Molecular Diagnosis of Enteric Fever: Progress and Perspectives

Zhou, Lei; Darton, Thomas C.; Waddington, Claire S.; Pollard, Andrew J.

doi:10.5772/30374

Cited by 5 publications

(6 citation statements)

References 125 publications

(139 reference statements)

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“…In order to be introduced into routine clinical practice, any novel enteric fever diagnostic has to outperform the current ‘gold-standard’ automated blood culture, either in terms of cost, sensitivity, specificity, ease of use or rapidity of diagnosis. Despite over a decade of effort, PCR diagnostics of typhoid have not been brought into routine clinical use, partially due to the technical hurdles caused by a low bacterial load of clinical specimens [ 8 ]. In this study we describe a novel blood culture PCR method for detection of S .…”

Section: Discussionmentioning

confidence: 99%

“…As was shown in our previous study, a 5 hour TSB-2.4% ox bile blood culture PCR assay could detect S . Typhi as low as 0.75 CFU/ml in blood [ 22 ], and increased the speed of a positive confirmatory diagnosis of typhoid in a human challenge study [ 8 ]. Recently, another study used a 6 hour BHI broth culture for bacterial enrichment in PCR detection of blood typhoidal and non typhoidal Salmonellae infections [ 23 ].…”

Section: Discussionmentioning

confidence: 99%

“…Molecular methods, especially polymerase chain reaction (PCR) based assays, have attracted much attention in last decade for diagnosis of enteric fever [ 8 ]. The low bacterial burden in blood of enteric fever patients (estimated as a median of 0.3 CFU/ml blood [ 9 ]) hinders the technical progress in this field.…”

Section: Introductionmentioning

confidence: 99%

“…In order to overcome this limitation, we and others have previously demonstrated the utility of a pre-culture incubation step prior to PCR amplification [ 22 , 23 ]. This approach has proved valuable for early diagnosis of typhoid fever [ 8 ].…”

Section: Introductionmentioning

confidence: 99%

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Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples

et al. 2016

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BackgroundEnteric fever remains an important cause of morbidity in many low-income countries and Salmonella Paratyphi A has emerged as the aetiological agent in an increasing proportion of cases. Lack of adequate diagnostics hinders early diagnosis and prompt treatment of both typhoid and paratyphoid but development of assays to identify paratyphoid has been particularly neglected. Here we describe the development of a rapid and sensitive blood culture PCR method for detection of Salmonella Paratyphi A from blood, potentially allowing for appropriate diagnosis and antimicrobial treatment to be initiated on the same day.MethodsVenous blood samples from volunteers experimentally challenged orally with Salmonella Paratyphi A, who subsequently developed paratyphoid, were taken on the day of diagnosis; 10 ml for quantitative blood culture and automated blood culture, and 5 ml for blood culture PCR. In the latter assay, bacteria were grown in tryptone soy broth containing 2.4% ox bile and micrococcal nuclease for 5 hours (37°C) before bacterial DNA was isolated for PCR detection targeting the fliC-a gene of Salmonella Paratyphi A.ResultsAn optimized broth containing 2.4% ox bile and micrococcal nuclease, as well as a PCR test was developed for a blood culture PCR assay of Salmonella Paratyphi A. The volunteers diagnosed with paratyphoid had a median bacterial burden of 1 (range 0.1–6.9) CFU/ml blood. All the blood culture PCR positive cases where a positive bacterial growth was shown by quantitative blood culture had a bacterial burden of ≥ 0.3 CFU/ ml blood. The blood culture PCR assay identified an equal number of positive cases as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood), but utilized only half the volume of specimens.ConclusionsThe blood culture PCR method for detection of Salmonella Paratyphi A can be completed within 9 hours and offers the potential for same-day diagnosis of enteric fever. Using 5 ml blood, it exhibited a lower limit of detection equal to 0.3 CFU/ml blood, and it performed at least as well as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood) of clinical specimens despite using half the volume of blood. The findings warrant its further study in endemic populations with a potential use as a novel diagnostic which fills the present gap of paratyphoid diagnostics.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples

et al. 2016

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“…To perform the culture-PCR assay, 5 mL of heparinised peripheral venous blood was collected at daily after challenge ( Table 1 ), and performed as previously described. 19 , 20 , 25 Briefly, blood was added to culture media (20 mL 3% (w/v) ox bile/tryptone soya broth containing 1.5 μL micrococcal nuclease) and incubated for 5 h (37 °C, 220 rpm New Brunswick, Excella e24). Bacteria were then concentrated by centrifugation at 6000 ×g for 20 min and the supernatant removed.…”

Section: Methodsmentioning

confidence: 99%

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

Darton

Zhou

Blohmke

et al. 2017

Journal of Infection

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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.

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Sensitive and rapid detection of pathogenic bacteria in small volumes using impedance spectroscopy technique

Pal

Sharma

Gupta

2016

Biosensors and Bioelectronics

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Molecular Diagnosis of Enteric Fever: Progress and Perspectives

Cited by 5 publications

References 125 publications

Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples

Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples

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

Sensitive and rapid detection of pathogenic bacteria in small volumes using impedance spectroscopy technique

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