Indan Journal of Medical Research Indan Journal of Medical Research Indan Journal of Medical Research
  Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login  
  Home Print this page Email this page Small font sizeDefault font sizeIncrease font size Users Online: 6038    

   Table of Contents      
Year : 2012  |  Volume : 136  |  Issue : 3  |  Page : 477-482

Contribution of hepatitis E virus in acute sporadic hepatitis in north western India

1 Department of Zoology, University of Rajasthan; Department of Gastroenterology, SMS Hospital, Jaipur, India
2 Department of Zoology, University of Rajasthan, Jaipur, India
3 Department of Gastroenterology, SMS Hospital, Jaipur, India
4 Department of Microbiology, SMS Medical College, Jaipur, India

Date of Web Publication4-Oct-2012

Correspondence Address:
Bharti Malhotra
Associate Professor of Microbiology & Nodal Officer Advanced Research & TB Lab, SMS Medical College, Jaipur 302 004
Login to access the Email id

Source of Support: None, Conflict of Interest: None

PMID: 23041743

Rights and PermissionsRights and Permissions

Background & objectives: Hepatitis E virus (HEV) causes acute viral hepatitis. Majority of the documented studies on hepatitis E have been focused on the incidence of this disease in northern and south central India. Limited data are available on HEV infection among acute sporadic hepatitis cases in north western India. The present study was undertaken to investigate the contribution of hepatitis E virus infection in sporadic hepatitis cases in Rajasthan and neighbouring States.
Methods: Seven hundred and thirty six patients suspected to have viral hepatitis were screened for the hepatotropic viral markers, hepatitis A, B, C and E by using commercial enzyme immunoassay kits with a high sensitivity and specificity. The acute nature of HEV infection was also confirmed by the detection of HEV RNA by nested RT-PCR.
Results: Hepatitis E was found to be the major cause of acute sporadic viral hepatitis (49.7%) in this region of India. Mixed infections of HEV-HAV (1.2%), HEV-HBV (6.1%), and HEV-HCV (1.7%) were also detected. No viral marker was detected in 32 per cent cases.
Interpretation & conclusion: HEV was found as the major aetiological agent of acute sporadic viral hepatitis in Rajasthan (north western India). It is important to screen primarily for all the common enterically and parenterally transmitted hepatotropic viral markers in acute sporadic viral hepatitis. There is a need to do additional serological and molecular tests to identify the aetiological agent in the cases of acute hepatitis.

Keywords: Acute hepatitis - HBV - HCV - HEV - hepatitis - PCR - sporadic viral hepatitis

How to cite this article:
Chandra NS, Sharma A, Rai RR, Malhotra B. Contribution of hepatitis E virus in acute sporadic hepatitis in north western India. Indian J Med Res 2012;136:477-82

How to cite this URL:
Chandra NS, Sharma A, Rai RR, Malhotra B. Contribution of hepatitis E virus in acute sporadic hepatitis in north western India. Indian J Med Res [serial online] 2012 [cited 2021 Sep 18];136:477-82. Available from:

Hepatitis E was first recognised during an epidemic of hepatitis in Kashmir valley in 1978 [1] . It is an enterically transmitted disease that spreads through faecal contamination of drinking water. It occurs both in the form of epidemics as well as sporadic infection in developing countries [2],[3],[4],[5] . According to the South East Asia Regional office of the World Health Organization (WHO), hepatitis E is widespread in developing countries, accounting for up to 90 per cent of all sporadic cases of acute viral hepatitis [6] . Hepatitis E virus affects young to middle aged adults and causes high mortality in pregnant women, 20-30 per cent as compared to 0.2-1 per cent in general population [7] . It has been implicated as an important aetiological agent for sporadic fulminant hepatic failure (FHF) in developing countries [8] .

Aetiology of acute viral hepatitis (AVH) cannot be differentiated on the basis of mode of presentation; confirmation is done serologically. Hepatitis E virus is an important hepatotropic virus that causes acute viral hepatitis. Most of the studies on prevalence of hepatitis E virus are from epidemic setting and some from sporadic setting. Therefore, the present study was undertaken to investigate the aetiology of AVH sporadic cases in Rajasthan and neighbouring States for hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis E virus (HEV). The acute nature of HEV infection was also confirmed by the detection of HEV RNA by nested RT-PCR.

   Material & Methods Top

Patients: From January 2006 to July 2008, serum samples were collected from 736 individuals (aged 10-70 yr) from consecutive adult acute hepatitis cases aged 14-60 yr and only 4 children (aged 10-14 yr) suspected of having viral hepatitis, attending the OPD and wards of the Department of Gastroenterology, Sawai Man Singh (SMS) Hospital, Jaipur, Rajasthan. SMS hospital is a tertiary care centre where patients come from not only Rajasthan but from neighbouring States of Haryana, Punjab, Uttar Pardesh and Gujarat also. The study was approved by the institutional ethics committee and informed written consent was taken from the patients. Patients with signs and symptoms of jaundice, fever, loss of appetite, abdominal pain, scleral icterus, altered sensorium, encephalopathy and fatigue were included in the study. A detailed history of each patient was recorded, including travel history, blood transfusion, food and water intake from outside sources.

Immunoassays: Blood samples were collected from all patients under aseptic conditions and centrifuged at 1200 g. Serum was separated and stored at -80°C for further analysis. The samples were screened using commercially available Micro ELISA for markers of hepatitis A (IgM anti-HAV, Adaltis, Spain), hepatitis B (HBsAg, Biorad Monalisa HBsAg plus, IgM anti HBc in HBsAg positive cases Abbott laboratories North Chicago, IL) hepatitis C (Innotest HCV ab IV, Belgium) and hepatitis E (EIAgen HEV IgM, Adaltis, Spain). On the basis of serological tests, viral hepatitis was classified as acute hepatitis A (presence of IgM anti-HAV), acute hepatitis B (presence of HBsAg & IgM anti HBc), and hepatitis C may be acute or chronic (presence of anti-HCV) or acute hepatitis E (presence of IgM anti HEV), acute hepatitis E and B (presence of anti IgM HEV, HBsAg, IgM anti HBc) and HBV carrier (HBsAg positive). The ELISA kit used for IgM anti HEV were coated with recombinant proteins for open reading frame (ORF) 1 and 2 with 98 per cent sensitivity and specificity. ELISA was performed as per manufacturers' protocol.

HEV RNA extraction and detection: HEV RNA detection was done in all 356 IgM anti-HEV positive cases and in 28 patients within first five days of illness but negative for all serological markers. Extracted RNA by Guanidinium thiocyanate (GITC) chloroform phenol method with minor modification [9] was subjected for cDNA synthesis. cDNA synthesis was carried out using MuLV RT enzyme, reverse primer (20 pmol/ml, Promega) (external anti-sense: 5'- CCG AAT TCA AAG GCA TCC ATG GTG TTT GAG AAT GAC- 3') RNase out (20 U/μl, Invitrogen, UK), 0.1M DTT and 5 μl templates at 42o C for one hour.

After cDNA synthesis PCR amplification was carried out using the specific previously validated primers selected from non-structural ORF-1 region (Gene Bank accession no. M-32400) [10] . The primers used were external sense: 5'- CCG GAT CCA CAC ACA TCT GAG CTA CAT TCG TGA GCT- 3', external anti-sense: 5'- CCG AAT TCA AAG GCA TCC ATG GTG TTT GAG AAT GAC- 3', internal sense: 5'- GGA ATT CGA CTC CAC CCA GAA TTA CTT- 3', and internal anti-sense 5'- GGA ATT CAC AGC CGG CGA TCA GGA CAG- 3'. These two sets of primers were designed to produce 343 bp segment of ORF1 region [10] . The thermal cycling conditions were initial denaturation 94 o C for 5 min followed by 30 cycles of denaturation for 30 sec at 94 o C, annealing for 30 sec at 59 o C and extension for 30 sec at 72 o C, as well as final extension for 7 min at 72 o C. The final PCR products of 384 patients (356 IgM antiHEV positive cases and 28 negative for all markers but with <5 day illness) were checked on 2 per cent gel electrophoresis stained with ethidium bromide (0.5 μg/ml) under UV transilluminator. For nested PCR, steps were taken to guard against carry over contamination by working in dedicated and physically separated rooms for sample preparation, master mix, amplification and electrophoresis. Each room had dedicated reagents, consumables, filter tips, micro pipettes, furniture, colour coded footwear and gowns, etc. which were not taken out to any other room. Samples were handled in biosafety cabinet. Positive and negative controls were included; sentinel negative controls were also used to check for carry over contamination. The first inner PCR product was handled in a separate safety cabinet from the outer final PCR product.

   Results Top

A total of 736 cases of acute sporadic hepatitis were seen over the study period. HEV was the most common cause followed by HBV, HAV and HCV. Of the 736 patients, 356 (48.3 per cent) patients with acute sporadic hepatitis were positive for IgM anti HEV alone (n=285) or in combination with other virus (n=71) by serology. In almost one third of the patients no viral marker could be detected [Table 1]. Among 356, 176 had acute viral hepatitis (AVH), 142 had fulminant hepatic failure (FHF) and 38 had subacute hepatic failure (SAHF). There was no mortality in 176 patients of AVH, while 16 (11.27%) FHF patients and 3 (7.8%) SAHF patients died. Of the 142 FHF patients, 24 (16.9%) were positive for HBs Ag, four for HAV IgM.
Table 1: The aetiology of acute sporadic hepatitis (n=736)

Click here to view

The age of presentation of patients with sporadic acute hepatitis E ranged from 10 to 65 yr (mean ± SD of 32.43 ± 11.07 yr). Maximum numbers of cases of HEV were seen in the age group 15-40 yr. Among 356 IgM anti HEV positive acute sporadic hepatitis E cases, 234 (65.73%) were males and 122 (34.26%) were females. Of the 122 consecutive females, 49 (40.16%) were pregnant. The mortality rate in pregnant females was 20.41 per cent (10/49) and in non-pregnant females was 4.10 per cent (3/73). 17 (34.69%) pregnancies ended in abortion or still birth and in 22 (44.89%) females pregnancy continued unaffected by AVH or normal delivery occurred during the disease course.

All 736 patients had abnormal liver function tests (LFT) suggestive of acute hepatitis; the tests were repeated at weekly intervals. The average duration of abnormal LFT was 11 to 15 days after onset of illness.

Serum samples from 356 IgM anti-HEV positive and 28 of 236 (11.86%) patients of acute sporadic hepatitis (within five days of illness and negative for all serological markers) were investigated for presence of HEV-RNA to confirm acute nature of HEV infection. Of the 356 IgM anti-HEV positives, 223 (62.64 %) and 10 out of 28 (35%) were found to be positive for HEV-RNA. Overall, 366 of 736 (49.72%) patients were found to be HEV positive by either ELISA or PCR.

   Discussion Top

Hepatitis E virus is a major hepatotropic virus for acute viral hepatitis. Hepatitis E exists as sporadic hepatitis with periodic resurgence. This infection is responsible for 30-70 per cent cases of acute sporadic hepatitis [11] and is the major cause of acute liver failure (ALF) [12] . It has been widely reported that HEV primarily affects young adults between 15-40 yr of age in endemic region [13] . Similar findings were observed in the present study.

In the present study, among the 736 serum samples obtained from acute sporadic hepatitis patients with jaundice, 366 (49.7%) were positive for HEV alone or in combination with other hepatitis viruses and responsible for about half of the acute viral hepatitis. HEV positivity ranging from 12.6-78.6 per cent has been reported by others from different parts of India [Table 2] [14],[15],[16],[17],[18],[19] . Positivity depends on sensitivity and specificity of kits used, population studied, type of samples whether sporadic or from outbreaks. Wide variation has been reported from 17-100 per cent in sensitivity of ELISA kits which depends on recombinant HEV antigens or synthetic peptides used in the kit corresponding to HEV epitopes, kits with ORF 2 sequence have shown little variation in geographically diverse strains in comparison to kits targeting ORF3. Variation may also occur with manufacturers lot and antibodies to different epitopes may differ in persistence [20],[21],[22] . The test may perform differently in endemic and non endemic situation as 33-40 per cent general population has been found to be anti HEV-IgG positive, so difficulty may occur to distinguish between present and past HEV infection [23],[24] . Moreover, in immunocompromised individuals antibodies may persist for 6-10 months. Therefore, anti HEV IgM alone may not be informative for diagnosis of acute sporadic HEV infections at times.
Table 2: Prevalence of hepatitis E virus in India reported in various studies

Click here to view

In the present study all anti-HEV IgM positive samples were tested for HEV RNA to confirm acute ongoing infection as it has been reported that PCR may be better indicator for acute HEV infection during the first week of illness than ELISA. In our study, 62.64 per cent anti-HEV IgM positive cases were positive for HEV-RNA also. This could be due to the fact that HEV viraemia is transient, occurs in first two wk of infection and declines after 1 wk of onset of Jaundice [25] . In patients with less than five days history of illness 80.7 per cent were positive for anti-HEV IgM and 85.3 per cent for HEV RNA as observed by us earlier also [26] . Clayson et al[25] observed 93 per cent positivity for HEV RNA and only 79 per cent for IgM anti-HEV while Kumar et al[14] reported only 9.4 per cent HEV RNA positivity in AVH cases but 100 per cent positivity in FHF cases in confirmed anti-HEV IgM positive samples. Detection of HEV RNA along with recombinant ORF2 ELISA for anti-HEV IgM could increase positive predictive value for diagnosis of acute HEV infection specially in endemic areas [14] . However, IgM antibody detection is a better choice for most diagnostic laboratories with serology facilities; HEV RNA testing may also be done in better equipped laboratories within one week of onset of symptoms when other serological tests are negative.

In endemic areas, infection with HEV has been seen in association with other hepatotropic viruses (HAV, HBV and HCV) as also observed in the present study [14],[27],[28],[29] . Dual infection with HAV, HBV and HCV in acute HEV patients was observed in 1.2, 6.1 and 1.7 per cent cases, respectively, without any squeal. All the HAV and HEV dual infections were possibly co-infections as these have a common route of transmission and all infected patients were less than 14 yr of age. Malathi et al[27] observed dual infection of HEV and HAV in 13.4 per cent patients of acute hepatitis in children and Kumar et al[28] observed 4.4 per cent patients of acute hepatitis in adults.

In our study, dual acute infection of HEV and HBV was reported in 4.61 per cent and acute HEV on chronic HBV infection was seen in 1.5 per cent of acute hepatitis cases. It could be due to two reasons; one, the population corresponds to meso endemic zone of HBV; second reason could be attributed to the reactivation of latent HBV due to clinical HEV. However, there is little data available to determine such type of infection. In a study from Chennai [29] , dual infection of HEV and HBV was observed in 5.4 per cent of adults patients of acute hepatitis. Anti-HEV IgM seropositivity rate ranging from 3.2 to 31 per cent in HBsAg carriers has been reported in different studies [14],[30] and hepatitis E has also been suspected to cause super-infection in carriers of hepatitis B [31] .

The HCV - HEV super-infection was observed in 1.7 per cent cases, all of whom had a history of prior exposure to procedures relating to parenteral route (7 had blood transfusion 4 had surgery and 2 were drug abusers). Studies from Delhi and Chandigarh [8],[28] reported dual infection of HEV - HCV in 11 and 7.4 per cent cases, respectively. HCV positivity varies in different geographical areas. Low positivity of HCV has been reported from Rajasthan earlier; 0. 28 per cent by Sood et al[32] from a new private hospital and 1.693 per cent by Sharma et al[33] from a tertiary care hospital. Mehta et al[34] from Jodhpur reported 0.27per cent HEV positivity in blood donors. Since our study was from known AVH cases, 5.1 per cent HCV positivity and 1.7 per cent HEV-HCV positivity are justified as per local trends of HCV infection.

In our study 32 per cent patients were found to be negative for all common hepatotropic viral markers by serological tests, further testing for HEV RNA in 28 samples negative for all serological test in patients with less than five days of illness helped in diagnosing HEV infection in additional patients. However, additional serological and molecular tests should be planned for identifying the aetiological agents in serologically negative cases for HAV, HBV, HCV and HEV to determine whether other viruses like hepatitis G (HGV), Sen Virus, TT virus, etc. exist in this part of India or variant of any other known pathogen.

   References Top

1.Khuroo MS. Study of an epidemic of non-A, non-B hepatitis. Possibility of another human hepatitis virus distinct from post-transfusion non-A, non-B type. Am J Med 1980; 68 : 818-24.  Back to cited text no. 1
2.Bradley DW. Hepatitis E: epidemiology, aetiology and molecular biology. Rev Med Virol 1992; 2 : 19-28.  Back to cited text no. 2
3.Labrique AB, Thomas DL, Stoszek SK, Nelson KE. Hepatitis E: an emerging infectious disease. Epidemiol Rev 1999; 21 : 162-79.  Back to cited text no. 3
4.Worm HC, Van der poel WH, Brandstatter G. Hepatitis E: an overview. Microbes Infect 2002; 4 : 657-66.   Back to cited text no. 4
5.Arankalle VA, Chobe LP, Jha J, Chadha MS, Banerjee K, Favorov MO, et al. Aetiology of acute sporadic non-A, non-B viral hepatitis in India. J Med Virol 1993; 40 : 121-5.  Back to cited text no. 5
6.Aggarwal R, Naik S. Epidemiology of hepatitis E: Current status. J Gastroenterol Hepatol 2009; 24 : 1484-93.  Back to cited text no. 6
7.Chandra V, Taneja S, Kalia M, Jameel S. Molecular biology and pathogenesis of hepatitis E virus. J Biosci 2008; 33 : 451-64.  Back to cited text no. 7
8.Nanda SK, Yalcinkaya K, Panigrahi AK, Acharya SK, Jameel S, Panda SK. Etiological role of hepatitis E virus in sporadic fulminant hepatitis. J Med Virol 1994; 42 : 133-7.  Back to cited text no. 8
9.Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162 : 156-9.  Back to cited text no. 9
10.Jameel S, Durgapal H, Habibullah CM, Khuroo MS, Panda SK. Enteric non-A, non-B hepatitis: epidemics, animal transmission, and hepatitis E virus detection by the polymerase chain reaction. J Med Virol 1992; 37 : 263-70.  Back to cited text no. 10
11.Panda SK, Thakral D, Rehman S. Hepatitis E virus. Rev Med Virol 2007; 17 : 151-80.  Back to cited text no. 11
12.Acharya SK, Panda SK, Saxena A, Gupta SD. Acute hepatic failure in India: a perspective from the East. J Gastroenterol Hepatol 2000; 15 : 473-9.  Back to cited text no. 12
13.Arankalle VA, Tsarev SA, Chadha MS, Alling DW, Emerson SU, Banerjee K, et al. Age-specific prevalence of antibodies to hepatitis A and E viruses in Pune, India, 1982 and 1992. J Infect Dis 1995; 171 : 447-50.   Back to cited text no. 13
14.Kumar S, Ratho RK, Chawla YK, Chakraborti A. The incidence of sporadic viral hepatitis in North India: a preliminary study. Hepatobiliary Pancreat Dis Int 2007; 6 : 596-9.   Back to cited text no. 14
15.Chadha MS, Walimbe AM, Chobe LP, Arankalle VA. Comparison of aetiology of sporadic acute and fulminant viral hepatitis in hospitalized patients in Pune, India during 1978-81 and 1994-97. Indian J Gastroenterol 2003; 22 : 11-5.   Back to cited text no. 15
16.Das K, Agarwal A, Andrew R, Frösner GG, Kar P. Role of hepatitis E and other hepatotropic virus in aetiology of sporadic acute viral hepatitis: a hospital based study from urban Delhi. Eur J Epidemiol 2000; 16 : 937-40.   Back to cited text no. 16
17.Jain A, Kar P, Madan K, Das UP, Budhiraja S, Gopalkrishna V, et al. Hepatitis C virus infection in sporadic fulminant viral hepatitis in North India: cause or co-factor. Eur J Gastroenterol Hepatol 1999; 11 : 1231-7.   Back to cited text no. 17
18.Bansal J, He J, Yarbough PO, Sen S, Constantine NT, Sen D. Hepatitis E virus infection in eastern India. Am J Trop Med Hyg 1998; 59 : 258-60.  Back to cited text no. 18
19.Sarguna P, Rao A, Sudha Ramana KN. Outbreak of acute viral hepatitis due to hepatitis E virus in Hyderabad. Indian J Med Microbiol 2007; 25 : 378-82.  Back to cited text no. 19
20.Mast EE, Alter MJ, Holland PV, Purcell RH. Evaluation of assays for antibody to hepatitis E virus by a serum panel. Hepatitis E Virus Antibody Serum Panel Evaluation Group. Hepatology 1998; 27 : 857-61.  Back to cited text no. 20
21.Ticehurst JR. Hepatitis E virus. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, editors. Manual of clinical microbiology. 7 th ed. Washington, DC: American Society for Microbiology; 1999.  Back to cited text no. 21
22.Longer CF, Denny SL, Caudill JD, Miele TA, Asher LV, Myint KS, et al. Experimental hepatitis E: pathogenesis in cynomolgus macaques (Macaca fascicularis). J Infect Dis 1993; 168 : 602-9.  Back to cited text no. 22
23.Lin CC, Wu JC, Chang TT, Chang WY, Yu ML, Tam AW, et al. Diagnostic value of immunoglobulin G (IgG) and IgM anti-hepatitis E virus (HEV) tests based on HEV RNA in an area where hepatitis E is not endemic. J Clin Microbiol 2000; 38 : 3915-8.  Back to cited text no. 23
24.Favorov MO, Fields HA, Purdy MA, Yashina TL, Aleksandrov AG, Alter MJ, et al. Serologic identification of hepatitis E virus infections in epidemic and endemic settings. J Med Virol 1992; 36 : 246-50.   Back to cited text no. 24
25.Clayson ET, Myint KS, Snitbhan R, Vaughn DW, Innis BL, Chan L, et al. Viremia, fecal shedding, and IgM and IgG responses in patients with hepatitis E. J Infect Dis 1995; 172 : 927-33.   Back to cited text no. 25
26.Chandra NS, Sharma A, Malhotra B, Rai RR. Dynamics of HEV viremia, fecal shedding and its relationship with transaminases and antibody response in patients with sporadic acute hepatitis E. Virol J 2010; 7 : 213-9.  Back to cited text no. 26
27.Malathi S, Mohanavalli B, Menon T, Srilatha P, Sankaranarayanan VS, Raju BB, et al. Clinical and viral marker pattern of acute sporadic hepatitis in children in Madras, South India. J Trop Pediatr 1998; 44 : 275-8.   Back to cited text no. 27
28.Kumar S, Ratho RK, Chawla YK, Chakraborti A. Virological investigation of a hepatitis E epidemic in North India. Singapore Med J 2006; 47 : 769-73.   Back to cited text no. 28
29.Mohanavalli B, Menon T, Mohan KV, Ramathilagam B, Dinakaran N, Malathi S, et al. Serological profile of hepatitis A-E viruses in sporadic acute viral hepatitis in adults in Madras. Indian J Gastroentrol 1996; 15 : A116.  Back to cited text no. 29
30.Khuroo MS, Rustgi VK, Dawson GJ, Mushahwar IK, Yattoo GN, Kamili S, et al. Spectrum of hepatitis E virus infection in India. J Med Virol 1994; 43 : 281-6.   Back to cited text no. 30
31.Khuroo MS, Deurmeyer W, Zargar SA, Ahanger MA, Shah MA. Acute sporadic non-A, non-B hepatitis in India. Am J Epidemiol 1983; 118 : 360-4.  Back to cited text no. 31
32.Sood S, Malvankar S. Seroprevalence of hepatitis B surface antigen, antibodies to the hepatitis C virus, and human immunodeficiency virus in a hospital-based population in Jaipur, Rajasthan. Indian J Commun Med 2010; 35 : 165-9.  Back to cited text no. 32
33.Sharma R, Sinha P , Bachiwal R, Rishi S. Seroprevalence of anti-hepatitis C virus antibody in a hospital-based population of Jaipur, Rajasthan. Indian J Commun Med 2007; 32 : 158-59.  Back to cited text no. 33
34.Mehta NM, Purohit A, Haag A, Mathur A, Joshi KC, Joshi R, et al. Co-infection rate of HIV and hepatitis C Virus (HCV) among blood donors in Rajasthan, India. Int Conf AIDS, 2002; 14 : abstract no. TuPeA4401.  Back to cited text no. 34


  [Table 1], [Table 2]


    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

  In this article
   Material & Methods
    Article Tables

 Article Access Statistics
    PDF Downloaded333    
    Comments [Add]    

Recommend this journal