected from challenge study participants suggests primary bacteraemia occurs 12-36 h after S. Typhi ingestion.
- Additional use of culture-PCR demonstrates the true attack rate after typhoid challenge is markedly higher (75%) than previously assumed (60%).

SummaryBackgroundImproved diagnostics for typhoid are needed; a typhoid controlled human infection model may accelerate their development and translation. Here, we evaluated a blood culture-PCR assay for detecting infection after controlled human infection with S. Typhi and compared test performance with optimally performed blood cultures.Methodology/Principal findingsCulture-PCR amplification of blood samples was performed alongside daily blood culture in 41 participants undergoing typhoid challenge. Study endpoints for typhoid diagnosis (TD) were fever and/or bacteraemia. Overall, 24/41 (59%) participants reached TD, of whom 21/24 (86%) had ≥1 positive blood culture (53/674, 7.9% of all cultures) or 18/24 (75%) had ≥1 positive culture-PCR assay result (57/684, 8.3%). A further five non-bacteraemic participants produced culture-PCR amplicons indicating infection; overall sensitivity/specificity of the assay compared to the study endpoints were 70%/65%. We found no significant difference between blood culture and culture-PCR methods in ability to identify cases (12 mismatching pairs, p = 0.77, binomial test). Clinical and stool culture metadata demonstrated that additional culture-PCR amplification positive individuals likely represented true cases missed by blood culture, suggesting the overall attack rate may be 30/41 (73%) rather than 24/41 (59%). Several participants had positive culture-PCR results soon after ingesting challenge providing new evidence for occurrence of an early primary bacteraemia.Conclusions/SignificanceOverall the culture-PCR assay performed well, identifying extra typhoid cases compared with routine blood culture alone. Despite limitations to widespread field-use, the benefits of increased diagnostic yield, reduced blood volume and faster turn-around-time, suggest that this assay could enhance laboratory typhoid diagnostics in research applications and high-incidence settings.

The culture-PCR assay for detection of fliC-D DNA in human blood samples.158