Je-Hyoung Kim1, Chom-Kyu Chong2, Mangalam Sinniah3, Jeyaindran Sinnadurai3, Hyun-Ok Song4, Hyun Park5. 1. Zoonosis Research Center, Department of Infection Biology, Wonkwang University School of Medicine, Iksan, Jeonbuk, Republic of Korea. 2. Department of Biochemistry, Division of Life Science, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea. 3. Hospital Kuala Lumpur, Jalan Pahang, Kuala Lumpur 50586 Malaysia. 4. Zoonosis Research Center, Department of Infection Biology, Wonkwang University School of Medicine, Iksan, Jeonbuk, Republic of Korea. Electronic address: hosong@wku.ac.kr. 5. Zoonosis Research Center, Department of Infection Biology, Wonkwang University School of Medicine, Iksan, Jeonbuk, Republic of Korea. Electronic address: hyunpk@wku.ac.kr.
Abstract
BACKGROUND: Dengue is a mosquito-borne disease that causes a public health problem in tropical and subtropical countries. Current immunological diagnostics based on IgM and/or nonstructural protein 1 (NS1) antigen are limited for acute dengue infection due to low sensitivity and accuracy. OBJECTIVES: This study aimed to develop a one-step multiplex real-time RT-PCR assay showing higher sensitivity and accuracy than previous approaches. STUDY DESIGN: Serotype-specific primers and probes were designed through the multiple alignment of NS1 gene. The linearity and limit of detection (LOD) of the assay were determined. The assay was clinically validated with an evaluation panel that was immunologically tested by WHO and Malaysian specimens. RESULTS: The LOD of the assay was 3.0 log10 RNA copies for DENV-1, 2.0 for DENV-3, and 1.0 for DENV-2 and DENV-4. The assay showed 95.2% sensitivity (20/21) in an evaluation panel, whereas NS1 antigen- and anti-dengue IgM-based immunological assays exhibited 0% and 23.8-47.6% sensitivities, respectively. The assay showed 100% sensitivity both in NS1 antigen- and anti-dengue IgM-positive Malaysian specimens (26/26). The assay provided the information of viral loads and serotype with discrimination of heterotypic mixed infection. CONCLUSIONS: The assay could be clinically applied to early dengue diagnosis, especially during the first 5 days of illness and approximately 14 days after infection showing an anti-dengue IgM-positive response.
BACKGROUND: Dengue is a mosquito-borne disease that causes a public health problem in tropical and subtropical countries. Current immunological diagnostics based on IgM and/or nonstructural protein 1 (NS1) antigen are limited for acute dengue infection due to low sensitivity and accuracy. OBJECTIVES: This study aimed to develop a one-step multiplex real-time RT-PCR assay showing higher sensitivity and accuracy than previous approaches. STUDY DESIGN: Serotype-specific primers and probes were designed through the multiple alignment of NS1 gene. The linearity and limit of detection (LOD) of the assay were determined. The assay was clinically validated with an evaluation panel that was immunologically tested by WHO and Malaysian specimens. RESULTS: The LOD of the assay was 3.0 log10 RNA copies for DENV-1, 2.0 for DENV-3, and 1.0 for DENV-2 and DENV-4. The assay showed 95.2% sensitivity (20/21) in an evaluation panel, whereas NS1 antigen- and anti-dengue IgM-based immunological assays exhibited 0% and 23.8-47.6% sensitivities, respectively. The assay showed 100% sensitivity both in NS1 antigen- and anti-dengue IgM-positive Malaysian specimens (26/26). The assay provided the information of viral loads and serotype with discrimination of heterotypic mixed infection. CONCLUSIONS: The assay could be clinically applied to early dengue diagnosis, especially during the first 5 days of illness and approximately 14 days after infection showing an anti-dengue IgM-positive response.
Authors: Kevin Vanneste; Linda Garlant; Sylvia Broeders; Steven Van Gucht; Nancy H Roosens Journal: BMC Bioinformatics Date: 2018-09-04 Impact factor: 3.169