Currently circulating genotypes of Hepatitis A virus in South West, East and North East India, 2017–2018

Abstract

Background

This study was carried out to understand the circulating genotypes of Hepatitis A virus (HAV) in South West, East and North East India during the period 2017–2018 as a part of acute febrile illness surveillance at the Manipal Institute of Virology.

Methods

Archived serum samples of 48 Hepatitis A confirmed cases were subjected to RNA extraction using QIAamp® Viral RNA Mini Kit (QIAGEN, Germany). The samples with molecular confirmation for HAV by reverse transcriptase real-Time PCR (Real Star® HAV RT-PCR Kit 2.0, Altona Diagnostics, GmbH, Hamburg, Germany) were further subjected to nested conventional PCR targeting the 5′ UTR region. The purified PCR products were sequenced using Big Dye Terminator Kit (Applied Biosystems, USA), in a 3500 XL genetic analyzer (Applied Biosystems, USA). The edited sequences by means of MEGA X (MEGA version 10.1) were compared with reference sequences in the NCBI nucleotide database.

Results

From states of Assam, Goa, Gujarat, Karnataka, Kerala, Maharashtra, Odisha, Tamil Nadu and Tripura, 139 Hepatitis A and 33 Hepatitis E cases were reported during the study period. The median age of the acute Hepatitis A cases was 19 years (IQR 12.8–24) and most of the affected individuals were students between 10 and 19 years (52.5%). In the present study, 14 samples from Assam, Goa, Gujarat, Karnataka, Odisha, Kerala, Maharashtra and Tamil Nadu were genotyped as genotype IIIA by nested conventional polymerase chain reaction.

Conclusion

The circulating HAV genotype in South West, North East and East India between 2017 and 2018 was IIIA.

Introduction

Hepatitis A virus (HAV) causes liver infection in about 1.5 million people annually even though most of the cases go undiagnosed and unreported.¹ The impoverished situation with inadequate sanitation leads to frequent epidemics in developing countries.

In low endemic areas, symptomatic infection often occurs as sporadic cases or small outbreaks among high-risk groups. Infection with HAV induces lifelong immunity. In highly endemic areas, almost all children acquire immunity because of childhood exposure. Humans are the only natural hosts and the main mode of transmission is the fecal oral route, which is often related to low education level, poor sanitation and poor personal hygiene in low-resource settings. Hepatitis A virus is a member of Picornaviridae family belonging to the Hepatovirus genus. This non-enveloped virus has a single-stranded RNA genome, which is classified into seven HAV genotypes based on 5′ UTR region comprising four human genotypes (I, II, III and VII) and three simian genotypes (IV, V and VI). Three human genotypes (I, II and III) were further subdivided as A and B. Genotypes IIIA, IA and IB were already reported from northern, southern, western and central regions of India.2, 3, 4

HAV is known to have one serotype only,² which led to the development of an effective vaccine in 1995. In developed countries, Hepatitis A has become the second most common travel-related vaccine preventable disease after enteric fever. The present cross-sectional study aimed to understand the currently circulating Hepatitis A genotypes in the South West, East and North East regions of India.

Materials and methods

Serum samples procured from acute hepatitis cases as a part of acute febrile illness study were screened for Anti HAV IgM (MP Biomedicals, GmbH, Germany, qualitative IgM ELISA) and subjected to molecular confirmation during the period 2017–2018. Cases tested positive for HAV were classified based on clinical features and laboratory parameters as acute viral hepatitis and acute fulminant liver failure. Cases with total bilirubin >2 mg/dl and serum alanine aminotransferase level >200 IU/L in the age group of 1–65 years were included after obtaining patient consent.

The serum samples with molecular confirmation of HAV RNA (Real Star® HAV RT-PCR Kit 2.0, Altona Diagnostics, GmbH, Hamburg, Germany) were chosen based on the date of sample collection, geographical areas and Ct (cycle threshold) value below 30 for sequencing. The extraction of RNA was carried out by QIAamp® Viral RNA Mini Kit (QIAGEN, Hilden, Germany) as per the manufacturer's instructions. Conventional nested PCR was carried out targeting the 5′ non-coding region.⁵ About 6 μl RNA was added to the first-round reaction mixture comprising 12.5 μl buffer mix (Applied Biosystems®, Foster city, USA), 2 μl each of primers (forward primer 3.8 nmol, reverse primer 3.26 nmol, SIGMA-ALDRICH, Missouri, USA), 1 μl of Enzyme mix 1X (Applied Biosystems® Foster city, USA) and 1.5 μl nuclease-free water. The amplification was carried out in ProFlex™ PCR system (Applied Biosystems® Foster city, USA). The reverse transcription was carried out at 45 °C for 30 min and initial denaturation at 95 °C for 5 min. The amplification of cDNA was carried out by 35 cycles of denaturation (95 °C for 30 s), primer annealing (55 °C for 45 s), extension (68 °C for 45 s) and a final extension at 68 °C for 7 min. About 1 μl of the first-round product was added to the reaction mixture containing 12.5 μl of buffer mix (Applied Biosystems® Foster city, USA), 2 μl each of primers (forward primer 4.84 nmol, reverse primer 6.32 nmol, SIGMA-ALDRICH, Missouri, USA), 1 μl of Enzyme mix 1X (Applied Biosystems® Foster city, USA) and 6.5 μl nuclease-free water. The initial reverse transcription was carried at 95 °C for 10 min followed by denaturation at 95 °C for 30 s, annealing at 45 °C for 30 s, extension (68 °C for 45 s) and a final extension at 68 C for 7 min. As shown in Fig. 1, the amplified PCR product was electrophoresed in ethidium bromide-free 1.8% (w/v) agarose gel (Merck, New Jersey, USA) and was purified using GenElute™ Gel purification Kit (SIGMA-ALDRICH, Missouri, USA). The purified PCR product was sequenced using Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems® Foster city, USA) on ABI 3500 XL Genetic Analyzer (Applied Biosystems® Foster city, USA) as per the manufacturer’s instruction. The sequences were analyzed using Sequencher 5.6 and compared with reference sequences in the NCBI database.

Fig. 1.

The gel electrophoresis picture showing conventional nested Hepatitis A PCR products. Eleven Lanes from left side show bands at 290 bp indicating samples positive for Hepatitis A RNA (HAV) when compared with 50 bp ladder in 1.8% w/v agarose gel (Merck, USA). NC is negative control with no RNA template was added. PC is positive control with added Hepatitis RNA template.

The phylogenetic tree was synthesized by means of MEGAX software aligning the samples with the reference nucleotides and using the maximum-likelihood method. The reliability of the tree was verified by the bootstrap method (1000 replicates).

This study was reviewed and approved by the Institutional Ethical Committee, Manipal Academy of Higher Education (IEC No: UEC/32/2013-14, MUEC/Renewal-08/2017).

Results

During the study period, serologically confirmed 139 Hepatitis A and 33 Hepatitis E cases were reported among the study participants from states of Assam, Goa, Gujarat, Karnataka, Kerala, Maharashtra, Odisha, Tripura and Tamil Nadu. A total of 107 and 32 HAV cases were reported in 2017 and 2018, respectively. Of the 48 serum samples, with molecular confirmation for HAV by reverse transcriptase real-time PCR (Real Star® HEV RT-PCR Kit 2.0, Altona Diagnostics, GmbH, Hamburg, Germany), 14 samples were selected based on the date of sample collection, geographical areas and Ct (cycle threshold) value below 30 for sequencing.

The highest number of cases was reported from Kerala (n = 49, 35.3%) followed by Karnataka (n = 28, 20.2%). The rest of the cases were reported from Odisha (n = 19, 13.7%), Goa (n = 14, 10.1%), Tamil Nadu (n = 10, 7.2%), Gujarat (n = 8, 5.7%), Assam (n = 8, 5.7%), Maharashtra (n = 2, 1.4%) and Tripura (n = 1, 0.7%). Among the 139 cases, there were 87 men (62.6%) and 52 women (37.4%). Below the age of 10 years, there were 14 confirmed cases (10.07%).

The majority of the enrolled cases belonged to the age group of 10–29 years (n = 102, 73.4%), as shown in Table 1. The median age of the HAV infected cases was 18 years (IQR 12.8–24). The most frequent symptoms were jaundice, malaise, nausea/vomiting, headache and arthralgia. Of the 139 patients, 73 (52.5%) and 66 cases (47.8%) belonged to low and middle socioeconomic status, respectively, as per the Modified Udai Pareek scale.⁶

Table 1.

Sociodemographic features of the study participants (n = 139).

Sociodemographic characteristics	Number (n)	Percentage (%)
Age groups (years)
1–5	7	5.0
6–12	21	15.2
13–19	46	33.1
20–35	55	39.6
36–45	7	5.0
46–55	1	0.7
56–65	2	1.4
Gender
Male	87	62.6
Female	52	37.4
Occupation
Unemployed	2	1.4
Homemaker	10	7.2
Unskilled	4	2.9
Semiskilled	16	11.5
Skilled	13	9.4
Students	73	52.5
Professionals	21	15.1
Socioeconomic status (SES)–modified Udai Pareek scale Risk factors
Low <40	73	52.5
Middle 40-70	66	47.5
High >70	0	0.0%
Travel history
Yes	32	23.1%
No	107	76.9%
Close contact with a clinical case
Yes	36	25.9%
No	103	74.1%
Water source for drinking purpose
Well water	75	53.9%
Piped water	17	12.3%
Others	47	33.8%

HAV infection was observed to be more prevalent among students (n = 73, 52.5%). About 26 patients (25.0%) gave a history of travel to neighbouring districts. Contact history with a case of jaundice was given by 24 study participants (23%). The main drinking water source was well water (n = 75, 53.9%) followed by piped water (n = 17, 12.3%) and water from rivers, ponds and streams.

All the study participants reported headache, nausea/vomiting and arthralgia, which is depicted in Table 2. Malaise, abdominal discomfort and diarrhoea were reported by 126 (91%), 81 (58%) and 3 (2%) enrolled patients, respectively. Only one case presented with skin rash (macule). Liver function parameters demonstrated total serum bilirubin above 6 mg/dl in 22 cases (15.8%). Serum alanine aminotransferase and aspartate aminotransferase more than 1000 IU/L were detected in 64 (46.1%) and 62 (44.6%) cases, respectively (Table 3).

Table 2.

Clinical features of study participants (n = 139).

Clinical features	Number (n)	Percentage (%)
Fever
Yes	139	100
No	0	0
Malaise
Yes	127	91.4
No	12	8.6
Nausea/vomiting
Yes	139	100
No	0	0
Abdominal discomfort
Yes	82	59.0
No	57	41.0
Arthralgia
Yes	139	100
No	0	0
Myalgia
Yes	139	100
No	0	0
Diarrhoea
Yes	4	2.9
No	135	97.1
Headache
Yes	139	100
No	0	0
Sore throat
Yes	22	15.8
No	117	84.2
Skin rash (macule)
Yes	1	0.7
No	138	99.3

Table 3.

Haematological and liver function parameters of enrolled cases (n = 139).

Laboratory parameters	Number (n)	Percentage (%)
Total bilirubin (mg/dl)
≤2	26	18.7
2.1–3	16	11.5
3.1–6	34	24.5
>6	22	15.8
Not available	41	29.5
Total protein (gm/dl)
≤6	29	20.9
6.1–8	51	36.7
>8	1	0.7
Not available	58	41.7
Serum albumin (g/dl)
≤2.7	1	0.7
2.8–3.5	17	12.2
>3.5	63	45.4
Not available	58	41.7
ALT (alanine aminotransferase IU/L)
≤200	9	6.5
201–500	5	3.6
501–1000	17	12.2
>1000	64	46.1
Not available	44	31.6
AST (aspartate aminotransferase IU/L)
≤200	18	12.9
201–500	9	6.5
501–1000	9	6.5
>1000	62	44.6
Not available	41	29.5
Hb:
Male
<12	14	10.1
12–16	61	43.9
>16	5	3.6
Not available	8	5.8
Female
<12	24	17.2
12–15	26	18.7
>15	1	0.7
Platelet (in lakhs)
<1.5	24	17.3
1.5–4.5	97	69.8
>4.5	4	2.9
Not available	14	10.0

In the present study, 14 samples from Assam, Goa, Gujarat, Karnataka, Odisha, Kerala, Maharashtra and Tamil Nadu were typed as HAV genotype IIIA. All the study strains had 99% similarity with strains (FJ360733, FJ360734, FJ360735) detected from different parts of India, spanning over a period of 50 years since 1955. About 99% similarity with recent strains (JX481908, JX481901 and JX481902) typed from Chandigarh, north India between 2007 and 2011 was observed, which is shown in Fig. 2. Our study strains had 99% sequence similarity to South Korean strains (MH128124) and strains circulating in France (FJ829484). All the 14 study strains were submitted in GenBank (MN171393–MN171406).

Fig. 2.

The figure depicts the phylogenetic tree representing different genotypes of HAV (IA, IB, IIA, IIB, IIIA and IIIB). Labelled sequences with black circles illustrating the study samples, whereas the remaining sequences represent the reference samples. Bootstrap values >60% are shown at the nodes.

Discussion

The present study observed a higher prevalence of Hepatitis A infection among children. About three-fourths of the patients were in the age group of 11–29 years. There was male gender preponderance and two-thirds cases with Hepatitis A infection were men. All the infected cases belonged to middle or low socioeconomic status. The majority of cases were from Kerala, South India, whereas the lowest number of cases was from Tripura. The main route of transmission was contaminated well water, which is very common in developing countries. Other sources of infection were water obtained from tankers, hand pumps, streams, public water taps and municipality taps. However, there are reports of isolation of fecal coliform bacilli and molecular detection HAV in the rural well water of developed countries.⁷ Contrary to annual reporting of few number of cases, a massive surge in symptomatic Hepatitis A cases, mainly among adults was reported from various regions in the United States in 2019. This was mainly attributed to homelessness and vaccination of high-risk individuals was recommended. Recently, 17 countries of Florida declared a national emergency for HAV.⁸

Many of the developed countries in Asia Pacific region such as Brunei, Japan, Republic of Korea and Singapore are showing S-shaped age–seroprevalence curve indicating a shift of low infection rate in last few decades making every one below 50 or 60 years susceptible to HAV infection and subsequent complications.⁹ In India, a higher seroprevalence of anti-HAV among students who practiced open defecation was observed compared with cases using a private toilet within the house.¹⁰ Meanwhile, the developed countries witness periodic outbreaks of HAV infections among individuals who experience homelessness and users of illicit drugs.¹¹ Our all representative study strains from South, West and North East India belonged to the genotype IIIA. This is in accordance with studies from Jaipur,¹² Jabalpur,⁴ Himachal Pradesh,¹³ Maharashtra,¹⁴ Tamil Nadu¹⁵ and Chandigarh.¹⁶ Co-circulation of genotypes IIIA and 1A was reported from Guwahati, North East India and Delhi.^3,17 Only one study from Pune, West India reported co-circulation of IIIA and IB in 2007.¹⁸ Currently, sub-genotype IA is the most prevalent circulating strain in China.¹⁹

Hepatitis A infection is endemic in Eastern Europe and Balkan region countries and studies from these areas have reported Hepatitis A sub-genotypes IA as the dominant strain. Even though IA was the most prevalent genotype in the Americas, IB was observed to be replacing genotype IA with an increasing number of IIIA between 2013 and 2018.²⁰ HAV outbreaks in developing countries are often attributed to ingestion of contaminated food and water as well as poor personal hygiene. Developed countries witness periodic outbreaks of HAV infection among homeless people, homosexuals or illicit drug users. Recent molecular studies targeting VP1/VP2 junction region of HAV resulted in the reclassification of genotype VII as a sub-genotype of genotype II. Molecular analysis of VP1 protein has to be carried out for early recognition of genetic changes of the virus in future.

Strength of the study

Most published articles report investigation of isolated outbreaks confined to rural or semi-urban areas. The present laboratory-based study carried out as a part of acute fever surveillance covers vast geographical areas in India. Molecular epidemiologic data are vital to confirm cases, differentiate outbreaks from sporadic cases, analyze various modes of transmission and identify populations at increased risk of infection. Meticulous epidemiologic surveillance with sustained public health laboratory support for HAV genotyping, facilitate early detection and monitoring of outbreaks in endemic countries like India.

As the present study was a retrospective study, we could not collect significant epidemiological information such as use of boiled water for drinking purposes, sewer leakage or distance between sewage tanks and water sources.

Ethics statement

Procedures performed in the study involving human subjects were carried out in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained for any experimentation with human subjects.

Disclosure of competing interest

The authors have none to declare.