Evaluation of in-house dengue real-time PCR assays in West Java, Indonesia

Abstract

Dengue is an infectious disease caused by infection of dengue virus (DENV) transmitted by Aedes aegypti and Aedes albopictus. In Indonesia, dengue commonly occurs with an increasing incidence rate annually. It is known that early detection of dengue infection is one of the keys to controlling this disease outbreak. Rapid and accurate early detection to diagnose dengue can be achieved by molecular tests, one of which is through a real-time PCR method. However, real-time PCR assay for dengue developed based on Indonesian DENV sequences has not been available. Therefore, we developed in-house dengue real-time PCR (SYBR- and TaqMan-based) assays and evaluated those assays in routine clinical testing in the community. These assays target the 3′ UTR region of the four DENV serotypes and was found to be specific for DENV. The most sensitive assay was the TaqMan assay with the LOD95% of 482 copy/ml, followed by the SYBR assay with the LOD95% of 14,398 copy/ml. We recruited dengue suspected patients from three primary health care services in West Java, Indonesia to represent the community testing setting. Dengue infection was examined using the two in-house real-time PCR assays along with NS1, IgM, and IgG rapid diagnostic tests (RDT). In total, as many as 74 clinical specimens of dengue suspected patients were included in this study. Among those patients, 21 were positive for TaqMan assay, 17 were positive for SYBR assay, nine were positive for NS1 test, six were positive for both IgG and IgM tests, and 22 were positive for IgG test only. Compared with our in-house TaqMan assay, the sensitivity of NS1 test, IgM test, and IgG test were 42.86%, 14.29%, and 28.57% respectively. Among these three RDT tests, NS1 showed 100% specificity. Thus, our study confirmed that NS1 test showed high specificity, indicating that a positive result of NS1 can be confidently considered a dengue case. However, NS1, IgM, and IgG tests with RDT are not enough to diagnose a dengue case. We suggest applying the high sensitivity and specificity rRT-PCR test as the gold standard for early detection and antibody test as a follow-up test for rRT-PCR negative cases.