Danish Nasar Khan

Co-positivity of anti-dengue virus and anti-Japanese encephalitis virus IgM in endemic area: co-infection or cross reactivity?

OBJECTIVE To report high co-positivity of anti-dengue virus (DV) and anti-Japanese encephalitis virus (JEV) IgM in an area endemic for both the viruses and to discuss the possibilities of co-infection. METHODS Serum samples from the patients who presented with fever, suspected central nervous system infection and thrombocytopenia, were tested for anti-DV IgM and anti-JEV IgM antibodies. Conventional reverse transcriptase polymerase chain reaction was done for detection of DV RNA and JEV RNA. RESULTS Of 1 410 patient sera tested for anti-DV and anti-JEV antibodies, 129 (9.14%) were co-positive for both. This co-positivity was observed only in those months when anti-JEV IgM positivity was high. Titers of both anti-DV IgM and anti-JEV IgM were high in most of the co-positive cases. Among these 129 co-positive cases, 76 were tested by conventional reverse transcriptase polymerase chain reaction for both flaviviruses, of which eight cases were co-positive for DV and JEV. CONCLUSIONS Co-infection with more than one flavivirus species can occur in hyperendemic areas.

Prevalence and genotypic characterization of human parvovirus B19 in children with hemato‐oncological disorders in North India

Human parvovirus B19 (B19V) has been associated with chronic anemia in immuno‐compromised patients. In the present study, the prevalence and genotype distribution of B19V in children from North India, suffering with hemato‐oncological disorders is reported. Children with aplastic anemia/leukemia/chronic hematological disorders, and healthy blood donors were enrolled in the study. Blood samples from cases and blood donors were analyzed for anti‐B19V IgM and anti‐B19V IgG antibodies by ELISA and for B19V‐DNA by PCR. B19V‐DNA positive samples were studied further for determination of viral load in samples and for B19V‐DNA sequence (VP1/VP2 overlapping region) analysis. Total 238 cases (103 leukemia, 77 aplastic anemia and 58 chronic hematological disorders) and 350 blood donors were enrolled in the study. Anti‐B19V IgM was positive in 16 (6.7%) cases, B19V‐DNA was detected in 13 (5.5%) cases and anti‐B19V IgG was positive in 127 (53.4%) cases. Total 223 (63.5%) blood donors were positive for anti‐B19V IgG, however, anti‐B19V IgM and B19V‐DNA was not detected in any blood donor. The prevalence of anti‐B19V IgG was significantly higher in children > 10 years of age. Viral load of B19V decreased with appearance of specific antibodies. Phylogenetic analysis of the VP1/VP2 overlapping region revealed that genotype 1 predominated in these patients (11/13, 84.6%), followed by genotype 3 (2/13, 15.4%). No genotype 2 was detected. All the genotype 1strains were sub‐typed as 1a, except four strains, which matched neither 1a nor 1b and formed a separate cluster. Both the genotype 3 strains were sub‐typed as 3b. J. Med. Virol. 87:303–309, 2015.

Human parvovirus B19 associated dilated cardiomyopathy.

We describe two children presenting with acute left ventricular dysfunction. Both cases had evidence of dilated cardiomyopathy, requiring inotropic support and were tested for cardiotropic viruses by conventional or real-time polymerase chain reaction using specific primers for enteroviruses, human parvovirus B19, Adenoviruses, Epstein-Barr virus (EBV), herpes simplex viruses 1 and 2 (HSV), human herpes virus-6 (HHV-6) and cytomegalovirus (CMV). IgG and IgM antibodies against parvovirus B19, EBV, HSV and CMV were also tested by ELISA. One case tested positive for parvovirus B19 infection and recovered completely within 6 months in absence of any specific therapy. The other case tested positive for parvovirus B19 infection in association with hypocalcaemia and was cured following standard heart failure therapy along with calcium and vitamin D supplementation. Sequence analysis of DNA products from both patients revealed genotype 3. To best of our knowledge this is first report of circulating genotype 3 from India.

Genotype 3b of human parvovirus B19 detected from hospitalized children with solid malignancies in a North Indian tertiary care hospital

Human parvovirus B19 (B19V) infection is known to cause serious consequences in immuno‐compromized individuals. The present cross sectional study was designed to estimate the prevalence and genotype distribution of B19V in children receiving chemotherapy for solid malignancies at a tertiary care hospital in North India during October 2013 to May 2015. Serum samples from all the patients were tested for anti‐B19V IgM and IgG antibodies and for B19V‐DNA as soon as received. Samples testing positive for B19V‐DNA were subjected to viral load estimation and to genotype determination by sequencing. Total 96 children were enrolled of which 9 (9.3%), 32 (33.3%), and 25 (26%) tested positive for anti‐B19V IgM, anti‐B19V IgG, and B19V‐DNA, respectively. The viral load of B19V‐DNA positive children ranged from 5.5 × 102 to 3.5 × 1012 copies/ml. Accordingly children were divided into three groups: group I, with acute infection (n = 25); group II, previously exposed (n = 27), and group III, negative for B19V infection or with inappropriate antibody response (n = 44). B19V positivity was significantly associated (P‐value < 0.0001) with a history of blood transfusion in the past 6 months, severe anemia (hemoglobin levels <6 gm%) and thrombocytopenia (platelets <150,000/cu.mm.). Sequence analysis of 21 of 25 DNA positive samples showed that all of them were Genotype 3b that clustered into three groups. All the sequences within each cluster were identical. The nucleotide identity of the sequences suggests a nosocomial outbreak of B19V during the study period. Children on chemotherapy for solid tumors should be routinely screened for B19V infection by both serology and PCR. J. Med. Virol. 88:1922–1929, 2016.

Observation on dengue cases from a virus diagnostic laboratory of a tertiary care hospital in North India

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.

Incidence and progression of Parvovirus B19 infection and molecular changes in circulating B19V strains in children with haematological malignancy: A follow up study

The present study was planned to estimate the incidence of human Parvovirus B19 infection and understand its progression in children suffering with hematological malignancy. The circulating B19V genotypes and viral mutations occurring in strains of B19V over one-year period were also studied. Children with malignancies were enrolled consecutively and were followed up for one-year period. Serum sample was collected at the time of enrolment and each follow up visit and was tested for anti B19V IgG and IgM as well as for B19V DNA. At least one B19V DNA positive sample from each patient was processed for sequencing. For patients positive for B19V DNA >1 time and at least 6 months apart, last positive sample from the same patient was also sequenced to study the nucleotide change over time. We have found very high incidence of B19V infection (100%) in the study population. All the patients tested positive for at least one B19V infection parameter (either antibodies or DNA) at least once, over one year of follow up. Cumulative percent positivity of anti B19V IgG, anti B19V IgM and B19V DNA was 85.3%, 45.2% and 72.1% respectively. Genotype 3b was reported, with occasional nucleotide change over one year period. DNA clearance was delayed in spite of appearance of IgG antibodies. Appearance of IgM class of antibodies was either delayed or absent. To conclude, children with haematological malignancies have high incidence of B19V infection with late and short lived serological response and persistence of DNA for long duration.

Aetiology of acute encephalitis syndrome in Uttar Pradesh, India from 2014 to 2016

Background & objectives: It is imperative to know the aetiology of acute encephalitis syndrome (AES) for patient management and policy making. The present study was carried out to determine the prevalence of common aetiological agents of AES in Uttar Pradesh (UP) state of India. Methods: Serum and/or CSF samples were collected from AES patients admitted at Gandhi Memorial and Associated Hospital, King Georges Medical University, Lucknow, a tertiary care centre, UP during 2014–16. Cerebrospinal fluid (CSF) and serum samples from cases were tested for IgM antibodies against Japanese encephalitis virus (anti-JEV), and dengue virus (anti-DENV) by ELISA; and for enterovirus, herpes simplex virus (HSV) and varicella zoster virus (VZV) by real-time PCR. Serum samples of cases having sufficient CSF volume, were also tested for anti-scrub typhus IgM antibodies and for Neisseria meningitides, Streptococcus pneumoniae and Haemophilus influenzae. Results: JEV and DENV (8% each) were the most common identified aetiology from the 4092 enrolled patients. Enterovirus, HSV and VZV, each were detected in <1% AES cases. Co-positivity occurred in 48 cases. Scrub typhus (31.8%) was the most common aetiology detected. Haemophilus influenzae and S. pneumoniae were detected in 0.97 and 0.94% cases, respectively, however, N. meningitides was not detected in any of the cases. About 40% of the JEV/DENV positive AES cases were adults. The gap between the total number of AES cases and those with JEV/ DENV infection increased during monsoon and post-monsoon seasons. Interpretation & conclusion: Scrub typhus, JEV and DENV are the main aetiological agents of AES in UP. DENV and JEV can no longer be considered paediatric diseases. The prevalence of non-JEV/DENV aetiology of AES increases in the monsoon and post-monsoon seasons.

Trend of Japanese encephalitis in Uttar Pradesh, India from 2011 to 2013.

As indicated by the sporadic Japanese encephalitis (JE) cases reported from the districts of Uttar Pradesh (UP), India, the disease is endemic in the state despite the fact that a JE vaccination programme has been ongoing in the state since 2006. Hence, the present study was undertaken to study the annual trend of JE in UP during January 2011 to December 2013. CSF and/or serum samples collected from acute encephalitis syndrome (AES) cases were referred to the virology laboratory at King Georges Medical University, Lucknow and were tested for anti-JEV IgM antibodies by JEV MAC-ELISA kit. The study reveals that 26·9%, 9·9% and 14·8% of AES cases were positive for anti-JEV IgM in the years 2011, 2012 and 2013, respectively. Of the total JE confirmed cases, 30% were adults. Males were more commonly affected than females. A distinct peak of JE was seen in the monsoon and post-monsoon season, although sporadic cases were also reported in other months. JE vaccination by district in UP is discussed. This study reports that the proportion of JE positives in AES cases is decreasing in UP although the number of AES cases has not decreased. The study also discusses the probable causes of this decrease, including JE vaccination and natural periodicity due to herd immunity.

Prevalence of Parvovirus B19V in Hematological Malignancies and Chronic Anemia

To the Editor: The present study was planned to estimate the prevalence of human Parvovirus B19 (B19V) infection in children suffering with hematological malignancies or chronic anemia. Such hospitalized children (<15 y) were enrolled consecutively from 2011 through 2014. King George’s Medical University’s institutional ethical clearance was obtained (no.52 E.C.M.IIA/P6). Anti B19V IgG and IgM antibodies (ELISA kits by NovaTech Immunodiagnostica, Dietzenbach, Germany) and B19V DNA (by real time PCR [1]) were estimated in serum samples of enrolled patients. Relevant laboratory parameters were noted from hospital records of cases. Statistical analysis was done using GraphPad Prism Version 5. Total 379 children were enrolled [Leukemia: 270 cases, chronic unexplained anemia: 109 cases; boys: 264(69.7%), girls: 115(30.3%)]. Mean age ± SD was 6.89 ± 4.17. A high prevalence (70.2%) of B19V infection (presence of any of the three markers) was seen in the study population. No statistically significant difference was seen in B19V positivity among patients of both disease groups. The anti B19V IgG positivity and total positivity of all the three markers of B19V infection increased but B19V DNA positivity decreased significantly with increasing age (Table 1). Data for hemoglobin levels, total leucocyte count, platelet count and bilirubin levels were available for only 186, 177, 173 and 132 cases respectively. The p values (95% CI) for severe anemia (Hemoglobin level < 8.0 g/dl), leucopenia (TLC < 4000/mm), thrombocytopenia (platelets <1.5 lac/mm) and jaundice (bilirubin level > 1.0 mg/dl) in cases with and without B19V infection were 0.516 (0.87–1.36), 0.17 (0.81– 3.27) , 0 .43 (0 .89–1.37) and 0.51 (0 .84–1.49) respectively. The present study describes high prevalence of B19V infection in immunocompromised children; also shown by earlier studies, varying from 34 to 45% [2–4]. Anti B19V IgG positivity increases with age; an observation reported earlier in healthy individuals [5]. The limitation of the present study was that we could not estimate B19V prevalence in age and sex matched healthy controls due to logistic reasons. We have however estimated earlier, B19V prevalence in blood donors, which indicated frequent B19V exposure in the Indian population [6]. B19V infection was not associated with anemia, thrombocytopenia, leucopenia or jaundice in contrast to earlier studies [7], probably because the patients had underlying diseases where these symptoms are features of disease per se. This indicates that B19V infection should be tested for in all hematological diseases patients irrespective of laboratory parameters, for appropriate patient management. To conclude, B19V infection is common in children with hematological diseases, with frequency increasing with age. Therefore, every such child should be evaluated for B19V infection even in the absence of its specific indications. * Amita Jain amita602002@yahoo.com

Molecular characterization of the rotavirus enterotoxin NSP4 gene of strains causing diarrhoea in children aged 0-5 years in northern India

Non structural protein 4 (NSP4) gene of Rotavirus encodes a multifunctional protein which has significant role in viral multiplication and pathogenesis of acute watery diarrhoea associated with rotaviral gastroenteritis. It is known as the first viral enterotoxin and mutations of the gene have been linked to altered pathogenesis. This study was planned to ascertain the genotypes and genetic variations of NSP4 gene in the rotavirus strains prevalent in this area. We collected consecutive diarrhoeal stools from equal no of children aged under five years hospitalized with diarrhoea in a period from January 2010 to June 2012 and tested them for rotavirus antigen by ELISA. NSP4 gene was amplified by RT-PCR and subsequently sequenced (Big-Dye terminator kit using 3130 ABI, Genetic analyzer) and genotyped by Rotavirus C software. Of the 260 samples, 58(22.3%) samples were positive by ELISA. We were able to amplify NSP4 gene by RTPCR from 45 strains of which 35 amplicons were selected for sequencing. Total 25(71.4%) strains belonged to genotype E1, 6 (17.1%) strains to genotype E2 and 4 (11.4%) matched with the genotype E6. Sequence analysis revealed changes in the nucleotides causing punctate mutations in the conserved regions, the Inter species variable domain (ISVD) and the enterotoxin region (amino acid 114-135). On evolutionary analysis of 33 strains amino acid at position 131 was found under positive selection.