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.

Figure 1

Real-time PCR amplification of S. Typhi and S. Paratyphi A in blood and bone marrow specimens from patients with culture confirmed enteric fever. The Ct values of amplification positive blood and bone marrow specimens have been converted by dilution factor to copies per ml of biological material (y axis). The median and quartile ranges of the number of copies per ml of biological sample are shown for amplification positive blood samples with S. Paratyphi A (n = 18), amplification positive blood samples with S. Typhi (n = 23) and amplification positive bone marrow samples with S. Typhi (n = 28). Statistical significance was calculated using a none-parametric student's t-test.