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

Abstract

Background: Improved 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 findings: Culture-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/significance: Overall 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.

Keywords: Controlled human infection model; Diagnostics; Febrile disease; Polymerase chain reaction; Salmonella Typhi; Typhoid fever.

Graphical abstract

Figure 1

STARD flowchart describing culture-PCR results in comparison with the reference standard (Blood culture, BC) for diagnosis of challenge study participants with typhoid infection after challenge. nTD, non-typhoid diagnosed; clinical dx, clinical diagnosis; micro dx, microbiological diagnosis.

Figure 2

Example of a challenge study participant who had several early positive culture-PCR results (yellow squares), in addition to early positive stool culture result (orange squares). This participant was subsequently diagnosed with typhoid infection based on both microbiological and clinical criteria. Red square, positive blood culture; grey squares, no sample collected; black line, oral temperature; dashed grey line, C-reactive protein level; shaded area, antibiotic treatment initiated.

Figure 3

Number of positive culture-PCR and blood culture samples collected after challenge by typhoid outcome. The 6 and 12 h positive results have been pooled into the 0.5 day group. The maximum number of TD samples/day exceeds the number of TD participants as more than one sample was collected per day following initiation of antibiotic treatment. TD, typhoid diagnosed; nTD, non-typhoid diagnosed; PCR, culture-PCR assay; BC, blood culture.