Shantanu Prakash

Molecular characterization of dengue viruses circulating during 2009–2012 in Uttar Pradesh, India

Dengue is the most rapidly spreading mosquito‐borne viral disease in the world; in India it has taken endemic proportion implicating all the four known dengue virus serotypes. Dengue infection is caused by a small, single stranded RNA virus comprising of four antigenically distinct virus serotypes designated as dengue virus type 1–4 (DENV‐1–4). On the basis of genomic variations, each serotype is classified further into its genotypes. Epidemiological studies have shown that the emergence of a newer dengue serotype/genotype after an interval always leads to a major outbreak; therefore a continuous epidemiological surveillance is needed to monitor the epidemiology of dengue viruses. The present study was planned to identify the serotype/genotype of dengue viruses circulating in Uttar Pradesh, India. Of 433 dengue suspected patients, tested by reverse transcriptase PCR (RT‐PCR), 136 were positive for dengue virus RNA. Of these, DENV‐1, 2, and 3 were detected in 26 (19.1%), 77 (56.6%), and 33 (24.3%) patients, respectively. Of 136 RT‐PCR positive samples, 24 samples were sequenced to identify their genotypes. For sequencing C–prM gene junction of dengue virus genome was chosen. Phylogenetic analysis of sequenced dengue strains revealed that all the 12 DENV‐1 strains were genotype III, all the eight DENV‐2 strains were genotype IV (Cosmopolitan genotype) and among four DENV‐3 strains, three were genotype III and one was genotype I. In conclusion, the co‐circulation of multiple dengue virus serotypes and genotypes is alarming in U.P., India. J. Med. Virol. 87: 68–75, 2015.

Prevalence of gyrA and B gene mutations in fluoroquinolone-resistant and -sensitive clinical isolates of Mycobacterium tuberculosis and their relationship with MIC of ofloxacin.

The study was done to know the prevalent mutations of gyrA and gyrB genes, and their significance with drug resistance in clinical isolates of Mycobacterium tuberculosis. A total of 100 ofloxacin- (OFX) resistant and 100 OFX-sensitive isolates of M. tuberculosis were consecutively selected from routine Tuberculosis laboratory. Drug resistance pattern of these isolates was recorded. MIC of OFX was tested in all these isolates by absolute concentration method. Quinolone resistance determining region (QRDR) of gyrA and gyrB genes of 320 and 428 bp, respectively, were amplified and sequenced. Sequencing data were analyzed by BLAST on NCBI with reference strain H37Rv. Of 100 OFX-sensitive isolates, 30 were pansusceptible, 28 were monoresistant, 10 were polyresistant and 32 were multidrug resistant (MDR). Among 100 OFX-resistant isolates, 19 were OFX monoresistant, 16 were polyresistant and 65 were MDR. Mutations in gyrA and gyrB genes were observed in 79% and 5% of OFX-resistant isolates, respectively. Most prevalent mutation was found at codon 94 in QRDR of gyrA gene. Double mutations found in gyrA gene and in both gyrA and gyrB genes signifies higher levels of OFX resistance. In one isolate, a substitution at codon 592 (Pro592Ser) was found as a novel mutation outside the QRDR of gyrB gene. Our findings support previous studies that the OFX resistance to M. tuberculosis is associated with mutations in the QRDR of gyrA gene; however, the level of OFX resistance may not be predicted based on the mutation patterns in the gyrA gene.

Intravenous methyl prednisolone in patients with solitary cysticercus granuloma: A random evaluation

PURPOSE To evaluate the role of intravenous methyl prednisolone in patients with solitary cysticercus granuloma with new-onset seizures. METHODS In this open-label, randomized, prospective, follow-up study, 52 patients with new-onset seizures and a single enhancing CT lesion of cysticercus were randomly divided in two groups to receive either intravenous methyl prednisolone for 5 days along with antiepileptic drug (n=25) or antiepileptic drug monotherapy (n=27) alone. The patients were followed up for at least for 9 months. Repeat CT scans were performed after 2 months. RESULTS After 2 months, lesion disappeared in 60% patients of intravenous methyl prednisolone group and 18.5% patients receiving only antiepileptic drug (p=0.001). As far as seizure recurrence was concerned, a lower number (16% versus 33%) of intravenous methyl prednisolone treated patient had recurrence, the difference was insignificant. CONCLUSION Intravenous methyl prednisolone therapy helps in early resolution of solitary cysticercus granuloma.

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.

Toll like receptor-4 gene polymorphisms in patients with solitary cysticercus granuloma

BACKGROUND Solitary cysticercus granuloma (SCG) of the brain is the most common type of neurocysticercosis in India. In this study, we evaluated TLR4 polymorphisms in patients with SCG. METHODS One-hundred-forty-three patients with SCG and 134 controls were enrolled. Assessment for TLR4 Asp299Gly and Thr399Ile polymorphism was done. TLR4 genotype was determined by PCR-sequencing chain termination method. The patients were followed for 6 months. RESULTS Asp/Gly (P=0.024) and Thr/Ile (P=0.004) genotypes were significantly associated with the SCG. The Gly (Asp/Gly plus Gly/Gly) genotype (P=0.025) and Ile (Thr/Ile plus Ile/Ile) genotype (P=0.008) were significantly associated with the SCG. Gly/Gly and Ile/Ile genotypes were not significantly associated with SCG (P=0.767 for Gly/Gly, P=0.936 for Ile/Ile). At 6 months, TLR4 299Asp/Gly (P=0.02) and 399Ile/Thr (P=0.023) polymorphisms were significantly associated with the calcification or persistence of SCG. CONCLUSIONS TLR4 polymorphisms are associated with the susceptibility to infection with SCG.

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.

Toll-like receptor-3 gene polymorphism in patients with Japanese encephalitis.

INTRODUCTION Japanese encephalitis (JE) is one of the most lethal mosquito-borne viral encephalitis seen in India. Toll-like receptors (TLRs) play a critical role in host defence mechanism against flaviviruses causing encephalitis. We assessed whether abnormalities in toll-like receptor (TLR3) increase the susceptibly for JE. METHOD A total of 103 JE patients (all from an endemic area) and 103 healthy control subjects were examined for TLR3 Leu412Phe polymorphism with the help of polymerase chain reaction (PCR) and genetic sequencing method. RESULTS A significantly higher frequency of Leu412Phe polymorphism was noted in JE patients as compared with healthy controls [mutant (TT) genotype, P-value=0.019; mutant (TT)+heterozygous (CT) genotype, P-value=0.013]. Furthermore, frequency of 412Phe allele (T) of TLR3 gene was significantly higher in patients with JE than in controls (P-value=0.001). There was no significant difference in the distribution of any of the TLR3 Leu412Phe (L412F) polymorphism genotype with death within 1month. CONCLUSION TLR3 gene polymorphism might confer host genetic susceptibility to JE in Indian population. TLR3 polymorphism did not affect the mortality.

Complete Genome Sequences of Two Isolates of Human Parvovirus 4 from Patients with Acute Encephalitis Syndrome

ABSTRACT Human parvovirus 4 (Parv4) is a relatively new virus. Association of this virus with any human disease is yet to be established. We detected human parvovirus 4 in the cerebrospinal fluid (CSF) of two patients presenting with acute encephalitis syndrome in northern India. This is the first report of the Parv4 genome sequence from northern India.

25-Hydroxy Vitamin D, Vitamin D Receptor and Toll-like Receptor 2 Polymorphisms in Spinal Tuberculosis: A Case-Control Study.

AbstractVitamin D deficiency and vitamin D receptor (VDR) gene abnormalities confer susceptibility to tuberculosis. Toll-like receptors (TLRs), such asTLR-2, are also important mediators of inflammatory response against Mycobacterium tuberculosis. We evaluated serum vitamin D, and VDR and TLR-2 gene polymorphisms in patients with spinal tuberculosis.This study comprised of 3 groups: spinal tuberculosis, pulmonary tuberculosis, and controls (each with 106 subjects). Enzyme-linked immunosorbent assay was used to measure vitamin D levels, and polymerase chain reaction-sequencing method was used to analyze VDR and TLR-2 gene polymorphisms. Patients were followed up for 6 months.Vitamin D deficiency was significantly more prevalent in patients with spinal tuberculosis (P < 0.001) and pulmonary tuberculosis (P = 0.011), versus controls. The heterozygous and mutant genotypes of VDR TaqI gene were significantly associated with spinal tuberculosis (P < 0.001; odds ratio [OR] 4.74 [2.45–9.18]) and pulmonary tuberculosis (P < 0.001; OR 3.52 [1.80–6.88]) when compared with controls. The heterozygous and mutant variants of VDR ApaI gene were significantly more common in patients with spinal tuberculosis in comparison with patients with pulmonary tuberculosis (P < 0.001; OR 2.90 [1.65–5.10]) and controls (P < 0.001; OR 6.56 [3.41–12.61]). We did not observe any significantly different results for TLR-2 gene polymorphisms. Vitamin D deficiency, VDR, and TLR-2 polymorphisms did not affect the 6-month disability.Vitamin D deficiency and VDR gene polymorphisms are significantly more prevalent in people with pulmonary and spinal tuberculosis. They may, in isolation or collectively, confer susceptibility to pulmonary and spinal tuberculosis.

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.