Use of dry blots for serotyping and genotyping of dengue viruses: A pilot study.

BACKGROUND & OBJECTIVES: Dengue (DEN) is a result of infection by one or multiple types of four dengue viruses known as Dengue virus (DENV) 1-4. Identifying circulating serotype and genotype is epidemiologically important, however, it is challenging in resource limited areas. Moreover, transporting samples from the collation site to the laboratory in appropriate condition is an exigent task. To overcome this, we evaluated the usefulness of dry blots of serum for DENV diagnosis, serotyping and genotyping. METHODS: Serum samples received for diagnosis were divided into parts; one was used for providing the diagnosis. Remaining sample was distributed in three parts (100 µl each), one part was used for molecular testing and two parts were mixed with RNAlater reagent® in equal volumes and was blotted on Whatman filter paper no 3. The blots were dried and stored at 4°C and 28°C and tested for presence of dengue RNA, serotypes and genotypes after 7 days of incubation. RESULTS: The diagnosis and serotyping results of serum sample and dry serum blots were in concordance. Out of 20 positive samples, 13 (65%) gave satisfactory sequencing results. Genotype III of DENV-1, Genotype IV of DENV 2 and Genotype I of DENV-4 were detected. INTERPRETATION & CONCLUSION: The results demonstrate that serum mixed with RNA protective solution and blotted on Whatman filter paper no 3 can be effectively used for diagnosis, serotyping and genotyping of DENVs. This will help in easy transportation, diagnosis and effective data generation in resource limited settings.

Expansion of the measles and rubella laboratory network, India.

Objective: To expand the measles and rubella laboratory network of India by integrating new laboratories. Methods: In collaboration with the World Health Organization (WHO), the Indian government developed a 10-step scheme to systematically expand the number of laboratories performing serological and molecular testing for measles and rubella. The Indian Council of Medical Research and WHO identified suitable laboratories based on their geographical location, willingness, preparedness, past performance and adherence to national quality control and quality assurance mechanisms. The 10-step scheme was initiated with training on measles and rubella diagnostic assays followed by testing of both measles and rubella serology and molecular unknown panels, cross-verification with reference laboratories and ended with WHO on-site accreditation. Findings: After extensive training, technical support, funding and monitoring, all six selected laboratories attained passing scores of 90.0% or more in serological and molecular proficiency testing of measles and rubella. Since 2018, the laboratories are a part of the measles and rubella network of India. Within 12 months of initiation of independent reporting, the six laboratories have tested 2287 serum samples and 701 throat or nasopharyngeal swabs or urine samples. Conclusion: The process led to strengthening and expansion of the network. This proficient laboratory network has helped India in scaling up serological and molecular testing of measles and rubella while ensuring high quality testing. The collaborative model developed by the Indian government with WHO can be implemented by other countries for expanding laboratory networks for surveillance of measles and rubella as well as other infectious diseases.

Molecular xenomonitoring of Dengue, Chikungunya and Zika infections: a year-round study from two Dengue endemic districts of central India.

BACKGROUND & OBJECTIVES: Infections caused by arboviruses and transmitted by Aedes species mosquitoes are a serious health concern. India is endemic for diseases like Dengue, Chikungunya and recently Zika has been reported from few states. Vector control is the only way to contain these diseases, however, data regarding vectors from central India is lacking; to fulfill the lacuna we conducted this study. METHODS: Entomological surveys were conducted from November 2017 to December 2018 for Aedes species in Dengue endemic areas of central India. The mosquitoes were identified, pooled and tested for the presence of Dengue, Chikungunya and Zika viruses by RT-PCR. The PCR products were sequenced to identify serotypes and genotypes of viruses. RESULTS: A total of 2991 adults of Aedes specimens were collected and tested. Ae. aegypti (94.6%) was found to be the most abundant species. Highest mosquito density was recorded in the monsoon periods. Dengue (n=5) and Chikungunya (n=4) virus were detected from pools of female Ae. aegypti. One pool of male Ae. aegypti was positive for Dengue virus-3 and Chikungunya virus. Zika virus was not detected from any pool. INTERPRETATION & CONCLUSION: The findings suggest that Ae. aegypti is the principal vector of Dengue and Chikungunya, which is capable to transmit these viruses vertically. The findings have epidemiological importance and will be helpful to program managers.

Laboratory surveillance of chikungunya in Madhya Pradesh, India (2016-2017).

Background & objectives: Chikungunya (CHIK) is a neglected, re-emerging arboviral disease. Limited information on CHIK-confirmed cases during interepidemic period is available from India. This surveillance study was conducted in Madhya Pradesh (MP), India, during the years 2016-2017, to provide information about CHIK cases. Methods: Blood samples collected from patients suspected having CHIK were tested by immunoglobulin (Ig) IgM ELISA or real time reverse transcription-polymerase chain reaction (rRT-PCR) for the detection of CHIK virus (CHIKV)-specific IgM antibodies or viral RNA, respectively. Partial envelope-1 gene sequencing was done. Clinical and demographic data were collected and analyzed. Results: Of the 4019 samples tested, 494 (12.2%) were found positive for CHIKV infection. The positivity was detected in both rural and urban areas. The mean age of CHIK-positive cases was 33.12±18.25 yr. Headache and joint pain were the most prominent symptoms, 34.6 per cent (171/494) of the CHIK cases required hospitalization and six patients with CHIKV infection died. The East/Central/South African genotype of CHIKV was found to be circulating in the study area. Interpretation & conclusions: Our study recorded a higher CHIK positivity during 2016-2017 in comparison to earlier reports from MP, India. A high proportion of CHIK cases required hospitalization and deaths were also reported, which indicated the severity of the disease in the study area. In-depth molecular analysis of the virus and other risk factors is essential to understand the trends in disease severity.

Viral hepatitis among acute hepatitis patients attending tertiary care hospital in central India.

Viral hepatitis is a considerable public health burden affecting millions of people throughout the world. The incidence of viral hepatitis varies greatly depending upon geographic locations, age and gender. Exploring the etiological spectrum and clinic-epidemiological profile of acute viral hepatitis (AVH) becomes essential for strategizing the preventive measures to control the diseases. An epidemiological data depicting AVH situation and its etiologies is missing from central India. With the aim of fulfilling this lacuna, the present analysis was done on samples tested over a period of 2 years from July 2015 to June 2017. Of the 1901 hepatitis cases, 597 individuals (31.4%) were positive for AVH infection and HEV was the predominant cause followed by HBV, HAV and HCV. Co-infections of hepatitis viruses were detected in 42 cases. Co-infection of HEV with HBV was the commonest pattern. Male preponderance was observed among AVH positive cases and the age group of 26-45 years was the most susceptible to the viral hepatitis infections, except hepatitis A, which was the most frequent among children. Two hundred patients (33.45%) required hospitalization and 51 deaths were attributed to AVH infections. The analysis for the first time reports intricacies and viral etiologies of AVH in central India. Regular diagnosis of AVH etiology and monitoring of cases will help in patient management and assist disease control programs to take policy decisions.

Molecular studies on dengue viruses detected in patients from Central India.

Purpose: Dengue viruses (DENVs), the causative agents of dengue (DEN), are classified into four serotypes and several genotypes. Identifying circulating serotypes and genotypes has clinical and epidemiological importance; however, limited information in this regard is available from Central India. This laboratory-based study was done to fill this lacuna. Materials and Methods: The samples collected in the acute phase of illness were subjected to DEN NS1 ELISA, and NS1-positive samples (n = 80) were subjected to serotyping; representative samples from each serotype were sequenced to identify genotypes. Results: Seventy-one (88.75%) samples could be serotyped. All the four DENV serotypes with dominance of DENV-3 (n = 33; 47%) were detected. DENV-4 was detected after a gap of 3 years. Cases with multiple DENV serotype infection were identified. Genotyping showed that DENV-1 belonging to genotype III, DENV-2 cosmopolitan (IV), DENV-3 genotype III lineage C and DENV-4 genotype I were in circulation in the year 2016. Conclusion: Our study documents the molecular characteristics of DENV circulating in the area. Detection of heterologous DENV serotype with dominance of DENV-3 emphasises the need for regular molecular monitoring.

Outbreaks of dengue in Central India in 2016: Clinical, laboratory & epidemiological study.

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.

Molecular and epidemiological analysis of pandemic and post-pandemic influenza A(H1N1)pdm09 virus from central India.

Influenza A(H1N1)pdm09 virus pandemic struck India in 2009 and continues to cause outbreaks in its post-pandemic phase. Diminutive information is available about influenza A(H1N1)pdm09 from central India. This observational study presents epidemiological and molecular findings for the period of 6 years. Throat swab samples referred from districts of Madhya Pradesh were subjected to diagnosis of influenza A(H1N1)pdm09 following WHO guidelines. Clinical and epidemiological data were recorded and analyzed. Hemagglutinin (HA) gene sequencing and phylogenetic analysis were performed. The H275Y mutation responsible for antiviral resistance was tested using allelic real-time RT-PCR. Out of 7365 tested samples, 2406 (32.7%) were positive for influenza A(H1N1)pdm09, of which 363 (15.08%) succumbed to infection. Significant trends were observed in positivity (χ2 = 50.8; P < 0.001) and mortality (χ2 = 24.4; P < 0.001) with increasing age. Mutations having clinical and epidemiological importance were detected. Phylogenetic analysis of HA gene sequences revealed that clade 7, 6A, and 6B viruses were in circulation. Oseltamivir resistance was detected in three fatal cases. Influenza A(H1N1)pdm09 viruses having genetic diversity were detected from central India and continues to be a concern for public health. This study highlights the need of year-round monitoring by establishment of strong molecular and clinical surveillance program.