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Background & Objectives: Dengue virus (DENV) causes outbreaks and sporadic cases in tropical and subtropical countries. Documenting intricacies of DEN outbreaks is important for future interventions. The objective of this study was to report clinical, laboratory and epidemiological features of DEN outbreaks reported in different districts of Central India in 2016., Methods: In 2016, outbreaks (n=4) suspected of DEN were investigated by rapid response team. Door-to-door fever and entomological surveys were conducted. Blood samples were collected and tested using NS1 or IgM ELISA; real-time reverse transcription-polymerase chain reaction was done to identify serotypes of DEN virus (DENV). NS1-positive samples were tested for the presence of IgG by ELISA. Clinical and demographic data were collected and analyzed., Results: Outbreaks occurred in both urban and rural areas in monsoon season and Aedes aegypti was identified as the vector. Fever, chills, headache and myalgia were the major symptoms; no fatality was recorded. Of the 268 DEN suspects, 135 (50.4%) were found serologically positive. DEN positivity was higher (n=75; 55.56%) among males and in the age group of 16-45 yr (n=78; 57.8%). DENV 3 followed by DENV 2 were detected as the major responsible serotypes. High attack rates (up to 38/1000) and low cumulative IgG prevalence (14.9%) were recorded in rural areas., Interpretation & Conclusions: Our study showed that DENV 3 was the major serotype responsible for outbreaks that occurred in monsoon. High attack rates and lower number of secondary infections in rural areas indicated that DENV is emerging in rural parts of Central India. Early diagnosis at local level and timely intervention by mosquito control activities are needed to avoid such outbreaks in future., Competing Interests: None

Background & Objectives: Dengue virus infection is endemic in India with all the four serotypes of dengue virus in circulation. This study was aimed to determine the geographic distribution of the primary and secondary dengue cases in India., Methods: A multicentre cross-sectional study was conducted at Department of Health Research / Indian Council of Medical Research (DHR)/(ICMR) viral research and diagnostic laboratories (VRDLs) and selected ICMR institutes located in India. Only laboratory-confirmed dengue cases with date of onset of illness less than or equal to seven days were included between September and October 2017. Dengue NS1 antigen ELISA and anti-dengue IgM capture ELISA were used to diagnose dengue cases while anti-dengue IgG capture ELISA was used for identifying the secondary dengue cases., Results: Of the 1372 dengue cases, 897 (65%) were classified as primary dengue and 475 (35%) as secondary dengue cases. However, the proportion varied widely geographically, with Theni, Tamil Nadu; Tirupati, Andhra Pradesh and Udupi-Manipal, Karnataka reporting more than 65 per cent secondary dengue cases while Srinagar, Jammu and Kashmir reporting as low as 10 per cent of the same. The median age of primary dengue cases was 25 yr [interquartile range (IQR 17-35] while that of secondary dengue cases was 23 yr (IQR 13.5-34). Secondary dengue was around 50 per cent among the children belonging to the age group 6-10 yr while it ranged between 20-43 per cent among other age groups., Interpretation & Conclusions: Our findings showed a wide geographical variation in the distribution of primary and secondary dengue cases in India. It would prove beneficial to include primary and secondary dengue differentiation protocol in the national dengue surveillance programme., Competing Interests: None

Background & Objectives: Dengue fever (DF) is associated with significant morbidity and mortality in the tropical and sub-tropical regions of the world. Since there are no effective antiviral drugs for treatment, clinicians often rely on the accurate diagnosis of dengue fever to begin supportive therapy at early stages of the illness. The objective of this study was to develop an in-house dengue virus serotype 2 (DENV-2) non-structural protein- 5 (NS5) based indirect ELISA., Methods: DENV-2 was raised in Vero cells and the viral proteins were separated and subsequently the NS5 protein was eluted. Serum samples from primary and secondary dengue fever patients; and acute and convalescent samples from Japanese encephalitis (JE) and West Nile virus (WNV) cases were used to validate the ELISA., Results: The assay was found to be 100 per cent specific in detecting DENV-2 specific antibodies from patient's serum. However, in terms of sensitivity, the assay could detect IgM antibodies only from 90 per cent of the primary dengue samples. The IgM/IgG ratio of the primary and secondary samples was 7.24 and 0.64, respectively., Interpretation & Conclusions: The results indicate that the DENV-2 NS5 ELISA is dengue group specific and can be used to differentiate dengue infection from other circulating Flavivirus infections. This NS5 ELISA can also be used to distinguish between primary and secondary dengue fever on the basis of IgM/IgG ratios. Further studies with larger sample sizes and different DENV serotypes are required to validate the ELISA., Competing Interests: None.

Background & Objectives: The epidemiology of dengue fever (DF) is complex in the Indian subcontinent as all the four serotypes are circulating. This study reports observations on dengue cases from a virus diagnostic laboratory of a north Indian tertiary care hospital catering to areas in and around Lucknow, Uttar Pradesh., Methods: Serum samples were obtained from suspected cases of dengue referred to the virus diagnostic laboratory during 2011 to 2013, and detailed history was taken on a pre-structured datasheet. All samples were tested for anti-dengue virus (DV) IgM antibodies and DV-non structural protein 1 antigen (NS1Ag) by ELISA. NS1Ag positive samples were tested further by conventional RT-PCR for DV-RNA detection and serotyping., Results: Of the 4019 suspected patients of dengue, 886 (22%) showed laboratory evidence of dengue virus infection. Of these, 19, 17 and 27 per cent were positive in 2011, 2012 and 2013, respectively. Children and adults were similarly affected by dengue in all the three years. Males were more commonly affected than females. The predominant DV serotype detected was DV-2, DV-1 and DV-3 in 2011, 2012 and 2013, respectively. DV-4 serotype was not detected. About half the cases positive for DV infection, showed symptoms of dengue with warning signs/ severe dengue. A distinct seasonality with increase in number of dengue cases in the post monsoon period was seen., Interpretation & Conclusions: Change in circulating serotype of dengue virus; a distinct adult dengue involvement; and a remarkable number of cases presenting with severe dengue manifestations are the main findings of this study.

Background & Objectives: Epidemiological interventions and mosquito control are the available measures for dengue control. The former approach uses serotype and genetic information on the circulating virus strains. Dengue has been frequently reported from Nepal, but this information is mostly lacking. The present study was done to generate a comprehensive clinical and virological picture of a dengue outbreak in Nepal during 2013., Methods: A hospital-based study involving patients from five districts of Nepal was carried out. Demographic information, clinical details and dengue serological status were obtained. Viral RNA was characterized at the molecular level by reverse-transcription polymerase chain reaction (RT-PCR), nucleotide sequencing and phylogenetic analysis., Results: From among the 2340 laboratory-confirmed dengue cases during the study period, 198 patients consented for the study. Clinically they had fever (100%), headache (59.1%), rashes (18.2%), retro-orbital pain (30.3%), vomiting (15.1%), joint pain (28.8%) and thrombocytopenia (74.3%). Fifteen (7.5%) of them had mucosal bleeding manifestations, and the rest were uncomplicated dengue fever. The patients were mostly adults with a mean age of 45.75 ± 38.61 yr. Of the 52 acute serum samples tested, 15 were positive in RT-PCR. The causative virus was identified as DENV serotype 2 belonging to the Cosmopolitan genotype., Interpretations & Conclusions: We report here the involvement of DENV serotype 2 in an outbreak in Nepal in 2013. Earlier outbreaks in the region in 2010 were attributed to serotype 1 virus. As serotype shifts are frequently associated with secondary infections and severe disease, there is a need for enhancing surveillance especially in the monsoon and post-monsoon periods to prevent large-scale, severe dengue outbreaks in the region.

Background & Objectives: Dengue (DEN) is a rapidly spreading arboviral disease transmitted by Aedes mosquitoes. Although it is endemic in India, dengue virus (DENV) infection has not been reported from tribal areas of Madhya Pradesh. Investigations were conducted to establish the aetiology of sudden upsurge of cases with febrile illness in June 2013 from tribal villages of Mandla district of Madhya Pradesh, India., Methods: The rapid response team of the National Institute for Research in Tribal Health, Jabalpur, conducted clinical investigations and field surveys to collect the samples from suspected cases. Samples were tested using molecular and serological tools. Collected mosquitoes were identified and tested for the presence of virus using semi nested reverse transcriptase-polymerase chain reaction (nRT-PCR). The sequences were analysed to identify serotype and genotype of the virus., Results: Of the 648 samples collected from 18 villages of Mandla, 321 (49.53%) were found to be positive for dengue. The nRT-PCR and sequencing confirmed the aetiology as dengue virus type 2. Eighteen per cent of patients needed hospitalization and five deaths were attributed to dengue. The virus was also detected from Aedes aegypti mosquito, which was incriminated as a vector. Phylogenetic analysis revealed that the dengue virus 2 detected belonged to cosmopolitan genotype of the virus., Interpretation & Conclusions: Dengue virus serotype 2 was detected as the aetiological agent in the outbreak in tribal villages of Mandla district of Madhya Pradesh. Conducive man-made environment favouring mosquitogenic conditions and seeding of virus could be the probable reasons for this outbreak. Urgent attention is needed to control this new threat to tribal population, which is already overburdened with other vector borne diseases.

Background & Objectives: In Odisha, several cases of dengue virus infection were detected for the first time in 2010, the importance of dengue as a serious mosquito-borne viral infection was felt only in 2011 with the reporting of many more positive cases. This retrospective three year study was done to find out the seroprevalence of dengue Ig m0 antibody and to know the predominant serotype of dengue virus among the patients suspected to have dengue virus infection in a tertiary care hospital in southern Odisha, India., Methods: Blood samples from clinically suspected dengue cases admitted in the Medicine and Paediatrics departments of a tertiary care hospital were collected. These were processed for detection of dengue specific IgM antibody, carried out by the ELISA method. Dengue IgM antibody positive serum samples were tested for serotypic identification., Results: Of the 5102 samples tested, 1074 (21.05%) were positive for dengue IgM. Maximum numbers of cases were found in 2012. Majority (47.86%) of cases were detected in the month of September. The most common affected age group was 11 to 20 yr. DENV1 and DENV2 were the detected serotypes., Interpretation & Conclusions: Rapid increase in the dengue cases in 2012 became a public health concern as majority of cases were affecting the young adolescents. Most of the cases were reported in post-monsoon period indicating a need for acceleration of vector control programmes prior to arrival of monsoon.

Background & Objectives: Leptospirosis, a spirochetal zoonosis, is underreported from the northern States of India. This study reports results of a 10-year retrospective sero-epidemiological survey of leptospirosis conducted in a tertiary care hospital in New Delhi, India., Method: A total of 1453 patients clinically suspected for leptospirosis were included and investigated initially by IgM ELISA. A proportion of these were subjected to culture, microscopic agglutination test (MAT) and polymerase chain reaction (PCR)., Results: Of the 1453 patients, 391 (26.90%) were positive serologically by IgM ELISA. Seropositive and seronegative patients revealed no significant difference in clinical features and laboratory parameters. Amongst the IgM seropositive cases, culture for leptospires was positive in 5 of 192 (2.6%), MAT in 50 of 138 (36.23%), PCR from blood and urine in 10 of 115 (8.7%) and 10 of 38 (26.31%) cases, respectively. In Leptospira spp. positive patients co-infections with viral hepatitis E, malaria and dengue fever were diagnosed in 27 cases., Interpretation & Conclusions: The overall seropositivity for leptospirosis was 26.9 per cent in our study. A decreasing trend in seropositivity was observed in recent years. Co-infections with malaria, dengue, hepatitis A and E were also seen. Since leptospirosis is a treatable disease, correct and rapid diagnosis may help in effective management of patients.

Background & Objective: Dengue haemorrhagic fever (DHF) is a severe illness caused by dengue viruses, the pathogenesis of which is not fully understood. Immune enhancement caused by cytokines, interferon and activated complement system is one of the hypotheses proposed to explain the haemorrhagic form of disease. This study was undertaken to investigate the role of pro-inflammatory cytokines tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) in dengue infection, and their possible association with disease severity., Methods: Blood samples were collected from 60 patients; 28 patients of DF and 32 of DHF (clinically categorized into DF or DHF as per WHO criteria) from September to November 2003 at Delhi. Fifteen healthy individuals were included as control. All the patients were confirmed positive for dengue infection by serology or by reverse transcriptase-polymerase chain reaction (RT-PCR). Serum levels of TNF-alpha and IFN-gamma were determined by ELISA., Results: The levels of both the cytokines were significantly elevated in the disease group as compared to the control group. Significantly higher levels of TNF-alpha; 258.02 pg/ml (P<0.005) were seen in patients having secondary infection, while patients with primary infection had higher level of IFN-gamma; 29.47 pg/ml (P<0.005). TNF-alpha was elevated in the later phase of illness, while IFN-gamma was elevated in early phase also., Interpretation & Conclusion: Overproduction of TNF-alpha during secondary infection may have a role in immunopathogenesis of DHF, however, study of other cytokines produced along with IFN-gamma;, during the course of dengue infection is required to know the specific role of IFN-gamma in DI.