Multiplex PCR Developed for Neglected Infectious Diseases

Diagnostic tests for these diseases depend on antibody detection. However, antibody detection is still limited by its tendency to return negative results during the early phase of these diseases.

Early clinical manifestations of scrub typhus, murine typhus, and leptospirosis, such as high fever, headache, muscular pain, and anorexia, are non-specific and usually diagnosed as acute undifferentiated febrile illness. These clinical manifestations range from mild, severe, to possibly fatal.

Scientists at the Chulalongkorn University (Bangkok, Thailand) and their colleagues used 83 EDTA blood samples taken from patients presented with acute undifferentiated febrile illness at various institutions. Patients included were over 18 years old, having acute fever (38 °C or higher), exhibiting non-specific symptoms, (headache, muscular pain, anorexia, and rash for 3–5 days), and tested negative for both influenza and dengue antigens.

The team developed a multiplex polymerase chain reaction PCR to combine three primer pairs that target specific genes encoding 56-kDa TSA of Orientia tsutsugamushi, 17-kDa antigen of Rickettsia typhi, and LipL32 of Leptospira interrogans and evaluate its performance in comparison to the standard serological tests. Multiplex PCR amplification was performed on a Thermal Cycler. Specificity of the multiplex PCR was tested with unrelated pathogens.

Multiplex PCR results were positive in 39 samples (47%), consisting of 22 samples of scrub typhus (26.5%), 11 samples of leptospirosis (13.25%), five samples of murine typhus (6.02%), and one sample of co-infection between scrub typhus and leptospirosis (1.2%). Twenty samples were detected by both serological methods and multiplex PCR. Nineteen samples were detected only by multiplex PCR but tested negative by standard serological methods. The diagnostic sensitivity and specificity of multiplex PCR were 100% and 70%, respectively, when serological methods were used as gold standard. Positive predictive value and negative predictive value of this assay were 51% and 100%, respectively.

The authors concluded that they had developed a novel multiplex PCR assay for identifying causative agents of scrub typhus, murine typhus, and leptospirosis in blood samples. This method is a rapid, sensitive, and specific diagnostic test. The multiplex PCR assay will become useful for the development of better health care and treatment of patients presented with acute undifferentiated febrile illness, particularly in endemic areas of these diseases. The study was published on July 8, 2019, in the journal PLOS Neglected Tropical Diseases.

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Chulalongkorn University