Development of TaqMan® Probe-based RT-qPCR Diagnostic Assay for Rapid Detection of Typhoid Fever

Abstract

Typhoid fever is an important worldwide contributor to illness and death, with an approximate annual prevalence of nearly 9-11 million cases, leading to approximately 110,000 deaths annually worldwide. According to WHO, Pakistan has the highest rate of typhoid fever among south Asian countries with 0.1 million cases annually. Typhoid fever is confirmed by laboratory diagnosis. Despite being recognized for over a century, typhoid fever lacks a definitive test diagnostic biomarker. Blood culture is used as a gold standard for diagnosis, but the sensitivity of blood culture is less than 50%. Bone marrow culture is not used because of the invasive nature. The Widal test quantifies the antibodies produced against flagella and LPS in the serum of patients suspected of having typhoid fever by agglutinating S. typhi cells. The earlier use of antibiotic medication can provide a false negative result in the Widal test. Diagnostic methods based on real time PCR have overcome these drawbacks of conventional methods by providing increased sensitivity for the detection of Salmonella typhi. Several genes have been targeted for the detection of Salmonella typhi in previous studies. This study is focused on developing a TaqMan® probe based Real-time multiplex PCR Assay for the detection of Salmonella typhi by targeting ttr, tviB, and staG genes. The assay was validated with bacterial and blood samples from suspected typhoid patients. All the cultured bacterial samples were positive by the developed Assay making the assay 100% sensitive and specific. In addition, out of 300 blood samples collected from suspected typhoid patients, 48 were positive by developed assay. Using blood culture as a standard diagnostic assay, developed PCR assay showed 100% sensitivity and 95% specificity. But to find the exact sensitivity and specificity, the assay needs to be compared with developed real-time PCR diagnostic kit.