R K Ratho

University of Rome Tor Vergata, Roma, Latium, Italy

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Publications (88)173.94 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Hepatitis A virus usually causes acute viral hepatitis (AVH) in the paediatric age group with a recent shift in age distribution and disease manifestations like acute liver failure (ALF). This has been attributed to mutations in 5'non-translated region (5'NTR) which affects the viral multiplication. The present study was aimed to carry out the molecular detection and phylogenetic analysis of hepatitis A virus strains circulating in north western India. Serum samples from in patients and those attending out patient department of Pediatric Gastroenterology in a tertiary care hospital in north India during 2007-2011 with clinically suspected AVH were tested for anti-hepatitis A virus (HAV) IgM antibodies. Acute phase serum samples were subjected to nested PCR targeting the 5'NTR region followed by sequencing of the representative strains. A total of 1334 samples were tested, 290 (21.7%) were positive for anti-HAV IgM antibody. Of these, 78 serum samples (< 7 days old) were subjected to PCR and 47.4% (37/78) samples showed the presence of HAV RNA. Children < 15 yr of age accounted for majority (94%) of cases with highest seropositivity during rainy season. Sequencing of 15 representative strains was carried out and the circulating genotype was found to be III A. The nucleotide sequences showed high homology among the strains with a variation ranging from 0.1-1 per cent over the years. An important substitution of G to A at 324 position was shown by both AVH and ALF strains. The cumulative substitution in AVH strains Vs ALF strains as compared to GBM, Indian and prototype strain in the 200-500 region of 5' NTR was comparable. Our results showed hepatitis A still a disease of children with III A as a circulating genotype in this region. The mutations at 5'NTR region warrant further analysis as these affect the structure of internal ribosomal entry site which is important for viral replication.
    The Indian Journal of Medical Research 02/2015; 141(2):213-20. · 1.66 Impact Factor
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    ABSTRACT: Tumour necrosis factor-α (TNF-α), a pro-inflammatory cytokine has been implicated in the pathophysiology of several viral infections. TNF-α promoter gene polymorphism is thus believed to play the modulating role in this disease pathogenesis. Several studies have shown the increased level of TNF-α in dilated cardiomyopathy (DCM). However, the role of the TNF-α promoter polymorphism is yet to be delineated in this regard. The present study for the first time tried to explore the association of TNF-α gene polymorphism with DCM of viral aetiology. Eighteen histopathologically proven DCM cases with viral genome positivity and 17 healthy controls were genotyped using polymerase chain reaction of TNF-α promoter gene followed by restriction fragment length polymorphism to determine the SNPs of -238G/A, -308G/A, -857C/T and -863C/A. Of the 18 DCM cases 4 (22.2%) were positive for adenovirus (AdV), 2 (11.1%) for enterovirus (EV) and 12 (66.7%) had co-infection. Six of the 18 DCM cases (35.3%) had -238G/A polymorphism, and 10 (55.5%) had -863 homozygous AA genotype. The association of these polymorphisms was statistically significant as compared to controls (P < 0.05). The present pilot study suggests the possible association of TNFα -238G/A and -863C/A polymorphism with DCM of viral aetiology.
    Indian Journal of Medical Microbiology 01/2015; 33(1):16-20. DOI:10.4103/0255-0857.148370 · 1.04 Impact Factor
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    ABSTRACT: To investigate an outbreak of fever with rash in an urbanized village in Chandigarh, India. Active case search was performed by house-to-house survey. The etiological agent of the outbreak was confirmed by serology. Spot map was done using Geographical Information System (GIS) technology. Out of 7742 persons screened, 12 were serologically confirmed rubella cases and 83 were epidemiologically linked cases. Overall attack rate was 1.1, more among the age group 1-4 years (4.9). An outbreak mimicking measles was investigated only to be confirmed as rubella.
    Indian pediatrics 11/2014; 51(11):897-9. DOI:10.1007/s13312-014-0523-8 · 1.01 Impact Factor
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    ABSTRACT: Rubella virus outbreaks usually occur when a large numbers of susceptible individuals accumulate. The disease presents clinically with fever and maculopapular rash. The present study reports the investigation of rubella outbreak in a modern and well-planned village near Chandigarh, North India. The blood samples were collected from 39 cases with febrile rash and from 15 age and sex matched healthy controls residing in the same locality and subjected for the detection of Rubella IgM and IgG antibodies by Enzyme linked immunosorbent assay. The throat swabs, urine and blood samples from acute cases were also collected and subjected to RT-PCR using the primers targeting the E1 region. The genetic characterization of the rubella virus was carried out to identify the circulating genotypes. In the present outbreak, 13 laboratory confirmed cases were reported. Rubella IgM antibodies were detected in 12/39 (30.7%) patients. Rubella RNA could be detected in 83.3% (5/6) of urine, 22.2% (2/9) of throat swabs, and 8.3% (1/12) of blood samples. The rubella genotype responsible for the present outbreak was identified as genotype 1a. This outbreak highlights the need for the introduction of rubella vaccine in the National Immunization Programme of India to prevent outbreaks and to aim towards the eradication of this disease. This study reports the presence of genotype 1a in North India for the first time and stresses the need for further molecular work to identify the circulating strains of the virus. J. Med. Virol. © 2014 Wiley Periodicals, Inc.
    Journal of Medical Virology 08/2014; 87(2). DOI:10.1002/jmv.24056 · 2.22 Impact Factor
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    ABSTRACT: Introduction:Dengue is one of the most important arboviral infections caused by one of the four dengue serotypes, 1-4.Objective:To study the applicability of different diagnostic methods in diagnosis of dengue viral infection.Materials and Methods:A total of 2101 blood samples were collected for confirmation of dengue viral infection. All the samples were tested by dengue-specific IgM ELISA, of which 111 were also tested for NS1 antigen detection and 27 acute samples (≤5 days) were further subjected for viral RNA detection by RT-PCR and isolation in C6/36 cell line. To detect the sensitivity of NS1 antigen for different dengue virus serotypes, four dengue serotype 1 and 12 dengue 3 were subjected for the NS1 antigen assay.Results:Most common age group affected was 16-45 years, with male to female ratio of 2.8:1. During first 3 days of illness virus isolation and RT-PCR were the most sensitive (83%) followed by NS1 antigen detection (75%) and IgM detection (37.5%). The positivity of IgM detection was found to be significantly higher as compared to NS1 detection during 4 to 5 days and also after 5 days of illness (P < 0.05). Dengue serotypes 1 and 3 were found to be co-circulated, dengue 1 being the predominant serotype.Conclusion:Virus isolation and RT-PCR were the most sensitive tests during the early period of illness whereas beyond third day, IgM antibody detection was found to be the most sensitive method of dengue diagnosis.
    Journal of global infectious diseases 07/2014; 6(3):109-13. DOI:10.4103/0974-777X.138504
  • Journal of Hepatology 04/2014; 60(1):S299. DOI:10.1016/S0168-8278(14)60852-7 · 10.40 Impact Factor
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    ABSTRACT: Human parvovirus B19 (PVB19) is linked to variety of diseases, including erythema infectiosum, transient aplastic crisis, fetal hydrops, cardiomyopathy and, recently, hepatitis and arthritis. Persistence of PVB19 in asymptomatic individuals has been reported in skin, synovium, myocardium and bone marrow. A higher level of PVB19 DNA has been observed in various tissues from cases of disease than in controls. Simultaneously, equal detection of PVB19 DNA has been shown in both cases and controls. Thus, it has become fundamental to study PVB19 DNA persistence in tissues that are unaffected by disease. This will help to better understand PVB19 DNA persistence in symptomatic and asymptomatic individuals and its possible pathogenic role in various diseases. A total of 70 adult autopsies were included and divided into seropositive (SP) and seronegative (SN) groups based on PVB19 IgG. Nested PCR for PVB19 DNA was carried out in myocardium, liver, kidney, and bone marrow. Of the 70 patients, 60 % belonged to the SP group and 40 % to the SN group. Seropositivity ranged from 50 % in the 12 to 20 year old group to 66.7 % in the 61 to 80 year old group. The viral genome was detected in 34.3 % of myocardium, 20 % of bone marrow, 10 % of kidney and 8.6 % of liver samples. There was no significant difference in the persistence rates between the SP and SN groups. The persistence of PVB19 DNA in various tissues ranged from 8.3 % to 36 % in the SP group and 10 % to 30 % in the SN group. The persistence of PVB19 DNA in all the tissues was low, and PVB19 serostatus had no influence on the persistence of PVB19 DNA.
    Archives of Virology 04/2014; 159(9). DOI:10.1007/s00705-014-2065-8 · 2.28 Impact Factor
  • Journal of hepatology. Supplement / EASL 04/2014; 60(1):S299.
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    ABSTRACT: The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low-speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs) were transfected with BLOCK-iT™ Fluorescent Oligo (scrambled siRNA) and lipofectamine complex using conventional and low-speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low-speed centrifugation significantly enhances the transfection efficiency of BLOCK-iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P < 0.0001), even at a low concentration of 40 picomoles without affecting the cell viability. Centrifugation enhanced transfection (CET) technique is simple, time-saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures.
    Indian journal of medical microbiology 04/2014; 32(2):164-8. DOI:10.4103/0255-0857.129806 · 1.04 Impact Factor
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    ABSTRACT: Hepatitis E virus (HEV) is the causative agent of hepatitis E. It can be asymptomatic, associated with acute self-limiting hepatitis or acute liver failure. The conventional diagnosis of HEV infection relies on anti-HEV IgM serology. The collection of blood samples by venepunture for laboratory confirmation is often difficult during an outbreak. Thus, testing the specimens of dried blood spots (DBS) on filter papers can prove to be a feasible alternative. The present study aimed to evaluate the applicability of anti-HEV IgM detection from DBS samples and the stability of anti-HEV IgM detection at varied time interval, at various storage temperatures. Paired blood and DBS sample were collected from 44 jaundiced patients and eight healthy controls during HEV outbreaks. The DBS were tested for anti-HEV IgM by available ELISA kit with in-house modifications. Three cut offs were determined, that is, the CO1: kit cut-off, CO2: mean of negative controls above 3SD and CO3: area under Receiver operating Curve. The sensitivity of anti-HEV IgM detection ranged from 86-91%. The maximum sensitivity (91%) and specificity (100%) was obtained using CO3. Maximum stability of anti-HEV IgM antibodies (100%) was observed till 65 days at 4°C. Storage at 37°C significantly reduced anti-HEV IgM positivity, wherein 42.85% sample became negative by 45 days. DBS showed good sensitivity and specificity for detecting anti-HEV IgM and can be considered an alternate to serum sample. Moreover, anti-HEV IgM was stable at 4°C, which makes DBS a preferred method for storage and transportation of the sample to reference laboratory. J. Med. Virol. © 2013 Wiley Periodicals, Inc.
    Journal of Medical Virology 04/2014; 86(4). DOI:10.1002/jmv.23874 · 2.22 Impact Factor
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    ABSTRACT: Background and aimsEvery year globally WHO reports 20 million Hepatitis E virus (HEV) infections. The disease occurs as sporadic cases or focused outbreaks and has potentials to cause massive epidemics. The reservoir of HEV during inter-epidemic period is not well characterized. The sporadic cases usually lack history of contact with clinically overt HEV patients. In the present context we evaluated the occurrence of subclinical HEV as a possible reservoir in endemic region.Methods Blood samples were collected from 67 apparently-healthy individuals and 10 acute viral hepatitis (AVH) patients during two HEV outbreaks in North India. The serum samples were tested for anti-HEV IgM, IgG, HEV-IgG avidity index, HEV viral-load and conventional-PCR followed by sequencing and phylogenetic analysis.ResultsA total of 14 (20.89%) apparently-healthy individuals showed the presence of anti-HEV IgM and IgG. Out of 14 based on HEV-IgG avidity index 9 (64.28%) had secondary-exposure, 4 (28.57%) had primary-exposure, while one patient had intermediate avidity. Subclinical subjects with primary-exposure had significantly higher anti-HEV IgM index as compared to secondary-exposure (p= 0.0028). Viral load in clinically jaundiced patients was significantly higher as compared to subclinical subjects (p<0.0001). Phylogenetic analysis showed HEV sequences retrieved from subclinical individuals clustered along with AVH patients, suggesting matched source. The significantly low viral-load in subclinical subjects hints towards the dose dependency for progression of clinical manifestation.Conclusion We document subclinical HEV with low level viremia occurs during outbreak settings and goes un-noticed, which helps maintaining the virus in nature possibly leading to its endemicity.This article is protected by copyright. All rights reserved.
    Liver international: official journal of the International Association for the Study of the Liver 04/2014; 35(3). DOI:10.1111/liv.12568 · 4.41 Impact Factor
  • International Journal of Infectious Diseases 04/2014; 21:315. DOI:10.1016/j.ijid.2014.03.1072 · 2.33 Impact Factor
  • International Journal of Infectious Diseases 04/2014; 21:315. DOI:10.1016/j.ijid.2014.03.1073 · 2.33 Impact Factor
  • International Journal of Infectious Diseases 04/2014; 21:329. DOI:10.1016/j.ijid.2014.03.1099 · 2.33 Impact Factor
  • Indian journal of dermatology, venereology and leprology 09/2013; 79(5):705-6. DOI:10.4103/0378-6323.116744 · 1.33 Impact Factor
  • Journal of global infectious diseases 07/2013; 5(3):121-2. DOI:10.4103/0974-777X.116878
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    ABSTRACT: Mumps is a vaccine-preventable disease that usually occurs as a self-limiting parotitis, but it can also lead to several life-threatening complications, including pancreatitis, meningitis, and encephalitis. The molecular epidemiology of the virus is poorly understood. The present study describes an outbreak of mumps virus infection in Punjab, India. The etiology was confirmed by serology and RNA detection to be mumps virus in 72 % of the cases and 50 % of contacts. This study, for the first time, revealed the mumps virus genotypes circulating in the Indian subcontinent as subtype G2 of genotype G.
    Archives of Virology 05/2013; 158(11). DOI:10.1007/s00705-013-1717-4 · 2.28 Impact Factor
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    ABSTRACT: Hepatitis E virus (HEV) is implicated in many outbreaks of viral hepatitis in the Indian subcontinent. The conventional diagnosis of such outbreaks rests on the detection of anti-HEV IgM antibodies. However, IgM antibodies develop after 4-5 days of infection. An early-diagnostic marker is imperative for timely diagnosis of the outbreak and also initiation of control measures. This study aimed to determine the use of hepatitis E virus antigen detection as an early diagnostic marker in an outbreak in comparison to anti-HEV IgM and RT-PCR analyses. Forty samples were collected during a suspected outbreak of viral hepatitis due to HEV. A total of 36 samples were positive for one or more HEV markers. The positivity for anti-HEV IgM, HEV antigen, and RT-PCR was 91.6%, 69.4%, and 47.2% respectively. RT-PCR and HEV antigen detection gave the highest positive results (100%) in the first 3 days of illness. Positive HEV PCR declined to 54% by Days 4-7, whereas HEV antigen and IgM detection were 88% and 100%, respectively. Sequencing of representative HEV samples indicated that the strains responsible for this outbreak belonged to genotype I, subtype 1a. HEV antigen was found to be an early diagnostic marker of acute infection. HEV antigen was detected in three additional cases in the early phase (1-3 days), and they had no detectable anti-HEV IgM antibodies. These three samples were also positive for HEV RNA. After Day 7, anti-HEV IgM was the main diagnostic indicator of infection. J. Med. Virol. © 2013 Wiley Periodicals, Inc.
    Journal of Medical Virology 05/2013; 85(5). DOI:10.1002/jmv.23529 · 2.22 Impact Factor
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    ABSTRACT: Background: Rubella is traditionally considered a childhood disease but has the potential to cause outbreaks in hospital set ups. It is important to know the susceptibility status of health care workers (HCWs) as to frame guidelines for their immunization and thus prevent hospital outbreaks. Participants: The rubella susceptibility status of 313 HCWs working in the institute was assessed. This study was initiated after we reported an outbreak due to rubella among HCWs of our institute. Materials and Methods: The serum samples were tested to determine Rubella IgG titres by enzyme linked immunosorbent assay (ELISA). Results: Overall, 48 (15.3%) subjects were found to be negative, thereby indicating their susceptibility to infection. Out of them, 29 (60.5%) were in contact with pregnant women during the course of their employment. There is a risk of nosocomial transmission of rubella from affected HCWs to their contacts especially pregnant women as many of the rubella infections are asymptomatic. Conclusion: Hence, we stress the need for vaccinating the HCWs at the start of their employment to contain the spread of infection and also to reduce the risk of outbreaks in work place.
    Indian Journal of Pathology and Microbiology 04/2013; 56(2):148-50. DOI:10.4103/0377-4929.118704 · 0.64 Impact Factor
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    ABSTRACT: Hepatitis E virus (HEV) is an important cause of hepatitis in developing nations. Disease spans from asymptomatic infection to acute viral hepatitis (AVH) and acute liver failure (ALF). Cell-mediated immunity (CMI) is less studied. Studies document CMI in HEV patients using [ 3 H]-thymidine incorporation (radioactive in nature). The aim of this study was to evaluate the antigenicity of recombinant HEV ORF 2 peptide (452-617 a.a) (pORF2) by non-radioactive MTT assay and detecting the proliferation indices of primary PBMC culture. A total of 27 laboratory confirmed HEV patients (16 AVH and 11 ALF) and 20 apparently healthy individuals (HC) were included. PBMCs were isolated, plated and stimulated with pORF2. After an incubation of 4 days, cells were looked for blastogenic transformation and subjected to MTT assay. PI of AVH, ALF and healthy controls were found to be 3.249 ± 0.219, 1.748 ± 0.076 and 0.226 ± 0.017, respectively. PI of AVH Vs HC, ALF Vs HC and AVH Vs ALF were found to be significantly higher ( P < 0.0001). This study demonstrates MTT to be an adaptable technique to evaluate CMI in HEV patients. Recombinant pORF2 was found to be antigenic in nature and PBMCs from AVH patients were immunologically more reactive than ALF patients.
    Indian Journal of Medical Microbiology 01/2013; 31(1):64-8. DOI:10.4103/0255-0857.108725 · 1.04 Impact Factor

Publication Stats

317 Citations
173.94 Total Impact Points

Institutions

  • 2014
    • University of Rome Tor Vergata
      Roma, Latium, Italy
  • 2002–2014
    • Biomedical Informatics Centre
      Chandigarh, Chandīgarh, India
  • 1997–2014
    • Postgraduate Institute of Medical Education and Research
      • • Department of Virology
      • • Department of Parasitology
      Chandigarh, Chandīgarh, India
  • 2010
    • Hannover Medical School
      Hanover, Lower Saxony, Germany
  • 2008
    • National and Kapodistrian University of Athens
      Athínai, Attica, Greece