ABSTRACT
BACKGROUND: Dengue virus serotyping is crucial from clinical management and epidemiological point of view. AIMS: To compare efficacy of two molecular detection and typing methods, namely, multiplex reverse transcription polymerase chain reaction (RT-PCR) and real-time Hybprobe assay using a panel of known dilution of four reference Dengue virus strains and a panel of sera collected from clinically suspected dengue patients. SETTINGS: This study was conducted at a tertiary-care teaching hospital in Delhi, India. MATERIALS AND METHODS: Dengue serotype specific virus strains were used as prototypes for serotyping assays. Viral load was quantified by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). Acute phase serum samples were collected from 79 patients with clinically suspected Dengue fever on their first day of presentation during September-October 2012. Viral RNA from serum and cell culture supernatant was extracted. Reverse transcription was carried out. Quantitative detection of DENV RNA from reference strain culture supernatants and each of the 79 patient samples by real-time PCR was performed using light cycler Taqman master mix kit. Serotyping was done by multiplex RT-PCR assay and Hybprobe assay. RESULTS: The multiplex RT-PCR assay, though found to be 100% specific, couldn't serotype either patient or reference strains with viral load less than 1000 RNA copies/ml. The Hybprobe assay was found to have 100% specificity and had a lower limit of serotype detection of merely 3.54 RNA copies/ml. CONCLUSIONS: HybProbe assay has an important role especially in situations where serotyping is to be performed in clinical samples with low viral load.
Subject(s)
Dengue Virus/classification , Dengue Virus/genetics , Genotyping Techniques/methods , Multiplex Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/methods , Serotyping/methods , Dengue/virology , Humans , India , RNA, Viral/blood , RNA, Viral/genetics , RNA, Viral/isolation & purification , Sensitivity and Specificity , Tertiary Care Centers , Viral LoadABSTRACT
Dengue is an important arboviral disease of tropical and subtropical regions, with significant morbidity and mortality. Dengue virus is antigenically classified into four serotypes, which are further classified into 4-5 genotypes based on their genetic diversity. Since genotypes vary in their virulence, their detection and analysis of spatial and temporal transition are essential. We utilized sequence information from the E-NS1 gene region for molecular and phylogenetic characterization of dengue viruses isolated from dengue patients between 2007 and 2009. All four serotypes and multiple genotypes were detected, with predominance and emergence of DENV-1 genotype V. Phylogenetic analysis revealed the emergence of DENV-1 genotype V from India for the first time, which has replaced the earlier circulating genotype III and genotype I. The circulation of multiple genotypes and genotype replacement is critical, since genotypes vary in their virulence, and this should be a point of concern for healthcare agencies.
Subject(s)
Dengue Virus/classification , Dengue Virus/genetics , Dengue/epidemiology , Dengue/virology , Viral Nonstructural Proteins/genetics , Base Sequence , Dengue Virus/isolation & purification , Disease Outbreaks , Genotype , Humans , India/epidemiology , Molecular Sequence Data , Phylogeny , RNA, Viral/genetics , Sequence Analysis, RNA , Viral Proteins/geneticsABSTRACT
Rotavirus gastroenteritis is a major cause of severe dehydrating diarrhea in children worldwide. Rotavirus G and P genotyping is essential for epidemiological surveillance and for better formulation of candidate rotavirus vaccines. Out of 862 diarrheal stool samples collected from hospitalized children aged < 2 years during February 2005 - March 2007, 318 (36.9%) were positive for rotavirus by ELISA. G and P genotyping was performed on 100 randomly selected positive samples using a seminested multiplex RT-PCR assay. The result of G genotyping indicates G1 (60%) was the most predominant VP7 type, followed by G2 (16%), G9 (8%) and G3 (3%). Two cases of G12 genotype were also observed. P genotypes identified were P[8] (40%) followed by P[4] (26%) and P[6] (17%). The most common G-P combinations were G1P[8] (26%), followed by G1P[4] and G1P[6]. Mixed infection involved 28% of strains. In this study the G1 and P[8] genotypes were the leading G and P types. Two cases with G12 genotype were also observed during the study.
