Development of multiplex PCR for neglected infectious diseases

PLoS Negl Trop Dis. 2019 Jul 8;13(7):e0007440. doi: 10.1371/journal.pntd.0007440. eCollection 2019 Jul.

Abstract

Scrub typhus, murine typhus, and leptospirosis are widely neglected infectious diseases caused by Orientia tsutsugamushi, Rickettsia typhi, and pathogenic Leptospira spp., respectively. Patients usually present with non-specific symptoms and therefore are commonly diagnosed with acute undifferentiated febrile illness. Consequently, patients face delayed treatment and increased mortality. Antibody-based serological test currently used as gold standard has limitations due to insufficient antibody titers, especially in the early phase of infection. In this study, we aimed to develop multiplex PCR to combine 3 primer pairs that target specific genes encoding 56-kDa TSA of O. tsutsugamushi, 17-kDa antigen of R. typhi, and LipL32 of L. Interrogans and evaluate its performance in comparison to the standard serological tests. Using EDTA blood samples of known patients, the sensitivity and specificity of our multiplex PCR was 100% and 70%, respectively. In addition, the assay was able to diagnose the co-infection of scrub typhus and leptospirosis. The assay may be useful in identifying causative agents during the early phase of these diseases, enabling prompt and appropriate treatment.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Bacterial / blood
  • Antigens, Bacterial / genetics*
  • DNA Primers / genetics
  • Fever / diagnosis
  • Fever / microbiology*
  • Humans
  • Leptospira / genetics
  • Leptospirosis / diagnosis
  • Mice
  • Multiplex Polymerase Chain Reaction / methods*
  • Neglected Diseases / diagnosis*
  • Neglected Diseases / microbiology*
  • Orientia tsutsugamushi / genetics
  • Rickettsia typhi / genetics
  • Scrub Typhus / diagnosis
  • Sensitivity and Specificity
  • Serologic Tests
  • Typhus, Endemic Flea-Borne / diagnosis

Substances

  • Antibodies, Bacterial
  • Antigens, Bacterial
  • DNA Primers

Grants and funding

The funder is as the Research Chair Grant, National Science an Technology Development Agency, Thailand, Chulalongkorn University graduate scholarship to commemorate to the 72nd anniversary of his majesty King Bhumibol Adulyadej, and a research group in the Ratchadaphiseksomphot Endowment Fund, Chulalongkorn University, Thailand. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.