title: Enhancing the sensitivity of Dengue virus serotype detection by RT-PCR among infected children in India.
abstract: Dengue surveillance relies on reverse transcription-polymerase chain reaction (RT-PCR), for confirmation of dengue virus (DENV) serotypes. We compared efficacies of published and modified primer sets targeting envelope (Env) and capsid-premembrane (C-prM) genes for detection of circulating DENV serotypes in southern India. Acute samples from children with clinically-diagnosed dengue were used for RT-PCR testing. All samples were also subjected to dengue serology (NS1 antigen and anti-dengue-IgM/IgG rapid immunochromatographic assay). Nested RT-PCR was performed on viral RNA using three methods targeting 654bp C-prM, 511bp C-prM and 641bp Env regions, respectively. RT-PCR-positive samples were validated by population sequencing. Among 171 children with suspected dengue, 121 were dengue serology-positive and 50 were dengue serology-negative. Among 121 serology-positives, RT-PCR detected 91 (75.2%) by CprM654, 72 (59.5%) by CprM511, and 74 (61.1%) by Env641. Among 50 serology-negatives, 10 (20.0%) were detected by CprM654, 12 (24.0%) by CprM511, and 11 (22.0%) by Env641. Overall detection rate using three methods sequentially was 82.6% (100/121) among serology-positive and 40.0% (20/50) among serology-negative samples; 6.6% (8/120) had co-infection with multiple DENV serotypes. We conclude that detection of acute dengue was enhanced by a modified RT-PCR method targeting the 654bp C-prM region, and further improved by using all three methods sequentially.
authors.affiliation: Division of Infectious Diseases, St. John's Research Institute, St. John's National Academy of Health Sciences, Koramangala, Bangalore, 560034, India. Electronic address: mail2fazil@gmail.com.
authors.forename: Syed Fazil
authors.initials: SF
authors.lastname: Ahamed
authors.affiliation: Division of Infectious Diseases, St. John's Research Institute, St. John's National Academy of Health Sciences, Koramangala, Bangalore, 560034, India. Electronic address: rosario.vivek@gmail.com.
authors.forename: Rosario
authors.initials: R
authors.lastname: Vivek
authors.affiliation: Division of Infectious Diseases, St. John's Research Institute, St. John's National Academy of Health Sciences, Koramangala, Bangalore, 560034, India. Electronic address: shalini.kotabagi@gmail.com.
authors.forename: Shalini
authors.initials: S
authors.lastname: Kotabagi
authors.affiliation: ICGEB-Emory Vaccine Centre, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India. Electronic address: kaustuvnayak@gmail.com.
authors.forename: Kaustuv
authors.initials: K
authors.lastname: Nayak
authors.affiliation: ICGEB-Emory Vaccine Centre, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India. Electronic address: chandeleanmol@gmail.com.
authors.forename: Anmol
authors.initials: A
authors.lastname: Chandele
authors.affiliation: ICGEB-Emory Vaccine Centre, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India; Department of Pediatrics, Emory University, 1760 Haygood Drive, Atlanta, GA, 30322, USA. Electronic address: murali.kaja@emory.edu.
authors.forename: Murali-Krishna
authors.initials: MK
authors.lastname: Kaja
authors.affiliation: Division of Infectious Diseases, St. John's Research Institute, St. John's National Academy of Health Sciences, Koramangala, Bangalore, 560034, India; International Vaccine Access Center, Johns Hopkins Bloomberg School of Public Health, 415 N Washington St, Baltimore 21231, USA. Electronic address: ashet1@jhu.edu.
authors.forename: Anita
authors.initials: A
authors.lastname: Shet
mesh_terms: D002648:Child; D017931:DNA Primers / Q000235:genetics; D003715:Dengue / Q000175:diagnosis* / Q000821:virology; D003716:Dengue Virus / Q000145:classification* / Q000302:isolation & purification*; D006801:Humans; D007194:India; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012703:Serotyping / Q000379:methods*; D015678:Viral Structural Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; D015678:Viral Structural Proteins
keywords: Comparison; Dengue virus; Detection; RT-PCR; Serology; Serotyping