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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.

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