Serotype-specific differences in the laboratory parameters among hospitalized children with dengue and genetic diversity of dengue viruses circulating in Tamil Nadu, India during 2017.

In 2017, Tamil Nadu, a southern state, had the second highest number of dengue cases from India. In the present study, the serotype-specific differences in the clinical manifestations and laboratory parameters among hospitalized children with dengue were investigated and molecular characterization of the circulating dengue virus (DENV) serotypes during 2017 in Tamil Nadu was performed. Eighty children with dengue-like symptoms consecutively admitted to a tertiary care hospital and positive for DENV NS1 antigen were investigated for DENV serotype utilizing a real-time reverse transcriptase based polymerase chain reaction assay. Complete envelope (E) gene sequencing of the DENV strains was performed. Seventy samples were positive for serotyping (25 DENV-1, 17 DENV-2, six DENV-3, and 22 DENV-4). DENV-4 infections were associated with elevated levels of liver enzymes; Alanine aminotransferase (P = .021) and aspartate aminotransferase (P = .001). However, none of the serotype was associated with any specific clinical features and severe dengue. Asian and American/African genotypes of DENV-1 were cocirculating. The circulating genotype was cosmopolitan for DENV-2 with multiple lineages, genotype III for DENV-3 and genotype I for DENV-4. Unique mutations were present in the 2017 DENV-4 isolates. The present study suggests the association of DENV-4 with elevated liver enzymes in children hospitalized for dengue. Further, the study reports the genetic diversity of DENV circulating in Tamil Nadu during 2017. The study calls for continuous monitoring of the circulating serotypes and genotypes at regional level in India which might result in a region wise database useful in predicting future outbreaks.

Spatio-temporal distribution analysis of circulating genotypes of dengue virus type 1 in western and southern states of India by a one-step real-time RT-PCR assay.

Dengue virus type 1 (DENV-1) Asian and American/African (AM/AF) genotypes were reported to be co-circulating in southern and western states of India based on envelope (E) gene sequencing of few representative samples. The objective of the present study was to develop a one-step real-time RT-PCR to discriminate between Asian and AM/AF genotypes of DENV-1 and investigate the spatio-temporal distribution of the DENV-1 genotypes in southern and western states of India. A one-step real-time RT-PCR to discriminate the Asian and AM/AF genotypes of DENV-1 was developed and validated using 40 samples (17 Asian and 23 AM/AF), for which the envelope (E) gene sequence data was available. DENV-2, DENV-3 and DENV-4 isolates, one each and DENV negative samples (n = 17) were also tested by the assay. Additional 296 samples positive for DENV-1 from selected Southern and Western states of India were genotyped using the real-time RT-PCR assay. Among the samples used for validation, the genotyping results were concordant with sequencing results for 39 samples. In the one discordant sample which was positive for AM/AF by sequencing, the genotyping assay tested positive for both Asian and AM/AF genotype. DENV-2, DENV-3 and DENV-4 isolates were not reactive in the assay. None of the DENV negative samples were positive (sensitivity 100% and specificity 98.2%). A total of 336 samples (40 samples with sequence data and 296 samples without sequence data) were used for spatio-temporal distribution analysis. The results revealed that the Asian genotype was the predominant genotype in Tamil Nadu and Kerala, the southern states. The AM/AF genotype was the predominant genotype in Maharashtra, a western state of India. In Nashik district of Maharashtra, Asian genotype was observed in 32.6% of DENV-1 samples during 2017 while the same decreased to 7.3% during 2018. In Pune district, Asian genotype was observed in 40.0% of DENV-1 samples during 2018 only. To conclude, a one step real-time RT-PCR has been developed for discriminating Asian and AM/AF genotypes of DENV-1. This assay can act as a complement to sequencing but not a substitute and can be utilized in resource limited settings for molecular surveillance of DENV-1. DENV-1 Asian genotype was the dominant genotype in South India while, AM/AF genotype was dominant in Western India.

Effect of delay in analysis on neonatal cerebrospinal fluid parameters.

OBJECTIVES: The effect of delayed analysis on cerebrospinal fluid (CSF) white blood cell (WBC) count and glucose has never been studied in neonates. DESIGN: Prospective cohort study. SETTING: Level III newborn unit. PATIENTS: Neonates undergoing lumbar puncture were enrolled after consent. CSF was analysed at baseline (30 minutes) for protein, WBC and glucose; and from the same sample for WBC and glucose after a lag of 2 h and 4 h after lumbar puncture. Those with traumatic/inadequate CSF were excluded. Subjects were classified in three groups (n = 20 each) based on baseline WBC count: no WBC, 1-30 WBC and >30 WBC/microl. Analysis was by repeated-measures ANOVA. RESULTS: There was a significant decline in mean (SD) CSF glucose from baseline to 2 h and 4 h (41.0 (19) to 38.3 (19) and 36.2 (20) mg/dl, respectively) and WBC count (36 (45) to 28.6 (38) and 23.8 (34) cells/microl, respectively; both p<0.001). CSF glucose and WBC declined in all three groups (p<0.001). High baseline CSF WBC (p<0.001) and protein (p<0.001) was associated with a more rapid decline in the levels of CSF WBC, but not glucose. True CSF parameters could be predicted from 4-h parameters: "baseline glucose 5.4 + 0.98 (4-h glucose)" (adjusted R(2) 97.2%, p<0.001) and "baseline WBC 1.3 (4-h WBC) +0.05 (protein)" (adjusted R(2) 98.8%, p<0.001). In group 3, a diagnosis of meningitis (based on pleocytosis) would be missed in 52.6% and 78.9% subjects at 2 h and 4 h, respectively. CONCLUSIONS: CSF WBC count and glucose decrease significantly with time. Reliance on WBC counts of delayed samples can result in underdiagnosis.