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Open Access Research article

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

Tran Vu Thieu Nga12, Abhilasha Karkey3, Sabina Dongol3, Hang Nguyen Thuy12, Sarah Dunstan14, Kathryn Holt5, Le Thi Phuong Tu12, James I Campbell14, Tran Thuy Chau12, Nguyen Van Vinh Chau2, Amit Arjyal3, Samir Koirala3, Buddha Basnyat3, Christiane Dolecek14, Jeremy Farrar14 and Stephen Baker14*

Author Affiliations

1 Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam

2 The Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam

3 Oxford University Clinical Research Unit, Patan Academy of Health Sciences, Kathmandu, Nepal

4 Wellcome Trust Major Overseas Programme, Ho Chi Minh City, Vietnam

5 Department of Microbiology and Immunology, The University of Melbourne, Victoria, Australia

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BMC Infectious Diseases 2010, 10:125  doi:10.1186/1471-2334-10-125

Published: 21 May 2010

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.