|Ahead of print
|Role of real-time polymerase chain reaction in diagnosing Hepatitis E, the commonest cause of acute hepatitis in adult patients seeking institutional care
Divya Namdeo1, Pratima Shrivastava2, Garima Garg2, Ashish K Vyas2, Ram K Nema1, Abhishek Singhai3, Shashwati Nema4, Debasis Biswas5
1 Regional Virology Lab, All India Institute of Medical Sciences, Bhopal, Madhya Pradesh, India
2 Department of Microbiology, All India Institute of Medical Sciences, Bhopal, Madhya Pradesh, India
3 Department of Medicine, All India Institute of Medical Sciences, Bhopal, Madhya Pradesh, India
4 Regional Virology Lab; Department of Microbiology, All India Institute of Medical Sciences, Bhopal, Madhya Pradesh, India
5 Regional Virology Lab; Departments of Microbiology, All India Institute of Medical Sciences, Bhopal, Madhya Pradesh, India
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|Date of Submission||25-Aug-2022|
|Date of Decision||31-Oct-2022|
|Date of Acceptance||01-Nov-2022|
|Date of Web Publication||31-Mar-2023|
| Abstract|| |
Background: This cross-sectional study was performed with the aim of determining the prevalence of hepatitis E virus (HEV) infection among acute hepatitis patients attending a tertiary care teaching hospital in a developing country and to determine the relative performance of prevalent diagnostic assays in establishing its diagnosis. Materials and Methods: A total of 46 adult patients were included in this study, all of whom presented with jaundice of <4 weeks' duration and elevation of AST and ALT above 500 U/L. The prevalence of HEV among patients with acute hepatitis was calculated on the basis of the proportion of recruited patients reacting positively in serum anti-HEV immunoglobulin M (IgM) and real-time polymerase chain reaction (RT-PCR) assays. Results: Among the recruited patients, 11 (23.91%) and 15 (32.6%) patients were positive for anti-HEV IgM and RT-PCR, respectively. The two tests demonstrated poor inter-test agreement, thereby implying the necessity of performing both tests for reliable diagnosis of acute HEV virus infection. We also observed a significant difference in the duration of illness between RT-PCR positive and negative patients (P = 0.008). The mean (±SD) duration of illness in the two groups was 8.6 (±3.50) and 11.66 (± 5.15) days, respectively. Combining the results of IgM ELISA and RT-PCR, we observed that 23 out of 46 patients (50%) had evidence of acute HEV virus infection among our patients. Conclusion: Our study suggests that HEV is the commonest cause of acute hepatitis in adult patients attending a tertiary care teaching hospital and that the diagnostic algorithm for its confirmation should include both IgM ELISA and RT-PCR assays.
Keywords: Anti-HEV IgM ELISA, hepatitis A, hepatitis B, hepatitis E, RT-PCR
|How to cite this URL:|
Namdeo D, Shrivastava P, Garg G, Vyas AK, Nema RK, Singhai A, Nema S, Biswas D. Role of real-time polymerase chain reaction in diagnosing Hepatitis E, the commonest cause of acute hepatitis in adult patients seeking institutional care. Indian J Pathol Microbiol [Epub ahead of print] [cited 2023 Jun 1]. Available from: https://www.ijpmonline.org/preprintarticle.asp?id=373318
| Introduction|| |
Worldwide hepatitis E virus (HEV) infection is estimated to cause 20 million infections and accounts for 3.3 million cases of acute hepatitis E every year. According to a World Health Organization (WHO) estimate, HEV led to approximately 44,000 deaths and was responsible for 3.3% of the mortality due to viral hepatitis. Though HEV is prevalent worldwide, the highest prevalence is observed in East and South Asia. Two epidemiological patterns of the disease have been observed. In developing countries with poor hygiene and sanitation, the predominant pattern manifests as outbreaks and sporadic cases following fecal contamination of drinking water. HEV genotypes 1 and 2 are mainly associated with this pattern. On the other hand, in developed countries like Europe and USA, HEV mainly occurs as sporadic cases originating from the ingestion of undercooked animal meat. In such settings, HEV 3 and HEV 4 are the main causal agents.
India is hyperendemic for HEV transmission, and though it is less contagious than hepatitis A virus (secondary attack rate among household contacts being 0.7%–2% compared to the same in HAV infection being 50%–75%), during an epidemic pregnant women have a higher likelihood of getting infected (12%–20%) compared to non-pregnant females and males (1%–2%).
Since the clinical and biochemical alterations are non-specific in acute hepatitis, the etiological diagnosis of HEV requires the performance of molecular and serological assays. Among the serological assays, serum immunoglobulin M (IgM) is detectable from 4 weeks after the onset of infection and persists till 6 months after the disease onset. IgA is produced after IgM and is noticeable till about 1-month post-infection. On the other hand, HEV RNA can be detected during the incubation period of infection and remains detectable till 4 weeks and 6 weeks in blood and stool, respectively. Moreover, in immunosuppressed patients, anti-HEV antibodies are often undetectable and detection of HEV RNA by real-time polymerase chain reaction (RT-PCR) demonstrates higher sensitivity. With the limited applicability of cell culture assays in routine diagnostics, the diagnostic algorithm for acute HEV infection mostly depends on anti-HEV IgM assay and RT-PCR. Traditionally, IgM enzyme-linked immunosorbent assay (ELISA) has been considered as the initial diagnostic test, which is supplemented by RT-PCR in case of negative ELISA results in clinically suspected patients., The sensitivity of ELISA has also been reported to be sub-optimal in immunocompromised patients. This lack of unanimity on the choice of the diagnostic test has impeded the development of a universal guideline regarding the diagnostic approach for acute HEV infection.
In view of this existing gap in knowledge regarding the true prevalence and appropriate diagnostic approach for acute HEV infection, the present study was designed to estimate the burden of HEV infection in the cohort of acute hepatitis patients attending a tertiary care teaching hospital in central India. Conducted in a developing country which is endemic for a number of feco-orally transmitted waterborne pathogens, this study aimed at determining the relative contribution of HEV in the syndrome of acute hepatitis and also to evaluate the relative diagnostic performance of the two commonly performed assays and thus inform the choice of an appropriate diagnostic algorithm for reliable diagnosis of acute HEV infection.
| Materials and Methods|| |
This prospective study was performed on patients aged 18 years or more, who presented to OPD/IPD of the Medicine Department of our tertiary care teaching hospital, between January 2019 and March 2021, with jaundice of <4 weeks' duration along with grossly deranged liver enzymes (aspartate aminotransferase [AST] and alanine aminotransferase [ALT] >500 U/l). The study protocol was duly approved by the Institutional Human Ethics Committee (IHEC), AIIMS Bhopal (vide letter Ref. no IHEC-LOP/2020/MD0109; dated 10 January 2020). Demographic and clinical details of the patients, including their age; gender; presenting manifestations and salient laboratory findings, were collected by the clinicians and entered in a pre-designed case record form after obtaining written informed consent.
The serum sample of every recruited patient was collected and stored in a sterile 2 mL screw cap tube. The aliquot for anti-HEV IgM ELISA was kept at −20°C, while the one for HEV RT-PCR was stored at −80°C till further analysis. An assay for anti-HEV IgM was performed by commercially available HEV IgM ELISA 3.0, (MP Biomedicals Asia Pacific Pte. Ltd.) as per the manufacturer's directions. Viral RNA was extracted from serum specimens using a commercial RNA extraction kit (Qiagen QIAamp Viral RNA Mini kit, Germany) and the extracted RNA was used for RT-PCR reaction. Taqman RT-PCR assay was developed for the detection of HEV RNA in serum samples. The primers and probe were selected from a highly conserved open reading frame-3 region of the HEV genome and were used for performing RT-PCR as described by Jothikumar et al. No template control, extraction control, and positive control were included in each run. All RT-PCR experiments were run in duplicate. Samples with Ct values <40 were considered positive.
The prevalence of HEV among patients with acute hepatitis was calculated on the basis of the proportion of recruited patients reacting positively in serum anti-HEV IgM and RT-PCR assays. The association of positive results in either test with different demographic, clinical, laboratory, and epidemiological features was analyzed using the Chi-square test using OpenEpi (2) software. Interest agreement between ELISA and RT-PCR tests was also calculated using OpenEpi software. Comparison of the duration of illness between the three sub-groups of acute HEV patients, viz., ELISA and RT-PCR positive; only RT-PCR positive and only ELISA positive, was performed using a one-way analysis of variance test. Comparison of Ct values between RT-PCR and ELISA positive patients, and RT-PCR and ELISA negative patients was done by unpaired t-test using statistical package for social sciences version 26.
| Results|| |
We recruited 46 adult patients (27 males), who presented to our tertiary care teaching hospital in central India during the study duration with manifestations suggestive of acute hepatitis, as defined by our inclusion criteria. The mean (±SD) age of patients was 39 (±14) years, with the majority of the patients belonging to the age group of 21–30 years and 51–60 years (30.43% each). The mean (±SD) duration of illness of the patients was 11 (±5) days. The demographic particulars and the profile of liver function tests of the recruited patients are shown in [Table 1].
|Table 1: Profile of demographic details and liver function tests of recruited patients|
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To determine the prevalence of HEV infection in this cohort of patients, we undertook both anti-HEV IgM ELISA and HEV RT-PCR assays. As shown in [Table 2], 11 and 15 of the 46 recruited patients were positive for ELISA and RT-PCR, respectively; with 20 patients demonstrating discordant results between the two assays. The two tests thus demonstrated poor inter-test agreement with a Kappa value of 0.06. Combining the results of both assays, we observed a prevalence of 50% (23/46) for acute HEV infection in our cohort.
We next determined the relative preponderance of the different hepatitis viruses in this cohort and found HEV to be the commonest cause of acute hepatitis in adult patients presenting to a tertiary care teaching hospital. Hepatitis B virus (HBV) was the next most common with a prevalence of 7.52% (6/42). Only one patient was found to be infected with the hepatitis A virus (HAV) (2.71%). Three of the recruited patients were co-infected with HBV and HEV, among whom two were detected by anti-HEV ELISA and one by RT-PCR.
Having found that IgM ELISA and RT-PCR were not mutually exclusive but complementary to each other in establishing the diagnosis of acute HEV infection, we next attempted to identify the clinical and epidemiological correlates of this condition. To identify potential predictors of acute HEV infection, we performed the univariate analysis with a range of parameters like relevant risk factors, comorbidities, and severity of illness and failed to find any significant association between positive results in ELISA and any of these risk factors. Similarly, no statistically significant association could be derived between positive results in IgM ELISA and RT-PCR test and any of the laboratory indicators of liver function test.
However, we observed a significant correlation between the modality of HEV diagnosis and the duration of illness; with the RT-PCR positive patients having a significantly shorter duration of illness compared to those negative for RT-PCR (P = 0.004) [Table 3]. We also compared the viral load in the two RT-PCR positive groups by comparing the mean Ct values between RT-PCR positive patients with and without ELISA positive results. However, we did not observe a significant difference between the two groups (P = 0.267) [Table 3].
|Table 3: Association of anti-HEV IgM and RT-PCR results with the duration of illness and Ct value|
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| Discussion|| |
In this paper, we report HEV to be the commonest cause of acute hepatitis in adult patients presenting to a tertiary care teaching hospital in a developing country followed by HBV and HAV, respectively. None of the demographic, clinical, and routine laboratory parameters or risk factors was found to be significant predictors of HEV infection, thereby necessitating the mandatory use of specific laboratory tests. Among the tests evaluated, viz., anti-HEV IgM ELISA and HEV RT-PCR were not found to demonstrate significant inter-test agreement, thereby implying the complementary nature of the two tests in achieving the diagnosis of this infection. The duration of illness was, however, significantly shorter in RT-PCR positive patients than in those positive in IgM ELISA.
In previously reported studies of sporadic acute hepatitis cases from India, conducted in settings similar to ours, the prevalence of HEV infection has been found to range from 8.9% to 94.1%.,,,,,,,,,,, This wide variation among the reported studies could be attributed to heterogeneity in the inclusion criteria, sample size, endemicity of the infection in the concerned geographical regions, and sensitivity of the employed diagnostic methods.
The relative frequency of infection with the different hepatitis viruses has also demonstrated marked variability. In a hospital-based study from Lucknow involving both pediatric (n = 143) and adult (n = 124) patients, co-infection of multiple hepatitis viruses was observed in 12.73% of the recruited cases and the commonest co-infection was with HAV and HEV (8.61% and 8.87% of the total and adult patients, respectively). Contrary to this study, we observed HEV to be overwhelmingly commoner than the other hepatitis viruses and co-infection with the waterborne HAV and HEV was not observed by us. This might be due to the difference in the clinical profile of the two study groups, with the majority of our patients being out-patients and none of them suffering from acute hepatic failure. The difference in the sample size of the two studies could also lead to such differences in relative frequencies of the hepatitis viruses. Similar to our study, a study reported from Bangalore in 2009 also observed HEV to be the commonest cause of acute viral hepatitis. But contrary to our findings, HAV was reported to be more commoner than HBV. As observed by the authors, this could be due to the increased incidence of HAV in children and the development of protective immunity to HAV in the majority of individuals by late adolescence. This could be the reason for encountering a lower frequency of HAV infection in our study, which was restricted to adults only. However, similar to our finding of 7.14% cases of co-infection from HEV with HBV, two other studies from India also reported similar co-infection rates in 6.1% and 3.8% of cases.,
In our prospective study, the inter-test agreement between IgM ELISA and RT-PCR result was unsatisfactory, consequently indicating the need to conduct both tests for effective diagnosis of HEV infection. Likewise, other authors have also compared both the tests and suggested dual performance of both the serology and molecular assays for diagnosis.,,,, A study from central India, showed that only 31 samples were positive by PCR out of 341 anti-HEV IgM ELISA positive samples.
We also observed a significant difference in the duration of illness among RT-PCR positive (n = 15) and negative (n = 31) patients, thereby hinting at the duration of illness in influencing the choice of diagnostic tests. The viremia in HEV-infected patients peaks, during the incubation period and the early symptomatic phase. HEV RNA is detectable in blood only till 3 weeks after the onset of symptoms and thus samples collected in the late phase of symptomatic illness are expected to be negative for HEV RNA. Similar to our findings, several studies have also reported that during the first week of acute HEV infection, PCR is a better assay than anti-HEV IgM ELISA.,, The complementary nature of the two diagnostic assays, as revealed in the present study and in earlier studies, suggests the need to review the current diagnostic algorithm, which relies mostly on anti-HEV IgM ELISA as the first-line test and in which the RT-PCR assay is indicated only in immunocompromised patients. In a shift from this approach, we surmise that the testing algorithm should be reflective of the natural course of the infection and thus be dictated by the duration of the patient's illness.
Our study suffered from several limitations. Firstly, this pilot study was performed on a limited number of subjects due to resource constraints. Secondly, we could not evaluate the diagnostic performance of the RT-PCR assay in stool samples, which might have led to the detection of additional cases in view of the more prolonged excretion of the virus in stool.,, Thirdly, being a cross-sectional study, the temporal dynamics of viremia could not be ascertained in RT-PCR positive samples. Further studies need to be undertaken to address the above-mentioned limitations and also to study the genotypic distribution, phylogenetic association, and molecular epidemiology of HEV infection in this geographical region.
| Conclusion|| |
Our study suggests that HEV is the commonest cause of acute hepatitis in adult patients attending a tertiary care teaching hospital in our population and that the diagnostic algorithm for its confirmation should include both IgM ELISA and RT-PCR assays, with the choice of test being influenced by the duration of presenting illness.
We acknowledge ICMR/DHR, Govt. India, for supporting the Regional Virology Lab, AIIMS Bhopal, where the study was conducted.
Declaration of patient consent
Written informed consent has been obtained from the patient(s) for their participation in the study.
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of All India Institute of Medical Sciences Bhopal, (vide letter Ref. no IHEC-LOP/2020/MD0109 dated 10 January 2020).
Conceptualization—DB; Methodology—AKV, DN, RKN; Validation—AKV, PS, GG; Formal analysis—DB, SN; Investigation—PS, DB, GG, Resources—DB, AS, Data curation—PS, DN, DB; Writing—original draft preparation—DN, PS Writing—review and editing—DB; Supervision—DB, SN; Project Administration—DB; Funding acquisition—DB. All authors have read and agreed to the published version of the manuscript.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Department of Microbiology, Principal Investigator, Regional Virology Lab, All India Institute of Medical Sciences, Bhopal, Madhya Pradesh - 462 020
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]
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