A novel multiplex real-time PCR for the detection of Salmonella Typhi, Salmonella Paratyphi A and Burkholderia pseudomallei in clinical samples

Abstract

Enteric fever, caused by infection with Salmonella Typhi or Paratyphi A, B, or C (typhoidal Salmonella) and melioidosis are among the most common bacterial causes of acute febrile illness in tropical and subtropical countries. The diseases are widely spread even in developed countries mainly affecting travellers returning from endemic areas. Melioidosis is an increasingly recognized fatal septicemic infection also mimicking tuberculosis. These pathogens are largely underreported due to lack of sensitive and specific diagnostic tools. In this study, an inhouse multiplex real-time PCR assay was developed for the simultaneous detection of Salmonella Typhi, S. Paratyphi A and B. pseudomallei using specific primers. The inhouse developed assay was evaluated on buffy coat and serum samples collected from patients with acute febrile illness at four different centres. The assay had a detection limit of less than 1 genome copy for S. Typhi and S. Paratyphi A and 18 genome copies per 10 µl of PCR input for B. pseudomallei. Among the 1101 samples tested by multiplex real-time PCR, one (0.09%) sample was positive for S. Typhi. One sample was positive for blood culture identified as S. Typhi but negative by real-time PCR. The samples were negative for S. Paratyphi A and B. pseudomallei. The multiplex real-time assay would be highly useful as a diagnostic aid for the syndromic approach and comprehensive diagnosis of enteric fever and melioidosis. The assay could improve the overall diagnostic capability and be a useful tool during outbreak investigations.