R K Ratho

Biomedical Informatics Centre, Chandigarh, Chandigarh, India

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Publications (100)214.27 Total impact

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    ABSTRACT: Hepatitis E (HEV) infection is diagnosed on the basis of serum anti-HEV IgM detection. In outbreaks, early diagnostic method is important for prompt control measures. This study compared three diagnostic methods in 60 serum samples collected in suspected HEV outbreaks. The suitability of saliva samples for antibody detection was also evaluated in 21 paired serum- saliva samples. The anti-HEV IgM, HEV-Ag and HEV-RNA were detected in serum samples of 52 (86.66%), 16 (26.66%) and 18 (30%) patients respectively. The concordance between serum and saliva IgM was found to be 76.91%. The positivity of PCR and HEV-Ag detection was 100% within one week of illness which declined to 5-10% thereafter. The outbreak was attributed to HEV Genotype 1, Subtype 1a and the clinical and environmental strains clustered together. HEV antigen and RNA were an early diagnostic marker with 96.66% concordance. Saliva samples can be used as an alternative in outbreak setting.
    No preview · Article · Dec 2015
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    ABSTRACT: Background: The clinical manifestations of Hepatitis E virus (HEV) range from self-limiting acute viral hepatitis (AVH) to acute liver failure (ALF). The varied clinical course is thought to be immune-mediated. Toll-like receptors (TLRs) play a central role in sensing and initiating innate antiviral-response and downstream signaling of TLRs modulates cytokine production, thereby playing an important role in determining the disease course. Objectives: The present study was designed to elucidate the role of TLRs and cytokine production in the immunopathogenesis of HEV. Study design: Peripheral blood mono-nuclear cells were separated from 50 AVH-HEV, 30 ALF-HEV patients and 50 healthy-controls. One-part of the PBMC was processed for RNA-extraction another pulsed with HEV-ORF2-peptide. Gene-expression levels of TLR (2-4, 7, and 8) were checked using semi-quantitative Real-time-PCR. Cytokine levels were analyzed using Cytokine-Bead-Array. TLR3-silencing experiments were performed and post-silencing cytokine levels were estimated. Results: TLR3 gene-expression in AVH was significantly higher than ALF (202.4±36.36 Vs 13.71±5.01; p<0.0001). Higher amount of both anti-and pro-inflammatory cytokines; IFNγ, TNF-α, IL10 and TGF-β were detected in the PBMC culture-supernatant of AVH Vs ALF (p<0.0001, p=0.0008, p=0.0002, p<0.0001 respectively). Post-silencing TLR3, significant decrease in IFNγ level was observed in the PBMC culture-supernatant (4.08±1.06 Vs 23.20±12.51; p=N0.0213). Conclusions: TLR3 and IFNγ were found to play an important role in HEV disease pathogenesis. Patients capable of expressing high levels of TLR 3 and robust IFNγ response are able to limit the disease and recover uneventfully; while the patients with lower expression of TLR3 and IFNγ progress to ALF.
    No preview · Article · Oct 2015 · Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology
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    ABSTRACT: Enteroviruses (EVs) and adenoviruses (AdVs) are two important etiological agents of viral myocarditis and dilated cardiomyopathy (DCM). Both these viruses share a common receptor, the coxsackievirus and adenovirus receptor (CAR), for their infection. However, the role of viral load and CAR expression in disease severity has not yet been completely elucidated. The present study aimed to determine viral load of EV and AdV in DCM patients and correlate them with the level of CAR expression in these patients. Sixty-three DCM cases and 30 controls, each of whom died of heart disease other than DCM and non-cardiac disease respectively, were included. Viral load was determined by TaqMan real-time PCR using primers and probes specific for the AdV hexon gene and the 5'UTR region of EV. The CAR mRNA level was semi-quantitated by RT-PCR, and antigen expression was studied by immunohistochemistry. A significantly high AdV load (p < 0.05) and CAR expression (p < 0.05) were observed in DCM cases versus controls, whereas the EV load showed no significant difference. The data suggests a clinical threshold of 128 AdV copies/500 ng of DNA for DCM, with 66.7 % sensitivity and 65 % specificity. A positive correlation between AdV load and CAR expression (p < 0.001) was also observed in DCM cases. The high adenoviral load and increased CAR expression in DCM and their association with adverse disease outcome indicates role of both virus and receptor in disease pathogenesis. Thus, the need for targeting both the virus and the receptor for treatment of viral myocarditis and early DCM requires further confirmation with larger studies.
    No preview · Article · Oct 2015 · Archives of Virology
  • M. P. Singh · C Chandran · A Sarwa · A Kumar · M Gupta · A Raj · R. K. Ratho
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    ABSTRACT: Purpose: Primary infection with a varicella-zoster virus (VZV) leads to chickenpox. Though the incidence of the disease has decreased in many developed countries due to the introduction of the varicella vaccine, outbreaks continue to occur in developing countries. Materials and Methods: The present study reports an outbreak of varicella in an urbanised village in the vicinity of Chandigarh City in North India in November 2013. The outbreak was confirmed by the detection of VZV IgM antibodies in serum samples of clinically suspected patients. Vesicular fluid samples were collected from 8 patients with active lesions and tested for VZV DNA by polymerase chain reaction. Blood samples were also collected from 17 healthy controls residing in the same locality and tested for the presence of VZV IgM and IgG antibodies. Results: A total of 18 cases occurred, and the majority of them (67%) were <15 years of age. Of 17 samples collected from patients with the clinically suspected disease, 13 (76.5%) showed the presence of VZV IgM antibodies. Of the healthy controls, 6 were VZV IgM positive and 4 of them developed symptomatic disease on follow-up. VZV DNA was positive in 5/8 (62.5%) of the patients. In one patient, VZV DNA was detected in the absence of an IgM antibody response. Conclusion: The introduction of varicella vaccine in the universal immunisation programme of India may help to prevent these outbreaks; however, the cost-benefit analysis needs to be carried out before making such policies. © 2015 Indian Journal of Medical Microbiology Published by Wolters Kluwer - Medknow.
    No preview · Article · Oct 2015 · Indian Journal of Medical Microbiology

  • No preview · Article · Sep 2015 · The Lancet Infectious Diseases
  • MP Singh · M Majumdar · K Goyal · PVM Laxmi · RK Ratho

    No preview · Article · Sep 2015 · Journal of Clinical Virology
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    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.
    Full-text · Article · Feb 2015 · The Indian Journal of Medical Research
  • B Mishra · M Sharma · S Sarkar · A Bahl · U N Saikia · R K Ratho
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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.
    No preview · Article · Jan 2015 · Indian Journal of Medical Microbiology
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    K Madhanraj · N singh · M gupta · MP singh · RK Ratho
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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.
    Full-text · Article · Nov 2014 · Indian pediatrics
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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.
    No preview · Article · Aug 2014 · Journal of Medical Virology
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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.
    Full-text · Article · Jul 2014 · Journal of global infectious diseases
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    Manasi Majumdar · R Ratho · Y Chawla · MP Singh

    Full-text · Article · Apr 2014 · Journal of Hepatology
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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.
    No preview · Article · Apr 2014 · Archives of Virology
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    J.L. Mathew · R.K. Ratho · N. Ahmed · S. Dutta

    Preview · Article · Apr 2014 · International Journal of Infectious Diseases
  • Manasi Majumdar · R Ratho · Y Chawla · MP Singh

    No preview · Article · Apr 2014 · Journal of hepatology. Supplement / EASL
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    M Majumdar · R Ratho · Y Chawla · M P Singh
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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.
    Full-text · Article · Apr 2014 · Indian journal of medical microbiology
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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.
    No preview · Article · Apr 2014 · Journal of Medical Virology
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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.
    No preview · Article · Apr 2014 · Liver international: official journal of the International Association for the Study of the Liver
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    Full-text · Article · Apr 2014 · International Journal of Infectious Diseases
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    M. Sharma · B. Mishra · U.N. Saikia · A. Bahl · R.K. Ratho

    Preview · Article · Apr 2014 · International Journal of Infectious Diseases

Publication Stats

495 Citations
214.27 Total Impact Points


  • 2002-2015
    • Biomedical Informatics Centre
      Chandigarh, Chandigarh, India
  • 1999-2015
    • Postgraduate Institute of Medical Education and Research
      • Department of Virology
      Chandigarh, Chandigarh, India
  • 2014
    • University of Rome Tor Vergata
      Roma, Latium, Italy
  • 2010
    • Hannover Medical School
      Hanover, Lower Saxony, Germany
  • 2008
    • National and Kapodistrian University of Athens
      Athínai, Attica, Greece