Detection of Typhoidal and Paratyphoidal Salmonella in Blood by Real-time Polymerase Chain Reaction

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Detection of Typhoidal and Paratyphoidal Salmonella in Blood by Real-time Polymerase Chain Reaction

by Sarah Lindsay November 1, 2015

Authors

Sharon M. Tennant, Deanna Toema, Farah Qamar, Najeeha Iqbal, Mary Adetinuke Boyd, Joanna M. Marshall, William C. Blackwelder, Yukun Wu, Farheen Quadri, Asia Khan, Fatima Aziz, Kumail Ahmad, Adil Kalam, Ehtisham Asif, Shahida Qureshi, Erum Khan, Anita K. Zaidi, and Myron M. Levine

Abstract

Background: The gold standard for diagnosis of enteric fever caused by Salmonella Typhi or Salmonella Paratyphi A or B is bone marrow culture. However, because bone marrow aspiration is highly invasive, many hospitals and large health centers perform blood culture instead. As blood culture has several limitations, there is a need for novel typhoid diagnostics with improved sensitivity and more rapid time to detection.

Methods: We developed a clyA-based real-time polymerase chain reaction (qPCR) method to detect Salmonella Typhi and Salmonella Paratyphi A simultaneously in blood. The sensitivity and specificity of this probeset was first evaluated in vitro in the laboratory and then in a typhoid-endemic population, in Karachi, Pakistan, and in healthy US volunteers.

Results: We optimized a DNA extraction and real-time PCR-based method that could reliably detect 1 colony-forming unit/mL of Salmonella Typhi. The probe set was able to detect clinical Salmonella Typhi and Salmonella Paratyphi A strains and also diarrheagenic Escherichia coli, but not invasive E. coli or other invasive bacteria. In the field, the clyA qPCR diagnostic was 40% as sensitive as blood culture. However, when qPCR-positive specimens were considered to be true positives, blood culture only exhibited 28.57% sensitivity. Specificity was ≥90% for all comparisons and in the healthy US volunteers. qPCR was significantly faster than blood culture in terms of detection of typhoid and paratyphoid.

Conclusions: Based on lessons learned, we recommend that future field trials of this and other novel diagnostics that detect typhoidal and nontyphoidal Salmonella employ multiple methodologies to define a “positive” sample.

 

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