|Year : 2020 | Volume
| Issue : 1 | Page : 46-51
Hepatitis E outbreak in Jaipur due to Genotype IA
Bharti Malhotra1, Farah Deba2, Pratibha Sharma1, Khushbu Trivedi1, Jitendra Tiwari3, Aradhana Chouhan1, Ruchi Singh4, Deepa Meena4, HN Verma5
1 Department of Microbiology and Immunology, SMS Medical College, Jaipur, Rajasthan, India
2 Department of Microbiology and Immunology, SMS Medical College; Department of Microbiology, Jaipur National University, Jaipur, Rajasthan, India
3 Department of Microbiology and Immunology, SMS Medical College, Jaipur; Government Medical College, Bharatpur, Rajasthan, India
4 State Microbiologist IDSP, Directorate of Medical and Health, Jaipur, Rajasthan, India
5 Department of Microbiology, Jaipur National University, Jaipur, Rajasthan, India
|Date of Submission||12-Nov-2019|
|Date of Decision||25-Nov-2019|
|Date of Acceptance||17-Jun-2020|
|Date of Web Publication||25-Jul-2020|
Dr. Bharti Malhotra
Department of Microbiology, SMS Medical College, Jawahar Lal Nehru Marg, Jaipur - 302 004, Rajasthan
Source of Support: None, Conflict of Interest: None
Purpose: Suddenly, many cases of fever with jaundice were reported from Sodala area at Jaipur. This outbreak of acute hepatitis at Jaipur Rajasthan was investigated for aetiology and subsequent phylogenetic analysis. Methods: Blood samples were collected from 106 symptomatic patients of acute hepatitis and 39 pregnant females (with or without symptoms of hepatitis) during an outbreak at Jaipur. The samples were tested for hepatitis A virus (HAV) and hepatitis E virus (HEV) by serological and molecular methods (polymerase chain reaction [PCR]). Sequencing of nested PCR product was done for phylogenetic analysis. Hepatitis B surface antigen (HBs antigen), anti-hepatitis C virus (HCV), anti-Leptospira and anti-scrub typhus IgM enzyme-linked immunosorbent assay (ELISA) was done for patients negative for HEV and HAV. Results: Among 106 symptomatic patients, HEV IgM was positive in 84/106 (79.2%) patients and HEV RNA in 72/106 (67.9%) patients. Among pregnant women, 6/39 (15.4%) were HEV IgM positive and 5/39 (12.8%) for HEV RNA. One (2.5%) pregnant woman died due to hepatitis. All the isolates belonged to genotype 1A of HEV. All HAV, HEV-negative samples were negative for HBs antigen, HCV antibody, Leptospira and scrub typhus IgM ELISA. Conclusion: The outbreak was due to HEV genotype 1A. The municipal water supply was contaminated and sanitary conditions and waste disposal were poor in the area. Boiling of drinking water, fixing the water supply pipes and frequent hand washing helped in controlling the outbreak.
Keywords: HEV, outbreak, water borne, viral hepatitis
|How to cite this article:|
Malhotra B, Deba F, Sharma P, Trivedi K, Tiwari J, Chouhan A, Singh R, Meena D, Verma H N. Hepatitis E outbreak in Jaipur due to Genotype IA. Indian J Med Microbiol 2020;38:46-51
|How to cite this URL:|
Malhotra B, Deba F, Sharma P, Trivedi K, Tiwari J, Chouhan A, Singh R, Meena D, Verma H N. Hepatitis E outbreak in Jaipur due to Genotype IA. Indian J Med Microbiol [serial online] 2020 [cited 2020 Aug 11];38:46-51. Available from: http://www.ijmm.org/text.asp?2020/38/1/46/290677
| ~ Introduction|| |
Hepatitis E virus (HEV) is the leading cause of acute viral hepatitis in the world and occurs primarily in Africa, Central Asia and Mexico. Acute viral hepatitis due to HEV is an enterically transmitted disease that spreads through faecal contamination of drinking water. Water-borne hepatitis E epidemics have been reported in India since the early 1950s. It is reported that 70% of the acute sporadic viral hepatitis cases in India are due to HEV infections. The highest rates of infection occur in regions with poor sanitation and among population of low socioeconomic status. In developed countries, hepatitis E occurs sporadically, and food-borne infections are more frequent than water-borne infections. The virus commonly infects young adults aged 15–40 years, and fewer cases are seen in children. Fulminant hepatitis caused by HEV may result in death in pregnant women with a case fatality rate (CFR) of 15%–25% in the third trimester. Miscarriages or premature delivery can also occur as a result of HEV infection in pregnant women. In India, several outbreaks of hepatitis E due to contamination of drinking water have been reported.,,,, The present study was undertaken when suddenly many cases of fever with jaundice were reported from Sodala area, Jaipur, Rajasthan. A team from Virus Research and Diagnostic Lab (VRDL) and Integrated Diseases Surveillance Program (IDSP) went to the site for investigation. On investigation, the outbreak was found to be due to HEV. We report here the outbreak investigation and the phylogenetic analysis of HEV.
The present study was conducted by a team from IDSP and Indian Council of Medical Research (ICMR) Grade 1 VRDL, Department of Microbiology, SMS Medical College, Jaipur. The study was approved by the ethics committee of the institute (MC/EC/2011/227).
Inclusion criteria: patients having signs and symptoms of acute hepatitis such as fever, jaundice, abdominal pain, anorexia and vomiting residing in Sodala and pregnant women residing in Sodala with or without symptoms.
A house-to-house survey was conducted in four colonies of Sodala area at Jaipur, Rajasthan, by the IDSP and VRDL team; patients' history and other details were collected and filled up in a VRDL pro forma for outbreak. A total of 106 samples were collected from patients of acute hepatitis during 3 December to 16 December 2014 at the peak of the outbreak. Samples from 39 pregnant females from the area, with or without symptoms, were taken as pregnant women are vulnerable for HEV infection with high CFRs. Among the 39 women, five were in the first trimester, ten were in the second trimester and 24 were in the third trimester. The following details were collected: number of household, family members in the house; the source of drinking water supply; the number of individuals who showed signs and symptoms of hepatitis and their age, sex and time of onset of the illness. Blood samples were transported maintaining cold chain to the Laboratory at SMS Medical College and processed within 2 h of collection. Serological and molecular investigations were done. The samples were screened for IgM antibody to HEV and hepatitis A virus (HAV) by using commercial IgM enzyme-linked immunosorbent assay (ELISA) kits (Dia. Pro Diagnostic Sesto San Giovanni, Milano, Italy) as per manufacturer's instructions. All patients negative for HAV and HEV were tested further for HBs antigen (J Mitra and Co, New Delhi India), anti-HCV (J Mitra India), anti-Leptospira (PanBio Queensland, Australia) and scrub typhus IgM ELISA (Inbios International Inc. Seattle Washington, USA ) as in recent past, some outbreaks of scrub typhus were reported from Rajasthan with similar clinical picture. Reports of liver function tests, alanine transaminase (ALT) and aspartate transaminase (AST) levels and total bilirubin were noted down.
For molecular detection of hepatitis, nucleic acid extraction was done by EasyMAG automated nucleic acid extraction system (bioMérieux Inc Marcy-l'Étoile, France). 50-μL RNA was eluted from 200-μL sample. One-step real-time polymerase chain reaction (PCR) was performed for the detection of both HAV and HEV viral RNA using broad-base primers described earlier. Briefly Master mix was prepared using AgPath one-step reverse transcription (RT)-PCR kit in a total reaction volume of 20 μL containing 12.5 μL 2 × RT PCR buffer, 1-μL Enzyme Mix and 1-μL Primer Probe Mix 1 (PPM) (0.4-μL 10-μM HEV forward primer, 0.4-μL 10-μM HEV reverse primer and 0.2-μL F6-carboxyfluorescein (6-FAM) -labelled probe) and PPM2 (0.4-μL HAV forward primer, 0.4-μL 10-μM HAV reverse primer, 0.2-μL VIC labelled probe and 4.5-μL nuclease-free water). 5 μL of nucleic acid was added to 20-μL amplification mixture, and real-time PCR (Applied Biosystems 7500Dx, USA) was carried out using the following cycling conditions: initial cDNA synthesis for 50°C at 30 min, initial denaturation at 95°C for 10 min followed by 45 cycles of amplification including at 95°C at 15 s and annealing for 55°C at 30 s.
Fifty real-time PCR-positive samples were subjected to sequencing; briefly 10-μL RNA was used for cDNA synthesis by high-capacity cDNA synthesis kit (Applied Biosystems, USA), and the rest was stored at −80°C. A 5 μL of cDNA was added to 20-μL amplification mixture (containing 2.5-μL KAPA Taq buffer A, 2-μL deoxyribonucleotide triphosphates and 0.25-μL Taq polymerase. 1.5 μL of 10 pmoL each of primers P1 and P2 were added and the final volume was made up to 25 μL using nuclease-free water). Nested PCR was done with primers targeting 343 bp non-structural ORF-1 region.
First-round amplification was performed with sense primer – HEV EF: 5'-CCG GAT CCA CAC ACA TCT GAG CTA CAT TCG TGA GCT-3' and anti-sense primer – HEV ER: 5'-CCG AAT TCA AAG GCA TCC ATG GTG TTT GAG AAT GAC-3', at 95°C for 10 min (initial denaturation) followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 1 min and extension at 74°C for 1 min with final extension at 74°C for 5 min. The second round of PCR was performed using PCR product of first PCR instead of cDNA in the Master mix as above and using the 2nd set of primers: sense primer HEV IF: 5'-GGA ATT CGA CTC CAC CCA GAA TTA CTT-3' and anti-sense primer HEV IR: 5'-GGA ATT CAC AGC CGG CGA TCA GGA CAG-3', following the same conditions as used for the first-round amplification in a Veriti thermal cycler (Applied Biosystems, USA). The amplified product was detected in 2% agarose gel. Samples positive from earlier runs of PCR were used as positive control, and RNA quality was checked by NanoQuant (Tecan Group Ltd. Männedorf Switzerland). For quantification, 2-μl RNA was added to the plate, and absorbance was measured at 260 nm. Human housekeeping gene RNaseP was used as extraction control. To avoid cross-contamination, the two steps of nested PCR were carried out in different rooms, different steps were processed in different areas of lab as per the concept of five rooms and filter tips were used in all steps. RNase Zap (Thermofisher Scientific Waltham, Massachusetts, United States) was used to avoid degradation of RNA.
Direct sequencing of PCR product was performed using Big-dye terminator v 3.1 cycle sequencing reaction kit on an ABI 3500 DX Genetic analyser (Applied Biosystems, USA). Amplified sequences of HEV RdRp region of ORF1 were subjected to sequence analysis using Basic Local Alignment Search Tool program of National Centre for Biotechnology Information for sequence similarity search. Genotype was determined by constructing phylogenetic tree, by neighbour-joining method using MEGA 5.1.
Water samples were collected from municipal supplies (one from each colony) and from tube wells (one from each colony) from the four colonies of Sodala. These were tested for faecal contamination to detect coliform and calculate the most probable number (MPN) using 100-ml water samples. The residents were instructed to use boiling water for consumption and chlorination was ensured and to wash hands with soap and water each time after defecation and before meals.
| ~ Results|| |
Among 145 samples collected, 106 were from symptomatic patients of acute hepatitis and 39 from pregnant females. Among the symptomatic patients, HEV IgM was positive in 84/106 (79.2%) patients, out of which, 48.8% (41/84) were male and 49.0% (43/84) were female. HEV RNA was detected in 72/106 (67.9%) patients by real-time PCR. A total of 70/106 (66.0%) samples were positive for HEV both by ELISA and real-time PCR, 14/106 (13.2%) were positive by ELISA but negative by real-time PCR and 2/106 (1.9%) samples were positive by real-time PCR but negative by ELISA [Table 1]. HAV IgM and HAV RNA were detected in 1/106 (0.9%) patient who was < 15 years of age and tested negative for HEV both for IgM and RNA.
|Table 1: Symptomatic hepatitis E virus cases detected by enzyme-linked immunosorbent assay and real-time polymerase chain reaction|
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HEV positivity was seen predominantly in adults, 83.3% (70/84), than in children (age < 15 years), 16.6% (14/84). The mean age of the patients was 28 years (range: 2–65 years). Details of positivity in different colonies of Sodala are given in [Figure 1] (in map taken from Google).
|Figure 1: Map of Sodala showing HEV-positive cases in different colonies|
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Among the 39 pregnant females, 6/39 (15.4%) were positive by ELISA and 5/39 (12.8%) by real-time PCR and 4/39 (10.3%) were positive by both methods. One patient in the third trimester of pregnancy died due to hepatitis, with a CFR of 2.5% (1/39) in pregnant women.
Patients negative for both HEV and HAV were also negative for HBs antigen, anti-HCV, anti-Leptospira and anti-scrub typhus IgM ELISA. On sequencing and phylogenetic analysis, all the isolates were found to be genotype IA [Figure 2]. The sequences were submitted to Genbank, and MF594459-MF594506, MF594516 and MF594517 accession numbers were assigned. AST (364.61 ± 45.11 U/L), ALT (485.90 ± 54.88 U/L) and bilirubin levels (6.5 ± 2.1 mg/dL) in positive patients were found to be increased.
Symptoms of acute hepatitis were observed in individuals who used drinking water from municipal piped water supply, and majority of them also complained of turbid water supply and leakage in water supply pipelines. Municipal water supply lines were crossing the open drain, leading to sewage contamination of drinking water. Most areas were overpopulated with lack of sanitation and proper sewage disposal (supplementary data). MPN count for coliforms was > 10/100 ml in all the water samples from municipal pipe lines, whereas that from tube well was satisfactory.
As per records, jaundice was reported in sporadic cases since September 2014 and suddenly the numbers increased in end November–early December 2014; the last jaundice case was seen on 7 February 2015 and the outbreak was declared over [Figure 3]. In total, 289 cases of jaundice were reported affecting 1190 households in four colonies with a population of 9720 persons. HEV attack rate was found to be 2.9% (289/9720), and the overall CFR was 0.3% (1/289).
| ~ Discussion|| |
Faecal contamination of drinking water has been known to be the major cause in most of the outbreaks of hepatitis E reported from India.,,, In the present study, we also observed water-borne outbreak of HEV caused due to sewage contamination of drinking water in Sodala area of Jaipur, India. Acute hepatitis cases were observed in those drinking water from municipal supply that were crossing the open drain (Amanishah nala), leading to sewage contamination of drinking water. There was lack of sanitation and proper sewage disposal, and this might have contributed to the outbreak. The high MPN for coliform in water samples confirmed the sewage contamination.
In the present study, anti-HEV IgM was detected in 79.2% of samples by ELISA and HEV RNA in 67.9% of samples by real-time PCR. Similar positivity was reported from Odisha, eastern India (73.2%) by IgM ELISA and 54.54% positivity by RT PCR and 80.7% positivity by HEV IgM ELISA at Shimla in outbreaks due to HEV. Whereas another study from Odisha reported much lower positivity (33.1%) in 139 cases from 17 outbreaks which occurred from January 2012 to September 2013. In our study, we observed only 289 jaundice cases with a case attack rate of 2.9%, while a study from Sambhalpur at Odisha reported 2954 jaundice cases with 1.6% attack rate and a study from Nellore reported 23,915 jaundice cases with 5.6% attack rate. Highest numbers of jaundice cases, 79,091, were reported from Kanpur with 3.7% attack rate. The CFR in our study was 0.3%, which is in lower range of varying CFR of 0.22% to 12% reported from India in different studies.,,,,,,,
In our study, 70/106 (66.0%) samples were found to be positive for HEV both by ELISA and real-time PCR. In a previous study, 62.6% cases were found to be positive both by ELISA and real-time PCR. Anti-HEV IgM may remain detectable up to 6 months after the onset of symptoms, while HEV viraemia is reported to be transient, which occurs in the first 2 weeks of infection and declines after 1 week of onset of jaundice, hence PCR confirms acute ongoing infection. In the present study, higher infection rate was found in adults (83.3%) than in children below 15 years of age (16.6%). This might be due to the fact that children mainly have subclinical infection and acquire immunity to HEV in early life because the virus is maintained in community as sporadic infection.,,
In the present study, we have used multiplex PCR targeting both HAV and HEV because recent studies have shown the HAV occurrence in adolescents and adults. Though HAV and HEV have a common faeco-oral route of transmission, HAV infection was found in only one patient whose age was <15 years. This might be due to the fact that HAV infection is predominant in children in developing countries like India.
In India, higher incidence of HEV infection has been reported in pregnant women.,, In our study, 15.3% of the pregnant women were found to be positive for anti-HEV IgM antibodies and death occurred in one patient in the third trimester of pregnancy, with a CFR of 2.5% in pregnant women. Our findings are in consistence with those of earlier studies that report that HEV infection is found to be more severe in pregnant women, often leading to fulminant hepatic failure and death in a significant proportion of patients.,, However, reports from Egypt, Europe and the USA have shown that the course and severity of viral hepatitis during pregnancy is not different from that in non-pregnant women. A study from South India also showed a lower incidence of HEV infection among pregnant women. The authors proposed that South Indian strains of HEV are stable and less virulent than North Indian strains, in which a subgenotypic shift contributed to increased virulence.
Sequencing of the nested PCR products revealed genotype 1A of HEV to be responsible for the outbreak in our study. Genotype 1 of HEV has been the predominant cause of sporadic hepatitis and outbreaks of hepatitis E in hyperendemic regions. Other studies have also reported the same genotype responsible for causing HEV outbreaks in India.,
There were many limitations in our study: we did not check for the presence of HEV RNA in water and sewage samples, nor in faeces of patients. All the cases of jaundice were not tested for aetiology because the IDSP has a policy of testing only a few samples to indicate the cause of outbreak. However, we tested negative samples for Leptospira and scrub typhus as we had an outbreak of scrub typhus presenting with similar clinical findings. The present investigations helped identify the problem areas and steps were taken to improve the sanitation and drinking water supplies, which led to the control of the outbreak. Moreover, major changes have been made in the landscape of Sodala area which has helped bring down the jaundice cases.
| ~ Conclusion|| |
HEV genotype 1A was responsible for the outbreak which was due to the contamination of water supplies. The outbreak was controlled by educating the public and ensuring proper water supplies by boiling and enhanced chlorination and improving sanitary habits and drainage system.
Financial support and sponsorship
This work was funded by the ICMR under project Establishment of Grade 1 VRDL.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]