Improving the Diagnosis of Scrub Typhus by Combining groEL Based Polymerase Chain Reaction and IgM ELISA

Abstract

Introduction: Scrub typhus, an acute febrile illness, caused by Orientia tsutsugamushi, is an important cause of pyrexia of unknown origin in regions of endemicity. This disease is mostly underdiagnosed or misdiagnosed, the reasons for this being a combination of factors which include clinical manifestations that can mimic other infections, lack of easy and reliable diagnostic methods and variation among strains in endemic areas. Hence, easy and reliable methods of diagnosis will contribute to rapid identification and treatment of the infection.

Aim: The aim of the study was to compare four different methods of detection of scrub typhus and to identify one single test or a combination of tests detecting maximum number of cases.

Materials and methods: One hundred and forty-five suspected scrub typhus cases were included in this study. Duration of fever and presence of eschar in each patient was noted down. Enzyme-Linked Immunosorbent Assay (ELISA) to detect Immunoglobulin M (IgM) antibodies and Polymerase Chain Reaction (PCR) to detect three genes of Orientia, namely, 56 kDa, 16S rRNA, and groEL were done on these samples. The results of each test were analyzed to identify the test picking up maximum number of positive samples. Statistical analysis was performed using Chi-square test. The level of significance was set at p<0.05.

Results: These tests showed that IgM ELISA (93%) and PCR (68%) picked up maximum number of positives. Statistical analysis performed using Chi-square test between the diagnostic assays showed that the p - value <0.001 was significant for IgM ELISA. Among the molecular markers, p-value was significant (<0.001) for groEL PCR. Further analysis of eschar positivity and duration of fever also showed that groEL PCR could detect DNA of the bacterium even in cases with 10 days of fever and this PCR was the best among the molecular markers used to detect the infection.

Conclusion: This study suggests that IgM detection by ELISA and conventional groEL PCR, either in combination or alone, depending on the duration of fever, would enhance the diagnosis of scrub typhus.

Keywords: Fever; Laboratory diagnosis; Orientia tsutsugamushi; Rickettsial infections.

[Table/Fig-2]:

Gel electrophoresis picture of amplicons obtained in groEL polymerase chain reaction (PCR). Lane 9 is the positive control and Lane 10 the negative control. Lanes 2, 3, 4, 6, 7 and 8 show amplification products in the PCR while Lane 5 shows sample with no amplification.

[Table/Fig-3]:

Percentage positivity of different diagnostic markers/assays of scrub typhus. The tests were performed on samples from suspected cases.