Severe disease during both primary and secondary dengue virus infections in pediatric populations.

Dengue is a global epidemic causing over 100 million cases annually. The clinical symptoms range from mild fever to severe hemorrhage and shock, including some fatalities. The current paradigm is that these severe dengue cases occur mostly during secondary infections due to antibody-dependent enhancement after infection with a different dengue virus serotype. India has the highest dengue burden worldwide, but little is known about disease severity and its association with primary and secondary dengue infections. To address this issue, we examined 619 children with febrile dengue-confirmed infection from three hospitals in different regions of India. We classified primary and secondary infections based on IgM:IgG ratios using a dengue-specific enzyme-linked immunosorbent assay according to the World Health Organization guidelines. We found that primary dengue infections accounted for more than half of total clinical cases (344 of 619), severe dengue cases (112 of 202) and fatalities (5 of 7). Consistent with the classification based on binding antibody data, dengue neutralizing antibody titers were also significantly lower in primary infections compared to secondary infections (P ≤ 0.0001). Our findings question the currently widely held belief that severe dengue is associated predominantly with secondary infections and emphasizes the importance of developing vaccines or treatments to protect dengue-naive populations.

Analysis of dengue specific memory B cells, neutralizing antibodies and binding antibodies in healthy adults from India.

BACKGROUND: The Indian population is facing highest dengue burden worldwide supporting an urgent need for vaccines. For vaccine introduction, evaluation and interpretation it is important to gain a critical understanding of immune memory induced by natural exposure. However, immune memory to dengue remains poorly characterized in this region. METHODS: We enumerated levels of dengue specific memory B cells (MBC), neutralizing (NT) and binding antibodies in healthy adults (n=70) from New Delhi. RESULTS: NT-antibodies, binding antibodies and MBC were detectable in 86%, 86.56% and 81.63% of the subjects respectively. Among the neutralizing positive subjects, 58%, 27%, 5% and 10% neutralized all four, any three, any two and any one dengue serotypes respectively. The presence of the neutralizing antibodies was associated with the presence of the MBC and binding antibodies. However, a massive interindividual variation was observed in the levels of the neutralizing antibodies (range, <1:50-1:30,264), binding antibodies (range, 1:3,000-1:134,000,) as well as the MBC (range=0.006%-5.05%). CONCLUSION: These results indicate that a vast majority of the adults are immune to multiple dengue serotypes and show massive interindividual variation in neutralizing/binding antibodies and MBCs - emphasizing the importance of monitoring multiple parameters of immune memory in order to properly plan, evaluate and interpret dengue vaccines.

Correction: A High Malaria Prevalence Identified by PCR among Patients with Acute Undifferentiated Fever in India.

[This corrects the article DOI: 10.1371/journal.pone.0158816.].

Enhancing the sensitivity of Dengue virus serotype detection by RT-PCR among infected children in India.

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.

Evaluation of a pan-serotype point-of-care rapid diagnostic assay for accurate detection of acute dengue infection.

The catastrophic rise in dengue infections in India and globally has created a need for an accurate, validated low-cost rapid diagnostic test (RDT) for dengue. We prospectively evaluated the diagnostic performance of NS1/IgM RDT (dengue day 1) using 211 samples from a pediatric dengue cohort representing all 4 serotypes in southern India. The dengue-positive panel consisted of 179 dengue real-time polymerase chain reaction (RT-PCR) positive samples from symptomatic children. The dengue-negative panel consisted of 32 samples from dengue-negative febrile children and asymptomatic individuals that were negative for dengue RT-PCR/NS1 enzyme-linked immunosorbent assay/IgM/IgG. NS1/IgM RDT sensitivity was 89.4% and specificity was 93.8%. The NS1/IgM RDT showed high sensitivity throughout the acute phase of illness, in primary and secondary infections, in different severity groups, and detected all 4 dengue serotypes, including coinfections. This NS1/IgM RDT is a useful point-of-care assay for rapid and reliable diagnosis of acute dengue and an excellent surveillance tool in our battle against dengue.

A High Malaria Prevalence Identified by PCR among Patients with Acute Undifferentiated Fever in India.

BACKGROUND: Approximately one million malaria cases were reported in India in 2015, based on microscopy. This study aims to assess the malaria prevalence among hospitalised fever patients in India identified by PCR, and to evaluate the performance of routine diagnostic methods. METHODS: During June 2011-December 2012, patients admitted with acute undifferentiated fever to seven secondary level community hospitals in Assam (Tezpur), Bihar (Raxaul), Chhattisgarh (Mungeli), Maharashtra (Ratnagiri), Andhra Pradesh (Anantapur) and Tamil Nadu (Oddanchatram and Ambur) were included. The malaria prevalence was assessed by polymerase chain reaction (PCR), routine microscopy, and a rapid diagnostic test (RDT) with PCR as a reference method. RESULTS: The malaria prevalence by PCR was 19% (268/1412) ranging from 6% (Oddanchatram, South India) to 35% (Ratnagiri, West India). Among malaria positive patients P. falciparum single infection was detected in 46%, while 38% had P. vivax, 11% mixed infections with P. falciparum and P. vivax, and 5% P. malariae. Compared to PCR, microscopy had sensitivity of 29% and specificity of 98%, while the RDT had sensitivity of 24% and specificity of 99%. CONCLUSIONS: High malaria prevalence was identified by PCR in this cohort. Routine diagnostic methods had low sensitivity compared to PCR. The results suggest that malaria is underdiagnosed in rural India. However, low parasitaemia controlled by immunity may constitute a proportion of PCR positive cases, which calls for awareness of the fact that other pathogens could be responsible for the febrile disease in submicroscopic malaria.

Norovirus Gastroenteritis in a Birth Cohort in Southern India.

BACKGROUND: Noroviruses are an important cause of gastroenteritis but little is known about disease and re-infection rates in community settings in Asia. METHODS: Disease, re-infection rates, strain prevalence and genetic susceptibility to noroviruses were investigated in a birth cohort of 373 Indian children followed up for three years. Stool samples from 1856 diarrheal episodes and 147 vomiting only episodes were screened for norovirus by RT-PCR. Norovirus positivity was correlated with clinical data, secretor status and ABO blood group. RESULTS: Of 1856 diarrheal episodes, 207 (11.2%) were associated with norovirus, of which 49(2.6%) were norovirus GI, 150(8.1%) norovirus GII, and 8 (0.4%) were mixed infections with both norovirus GI and GII. Of the 147 vomiting only episodes, 30 (20.4%) were positive for norovirus in stool, of which 7 (4.8%) were norovirus GI and 23 (15.6%) GII. At least a third of the children developed norovirus associated diarrhea, with the first episode at a median age of 5 and 8 months for norovirus GI and GII, respectively. Norovirus GI.3 and GII.4 were the predominant genotypes (40.3% and 53.0%) with strain diversity and change in the predominant sub-cluster over time observed among GII viruses. A second episode of norovirus gastroenteritis was documented in 44/174 (25.3%) ever-infected children. Children with the G428A homozygous mutation for inactivation of the FUT2 enzyme (se428se428) were at a significantly lower risk (48/190) of infection with norovirus (p = 0.01). CONCLUSIONS: This is the first report of norovirus documenting disease, re-infection and genetic susceptibility in an Asian birth cohort. The high incidence and apparent lack of genogroupII specific immunity indicate the need for careful studies on further characterization of strains, asymptomatic infection and shedding and immune response to further our understanding of norovirus infection and disease.

Characterization of hepatitis E virus from sporadic hepatitis cases and sewage samples from Vellore, south India.

BACKGROUND: Hepatitis E virus (HEV) is endemic in India and causes epidemics and sporadic cases. However, the exact transmission route for sporadic hepatitis E remains unclear. This study investigated HEV in sporadic hepatitis cases and sewage samples, as sewage is the major source of contamination of water in developing countries. METHODS: Monthly sampling and testing for HEV in sewage samples from Vellore, India was carried out for 1 year (November 2009-October 2010) and plasma and/or fecal samples from sporadic hepatitis cases presenting to a hospital in Vellore during 2006-2010 were tested for HEV RNA. A total of 144 raw sewage samples and 94 samples from sporadic hepatitis cases were tested for HEV RNA using RT-PCR. RESULTS: The prevalence of HEV RNA in sewage and sporadic cases was 55.6% and 9.6%, respectively. HEV strains isolated from sewage showed 94-100% nucleotide sequence similarity with the HEV strains isolated from the sporadic hepatitis cases. HEV RNA in sewage was identified more often during the summer (81.2%) than the monsoon season (14.5%) (p < 0.001). CONCLUSION: This study indicates that sewage may be a source of contamination for sporadic hepatitis and also underscores the need for preventive measures to protect drinking water from sewage contamination, particularly in the summer. GENBANK ACCESSION NUMBERS: HEV strains isolated from this study were deposited in GenBank under accession numbers JF972766-JF972773, JN705651-JN705659 and JN705660-JN705662.

Hepatitis E virus infections in swine and swine handlers in Vellore, Southern India.

Hepatitis E virus (HEV) in industrialized countries is zoonotically transmitted, and swine act as a major reservoir of HEV. Serum samples from 102 swine and plasma from 34 swine handlers in Vellore, India were tested by using a reverse transcription-polymerase chain reaction to detect and genotype HEV. We measured levels of IgG against HEV in swine handlers and in age and geographically matched controls from rural and urban populations in Vellore. HEV was amplified from two pigs and both viruses belonged to genotype 4. No HEV RNA was amplified from any swine handler, but 94.1% of swine handlers were positive for antibodies against HEV, a seroprevalence rate significantly higher than in rural and urban controls. The HEV genotype circulating in swine in India is different from that of humans, but the higher antibody levels in swine handlers support the possibility that zoonotic infections may occur.

Investigation of an epidemic of Hepatitis E in Nellore in south India.

OBJECTIVE: To determine the incidence of acute hepatitis because of hepatitis E virus (HEV) and the source of the epidemic in Nellore in south India in 2008. METHODS: Anti-HEV IgM ELISA and RT-PCR for HEV-RNA were carried out on blood and stool samples from patients with acute hepatitis presenting to different hospitals in the city. The city was divided into 33 clusters, and 20 families from each cluster were systematically interviewed to determine the incidence of hepatitis E in the city. The survey was conducted on 2685 residents of 673 households from 24th November to 4th December 2008. RESULTS: The overall incidence of hepatitis was 5.7% (152/2685), i.e. an estimated 23,915 persons in the city were affected. There were two deaths because of acute hepatitis in the population surveyed translating to an estimated 315 deaths. Men had higher attack rates than women (7.8%vs. 3.5%) and young adults compared to children under 5 years (6.9%vs. 2.9%). Families drinking water from the pumping station at Bujjamarevu had the highest attack rate of 54.5% (39.8-69.2%). HEV IgM antibodies were present in 80/100 plasma samples tested. HEV-RNA was detected in 43/100 individuals tested, and isolates were characterized as genotype 1 by sequencing. CONCLUSION: Sewage draining into the river close to the pumping stations and broken pipelines crossing sewage drains may have triggered this large outbreak.

Seroprevalence of IgG antibodies to hepatitis E in urban and rural southern India.

Hepatitis E virus (HEV) is an important cause of sporadic and epidemic hepatitis E infection in northern India. Sera, collected from different age groups in rural (n=1144) and urban (n=1135) areas using a probability proportional to size survey, were tested using an ELISA for IgG antibodies. Antibodies increased with age in both populations, but the urban population had higher exposure in all age groups (Mann-Whitney U test, P<0.001 for all age groups except children <5 years). These results indicate that urban populations with higher density and common water supplies may be at greater risk of hepatitis E.