Standardization and Evaluation of an In-house Multiplex Real-time Polymerase Chain Reaction for Simultaneous Detection of Pathogenic Species of Brucella, Rickettsia, and Leptospira in Patients with Acute Febrile Illness

Abstract

Background and Objectives: Acute febrile illness is caused by a wide range of etiologies. Brucella, Rickettsia and Leptospira as a cause of acute febrile illness have not been well studied. Identification by culture is laborious and diagnosis is often based on serological tests. We aimed to develop an in-house multiplex real-time PCR assay for the simultaneous detection of Brucella, Rickettsia and Leptospira and evaluate on samples collected from patients presenting with acute febrile illness. Methods: Samples (n=1101) were collected from patients presenting with acute febrile illness from different regions. An inhouse multiplex real-time PCR was developed for the specific detection of Brucella species, Rickettsia species and pathogenic species of Leptospira. The assay was evaluated on clinical samples. IgM ELISA was carried out on randomly selected samples (n=178). Results: The detection limit of the multiplex real-time PCR assay was 6.3, 43.7 and 1.2 genome copies per 10 μl of PCR input for Brucella, Rickettsia and Leptospira respectively. Among 1101 samples, Leptospira was positive in 1.36% samples and Rickettsia was positive in 0.36% samples. Among random samples, 37.1% of samples were positive for Brucella IgM, 19.6% were positive for Rickettsia IgM and 11.2% were positive for Leptospira IgM. Interpretation and Conclusions: The study showed a high seroprevalence of Brucellosis and Rickettsiosis among the samples. The in-house multiplex real-time PCR assay will be a useful tool in the syndromic diagnosis for a specific and comprehensive laboratory diagnostic testing.