The sensitivity of real-time PCR amplification targeting invasive Salmonella serovars in biological specimens

Tran V.T. Nga, Abhilasha Karkey, Sabina Dongol, Hang N. Thuy, Sarah Dunstan, Kathryn Holt, Le T.P. Tu, James I. Campbell, Tran T. Chau, Nguyen V.V. Chau, Amit Arjyal, Samir Koirala, Buddha Basnyat, Christiane Dolecek, Jeremy Farrar, Stephen Baker

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Abstract

Background: PCR amplification for the detection of pathogens in biological material is generally considered a rapid and informative diagnostic technique. Invasive Salmonella serovars, which cause enteric fever, can be commonly cultured from the blood of infected patients. Yet, the isolation of invasive Salmonella serovars from blood is protracted and potentially insensitive.Methods: We developed and optimised a novel multiplex three colour real-time PCR assay to detect specific target sequences in the genomes of Salmonella serovars Typhi and Paratyphi A. We performed the assay on DNA extracted from blood and bone marrow samples from culture positive and negative enteric fever patients.Results: The assay was validated and demonstrated a high level of specificity and reproducibility under experimental conditions. All bone marrow samples tested positive for Salmonella, however, the sensitivity on blood samples was limited. The assay demonstrated an overall specificity of 100% (75/75) and sensitivity of 53.9% (69/128) on all biological samples. We then tested the PCR detection limit by performing bacterial counts after inoculation into blood culture bottles.Conclusions: Our findings corroborate previous clinical findings, whereby the bacterial load of S. Typhi in peripheral blood is low, often below detection by culture and, consequently, below detection by PCR. Whilst the assay may be utilised for environmental sampling or on differing biological samples, our data suggest that PCR performed directly on blood samples may be an unsuitable methodology and a potentially unachievable target for the routine diagnosis of enteric fever.

Original languageEnglish
Article number125
Number of pages9
JournalBMC Infectious Diseases
Volume10
DOIs
Publication statusPublished - 21 May 2010
Externally publishedYes

Cite this

Nga, Tran V.T. ; Karkey, Abhilasha ; Dongol, Sabina ; Thuy, Hang N. ; Dunstan, Sarah ; Holt, Kathryn ; Tu, Le T.P. ; Campbell, James I. ; Chau, Tran T. ; Chau, Nguyen V.V. ; Arjyal, Amit ; Koirala, Samir ; Basnyat, Buddha ; Dolecek, Christiane ; Farrar, Jeremy ; Baker, Stephen. / The sensitivity of real-time PCR amplification targeting invasive Salmonella serovars in biological specimens. In: BMC Infectious Diseases. 2010 ; Vol. 10.
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title = "The sensitivity of real-time PCR amplification targeting invasive Salmonella serovars in biological specimens",
abstract = "Background: PCR amplification for the detection of pathogens in biological material is generally considered a rapid and informative diagnostic technique. Invasive Salmonella serovars, which cause enteric fever, can be commonly cultured from the blood of infected patients. Yet, the isolation of invasive Salmonella serovars from blood is protracted and potentially insensitive.Methods: We developed and optimised a novel multiplex three colour real-time PCR assay to detect specific target sequences in the genomes of Salmonella serovars Typhi and Paratyphi A. We performed the assay on DNA extracted from blood and bone marrow samples from culture positive and negative enteric fever patients.Results: The assay was validated and demonstrated a high level of specificity and reproducibility under experimental conditions. All bone marrow samples tested positive for Salmonella, however, the sensitivity on blood samples was limited. The assay demonstrated an overall specificity of 100{\%} (75/75) and sensitivity of 53.9{\%} (69/128) on all biological samples. We then tested the PCR detection limit by performing bacterial counts after inoculation into blood culture bottles.Conclusions: Our findings corroborate previous clinical findings, whereby the bacterial load of S. Typhi in peripheral blood is low, often below detection by culture and, consequently, below detection by PCR. Whilst the assay may be utilised for environmental sampling or on differing biological samples, our data suggest that PCR performed directly on blood samples may be an unsuitable methodology and a potentially unachievable target for the routine diagnosis of enteric fever.",
author = "Nga, {Tran V.T.} and Abhilasha Karkey and Sabina Dongol and Thuy, {Hang N.} and Sarah Dunstan and Kathryn Holt and Tu, {Le T.P.} and Campbell, {James I.} and Chau, {Tran T.} and Chau, {Nguyen V.V.} and Amit Arjyal and Samir Koirala and Buddha Basnyat and Christiane Dolecek and Jeremy Farrar and Stephen Baker",
year = "2010",
month = "5",
day = "21",
doi = "10.1186/1471-2334-10-125",
language = "English",
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Nga, TVT, Karkey, A, Dongol, S, Thuy, HN, Dunstan, S, Holt, K, Tu, LTP, Campbell, JI, Chau, TT, Chau, NVV, Arjyal, A, Koirala, S, Basnyat, B, Dolecek, C, Farrar, J & Baker, S 2010, 'The sensitivity of real-time PCR amplification targeting invasive Salmonella serovars in biological specimens', BMC Infectious Diseases, vol. 10, 125. https://doi.org/10.1186/1471-2334-10-125

The sensitivity of real-time PCR amplification targeting invasive Salmonella serovars in biological specimens. / Nga, Tran V.T.; Karkey, Abhilasha; Dongol, Sabina; Thuy, Hang N.; Dunstan, Sarah; Holt, Kathryn; Tu, Le T.P.; Campbell, James I.; Chau, Tran T.; Chau, Nguyen V.V.; Arjyal, Amit; Koirala, Samir; Basnyat, Buddha; Dolecek, Christiane; Farrar, Jeremy; Baker, Stephen.

In: BMC Infectious Diseases, Vol. 10, 125, 21.05.2010.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - The sensitivity of real-time PCR amplification targeting invasive Salmonella serovars in biological specimens

AU - Nga, Tran V.T.

AU - Karkey, Abhilasha

AU - Dongol, Sabina

AU - Thuy, Hang N.

AU - Dunstan, Sarah

AU - Holt, Kathryn

AU - Tu, Le T.P.

AU - Campbell, James I.

AU - Chau, Tran T.

AU - Chau, Nguyen V.V.

AU - Arjyal, Amit

AU - Koirala, Samir

AU - Basnyat, Buddha

AU - Dolecek, Christiane

AU - Farrar, Jeremy

AU - Baker, Stephen

PY - 2010/5/21

Y1 - 2010/5/21

N2 - Background: PCR amplification for the detection of pathogens in biological material is generally considered a rapid and informative diagnostic technique. Invasive Salmonella serovars, which cause enteric fever, can be commonly cultured from the blood of infected patients. Yet, the isolation of invasive Salmonella serovars from blood is protracted and potentially insensitive.Methods: We developed and optimised a novel multiplex three colour real-time PCR assay to detect specific target sequences in the genomes of Salmonella serovars Typhi and Paratyphi A. We performed the assay on DNA extracted from blood and bone marrow samples from culture positive and negative enteric fever patients.Results: The assay was validated and demonstrated a high level of specificity and reproducibility under experimental conditions. All bone marrow samples tested positive for Salmonella, however, the sensitivity on blood samples was limited. The assay demonstrated an overall specificity of 100% (75/75) and sensitivity of 53.9% (69/128) on all biological samples. We then tested the PCR detection limit by performing bacterial counts after inoculation into blood culture bottles.Conclusions: Our findings corroborate previous clinical findings, whereby the bacterial load of S. Typhi in peripheral blood is low, often below detection by culture and, consequently, below detection by PCR. Whilst the assay may be utilised for environmental sampling or on differing biological samples, our data suggest that PCR performed directly on blood samples may be an unsuitable methodology and a potentially unachievable target for the routine diagnosis of enteric fever.

AB - Background: PCR amplification for the detection of pathogens in biological material is generally considered a rapid and informative diagnostic technique. Invasive Salmonella serovars, which cause enteric fever, can be commonly cultured from the blood of infected patients. Yet, the isolation of invasive Salmonella serovars from blood is protracted and potentially insensitive.Methods: We developed and optimised a novel multiplex three colour real-time PCR assay to detect specific target sequences in the genomes of Salmonella serovars Typhi and Paratyphi A. We performed the assay on DNA extracted from blood and bone marrow samples from culture positive and negative enteric fever patients.Results: The assay was validated and demonstrated a high level of specificity and reproducibility under experimental conditions. All bone marrow samples tested positive for Salmonella, however, the sensitivity on blood samples was limited. The assay demonstrated an overall specificity of 100% (75/75) and sensitivity of 53.9% (69/128) on all biological samples. We then tested the PCR detection limit by performing bacterial counts after inoculation into blood culture bottles.Conclusions: Our findings corroborate previous clinical findings, whereby the bacterial load of S. Typhi in peripheral blood is low, often below detection by culture and, consequently, below detection by PCR. Whilst the assay may be utilised for environmental sampling or on differing biological samples, our data suggest that PCR performed directly on blood samples may be an unsuitable methodology and a potentially unachievable target for the routine diagnosis of enteric fever.

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U2 - 10.1186/1471-2334-10-125

DO - 10.1186/1471-2334-10-125

M3 - Article

VL - 10

JO - BMC Infectious Diseases

JF - BMC Infectious Diseases

SN - 1471-2334

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