Use of whole blood over plasma enhances the detection of dengue virus RNA: possible utility in dengue vaccine trials.

In the present study, the utility of viral RNA isolated from whole blood over plasma for detection of dengue virus (DENV) was investigated in 80 samples referred for serotyping by DENV serotype-specific one-step real-time RT-PCR. DENV RNA was detected in 71.25% of the whole blood samples compared to 46.25% in the corresponding plasma samples. In secondary infections, DENV RNA was detected in 83.3% of whole blood samples, while it was detected in 40.5% of plasma samples (P = 0.0001). Non-structural protein 1 (NS1) antigen was detected in only 54.8% of the secondary infections. The detection rate of DENV RNA in whole blood is higher than in plasma. We suggest that one-step real-time RT-PCR using RNA from whole blood combined with an NS1 ELISA should be the choice for dengue diagnosis in dengue vaccine trials.

Seroprevalence of dengue in a rural and an urbanized village: A pilot study from rural western India.

BACKGROUND & OBJECTIVES: Dengue is highly prevalent in tropical and subtropical regions. The prevalence of dengue is influenced by number of factors, i.e. host, vector, virus and environmental conditions including urbanization and population density. A cross sectional study was undertaken to determine the seroprevalence of dengue in two selected villages that differed in the level of their urbanization and population density. METHODS: Two villages with demographically well-defined populations close to Pune, a metropolitan city of western India, were selected for the study. Age stratified serosurvey was carried out during February to May 2011 in the two villages-a rural village A, located 6 km from the national highway with a population density of 159/km2 ; and an urbanized village B, located along the highway with a population density of 779/km2 . Assuming a low seroposi- tivity of 10%, 702 serum samples were collected from village A. Sample size for village B was calculated on the basis of seropositivity obtained in village A, and 153 samples were collected. Serum samples were tested for the presence of dengue virus (DENV)-specific IgG. Simple proportional analyses were used to calculate and compare the seroprevalence. RESULTS: Of the 702 samples collected from village A, 42.8% were found positive for anti-DENV IgG. A significantly higher seropositivity for DENV (58.8%) was found in village B. In village A, there was an age dependent increase in seroprevalence; whereas, in village B, there was a steep increase from 17% positivity in 0-10 yr age group to 72% in the 11-20 yr age group. The seroprevalence was almost similar in the older age groups. INTERPRETATION & CONCLUSION: The observations suggested that prevalence of dengue is probably associated with urbanization and host population density. Areas that are in the process of urbanization needs to be monitored for prevalence of dengue and its vector, and appropriate vector control measures may be implemented.

Profile of killer cell immunoglobulin-like receptor and its human leucocyte antigen ligands in dengue-infected patients from Western India.

Killer cell immunoglobulin-like receptors (KIRs) regulate the activation of natural killer cells (NKs). Qualitative and quantitative differences in the type and the number of KIRs expressed on NK cells affect its activation which would influence the outcome of the disease. In this study, 114 hospitalized cases of dengue [82 dengue fever (DF) and 32 dengue haemorrhagic fever (DHF) cases] and 104 healthy controls (HC) without no known history of hospitalization for dengue-like illness were investigated for their KIR gene profile to find out the association of KIR genes with dengue disease severity. KIR gene profile was investigated using duplex sequence-specific priming polymerase chain reaction-based typing system. The results revealed a higher frequency of KIR3DL1 gene [P = 0.0225; odds ratio (OR) 4.1 95% confidence interval (CI) 1.1-14.8] and lower frequency of KIR3DS1/3DS1 genotype [P = 0.0225; OR 0.24 95% CI (0.068-0.88)] in DF cases compared to HC. Immunoglobulin-like receptor gene frequencies were not different between DHF and DF or HC. The results suggest that KIR3DL1/KIR3DS1 locus might be associated with the risk of developing DF.

Association of combinations of interleukin-10 and pro-inflammatory cytokine gene polymorphisms with dengue hemorrhagic fever.

Pro-inflammatory and anti-inflammatory cytokines have been shown to play an important role in dengue disease pathogenesis. In the present study, to find out whether single nucleotide polymorphisms (SNPs) in the pro-inflammatory and anti-inflammatory cytokine genes are associated with dengue disease severity, SNPs in TNF, IFNG, IL1B, IL8, IL0, IL17A and IL17F genes were investigated using polymerase chain reaction based methods in 132 dengue (DEN) cases [87 dengue fever (DF), 45 dengue hemorrhagic fever (DHF) cases] and 108 apparently healthy controls (HC) from Pune, Maharashtra, western India. Under recessive genetic model (C/C vs. T/T+T/C), the TNF rs1799964 C/C genotype was significantly associated with DEN [P=0.014, OR with 95% CI 3.07 (1.18-7.98)]. Frequency of T/C genotype of IL17F rs763780 was significantly lower in DEN group as compared to HC [P=0.033, OR with 95% CI 0.43 (0.19-0.95)]. Under overdominant genetic model (A/T vs. A/A+T/T), IL8 rs4973 A/T genotype was negatively associated with DHF compared to HCs [p=0.029, OR with 95% CI 0.43 (0.20-0.93)]. Under overdominant genetic model, A/G genotype of IL10 rs1800871 was significantly negatively associated with DHF compared to DF cases [p=0.014, OR with 95% CI 0.35 (0.15-0.84)]. Significantly higher frequency of the combined genotype IL10 A/A-IFNG A/T and lower frequency of the combined genotypes IL10 A/G-IL1B A/A, IL10 A/G-IL8 A/T and IL10 A/G-IL17F T/T were observed in DHF cases compared to DF. The results suggest that heterozygous genotypes of IL8 rs4973 and IL10 rs1800871 are associated with reduced risk of DHF. Combinations of IL10 rs1800871 and pro-inflammatory cytokine genotypes influence the risk of DHF.

Polymorphisms in RNA sensing toll like receptor genes and its association with clinical outcomes of dengue virus infection.

Functional polymorphisms in RNA recognizing toll like receptors (TLR) 3, 7, 8 and toll-interleukin-1 receptor domain containing adapter protein adapter (TIRAP) coding genes were investigated in 120 dengue cases [87 dengue fever (DF) cases and 33 dengue hemorrhagic fever (DHF) cases] and 109 healthy controls (HC) to identify their association with clinical outcomes of dengue virus infection. Results revealed significantly lower frequency of TLR3 rs3775291 T allele [DHF vs. DF P = 0.015 odds ratio (OR) with 95% confidence interval (CI) 0.390 (0.160­0.880); DHF vs. HC P = 0.018 OR with 95% CI 0.410 (0.170­0.900)] and 'T' allele carriers [DHF vs. DF P = 0.008 OR with 95% CI 0.288 (0.115­0.722); DHF vs. HC P = 0.040 OR with 95% CI 0.393 (0.162­0.956)] and higher frequency of TIRAP rs8177374 'C/T' genotype [DHF vs. HC P = 0.020 OR with 95% CI 2.643 (1.167­5.986)] in DHF. Higher frequency of TLR8 rs3764879­rs3764880 haplotype C-A was observed in male DF cases compared to male HC [P = 0.025 OR with 95% CI 2.185 (1.101­4.336)]. The results suggest that TLR3 and TIRAP gene variants influence the risk for DHF.

Association of vitamin D receptor gene polymorphisms with clinical outcomes of dengue virus infection.

Vitamin D is known to affect pathogenesis of dengue through modulation of immune responses. Vitamin D exerts its effects through vitamin D receptor (VDR). The functioning of VDR is affected by the gene polymorphisms in the coding (rs2228570) and 3'untranslated region (UTR) (rs1544410, rs7975232 and rs731236). In the present study, VDR gene polymorphisms were investigated in 112 dengue infected patients (83 dengue fever (DF) and 29 dengue hemorrhagic fever cases (DHF)) and 105 apparently healthy controls (HCs) using polymerase chain reaction based restriction fragment length polymorphisms methods. HCs had no documented evidence of symptomatic dengue. Results revealed significantly lower frequency of 'C' allele of rs7975232 in all dengue patients (DEN) as compared to HCs [(P corrected (Pc)=0.014, Odds ratio (OR) 0.51]. The frequency of C/C genotype of rs7975232 was significantly lower in DEN and DF cases compared to HCs (DEN vs. HCs: Pc=0.0184, OR 0.24; DF cases vs. HCs: Pc=0.028, OR 0.21). The frequency of T allele of rs2228570 in a dominant mode was significantly higher in DHF cases as compared to DF cases (P=0.034 OR 2.58). A significantly lower frequency of the haplotype G-C-T (Pc=0.0135) and higher frequency of the haplotype G-A-T (Pc=0.000085) was observed in DEN and DF cases as compared to HCs. The results suggest that the 3'UTR haplotypes of VDR gene are differentially associated with risk of symptomatic dengue requiring hospitalization. The 'T' allele of rs2228570 polymorphism in a dominant mode of inheritance is associated with DHF.