|Year : 2018 | Volume
| Issue : 3 | Page : 352-356
Hepatitis C virus infection in a tertiary care hospital in Mumbai, India: Identification of a mixed and novel genotype
Priya Madan Yabaji1, Aruna Shankarkumar1, Akash Shukla2, Shobna Bhatia3
1 Department of Transfusion Transmitted Diseases, National Institute of Immunohaematology (ICMR), Mumbai, Maharashtra, India
2 Department of Gastroenterology, Lokmanya Tilak Municipal General Hospital and Lokmanya Tilak Municipal Medical College, Mumbai, Maharashtra, India
3 Department of Gastroenterology, KEM Hospital, Mumbai, Maharashtra, India
|Date of Web Publication||14-Nov-2018|
Dr. Aruna Shankarkumar
Department of Transfusion Transmitted Diseases, National Institute of Immunohaematology (ICMR), 13th Floor, New Multistoreyed Bldg, KEM Hospital Campus, Parel, Mumbai - 400 012, Maharashtra
Source of Support: None, Conflict of Interest: None
Purpose: Hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). HCV being a ribonucleic acid virus has considerable sequence variability. Assessment of viral load and genotype is necessary for designing treatment strategies and monitoring for viral resistance among HCV-infected cases. HCC is the most common form of liver cancer, often occurring in people with chronic hepatitis B or C. We undertook this study to observe genotype distribution of the virus in HCV patients from Mumbai. Materials and Methods: Between January 2017 and December 2017, the study was conducted on 120 chronic hepatitis outpatients from a tertiary care hospital, Mumbai, after obtaining ethics approval. All these diagnosed cases of HCV were subjected to molecular diagnosis in a research institute, Mumbai, by real-time polymerase chain reaction-based techniques. Results: Males were more preponderant than females with HCV infection, and the highest number of HCV-infected cases was observed in the age group of 41–50 years. Genotype 3 (n = 70; 58.3%) accounted for the highest number of cases followed by genotypes 1b (n = 29; 24.2%) and then 1a (n = 14; 11.7%). Mixed genotypes 1b + 3 and individual genotype 4 were found in two cases each (1.7%). A total of three samples (2.5%) were found with untypeable genotype. Conclusion: The major HCV genotype observed was 3 which is difficult to treat with direct-acting antivirals, owing to the more rapid progression of liver disease, increased rates of steatosis (non-alcoholic fatty liver disease), a higher risk for cancer (HCC). We believe this study is the first one to address the prevalence of mixed genotypes and untypeable genotype from India.
Keywords: Genotypes, hepatitis C virus, India, mixed genotypes, viral load
|How to cite this article:|
Yabaji PM, Shankarkumar A, Shukla A, Bhatia S. Hepatitis C virus infection in a tertiary care hospital in Mumbai, India: Identification of a mixed and novel genotype. Indian J Med Microbiol 2018;36:352-6
|How to cite this URL:|
Yabaji PM, Shankarkumar A, Shukla A, Bhatia S. Hepatitis C virus infection in a tertiary care hospital in Mumbai, India: Identification of a mixed and novel genotype. Indian J Med Microbiol [serial online] 2018 [cited 2019 Oct 22];36:352-6. Available from: http://www.ijmm.org/text.asp?2018/36/3/352/245385
| ~ Introduction|| |
Hepatitis C virus (HCV) infects 3% of the world population, and its prevalence in our country is about 0.8%–1.5%. Countries with high rates of HCV infection include Egypt (>10%) and most other African countries (>3%). HCV spreads through contact with infected blood and shared needles, particularly among drug users. Those who are at risk include healthcare workers exposed to infected blood through accidental needle prick, long-term dialysis cases, child born to HCV-infected mother and individuals with multiple sex partners.
Hepatitis C can present as acute or chronic hepatitis. Most of the cases of acute hepatitis C are asymptomatic and hence unaware of the underlying infection. Approximately, 20%–30% of infected cases automatically resolve the virus and remaining 75%–80% of cases lead to chronic hepatitis. About 10%–25% of people with chronic hepatitis C will develop cirrhosis, a process that usually takes 20–30 years. People with cirrhosis are at increased risk of developing hepatocellular carcinoma (HCC). Unlike hepatitis B virus (HBV), there is no vaccine available against HCV. HCC is the most common form of liver cancer, often occurring in people with chronic hepatitis B or C. The only effective way to reduce HCV infection is to provide HCV-negative blood to the recipient.
HCV is an enveloped, single positive-stranded ribonucleic acid (RNA) virus from genus Hepacivirus, family Flaviviridae with a diameter of 50 nm. The genome consists of a single open reading frame which is 9600 nucleotide bases long. There is considerable sequence diversity because of RNA virus. HCV strains are classified into six genotypes on the basis of phylogenetic and sequence analyses of the whole viral genome. HCV genotypes differ at 30%–35% of nucleotides. Within each genotype, HCV is further classified into subtypes that differ at <15% of nucleotides. The high sequence variability of the HCV genome has hindered progress in vaccine development. Genotyping of HCV is crucial as it is a part of the pre-treatment process for patient management. It is also helpful in understanding the epidemiology, biological features of the virus as well as in inspecting the outbreak of infection.
The global epidemiology of HCV is well established. Although millions in India are affected by hepatitis C, the country has no accurate data regarding its prevalence as well as policies to support patients. India lacks HCV surveillance system as a consequence and there is complete lack of knowledge about the actual number of people living with HCV-related liver diseases. The present study was performed to identify the distribution pattern of HCV genotypes and pre-treatment HCV RNA viral load in HCV-infected patients in a tertiary care hospital, Mumbai.
| ~ Materials and Methods|| |
The present study was approved by ethics committees of King Edward Memorial (KEM) Hospital, Mumbai, and National Institute of Immunohaematology (NIIH), Mumbai. The study was carried out in NIIH. This study included 120 patients with HCV infection who were referred by KEM hospital, Mumbai from January 2017 to December 2017. All were naive for antiviral therapy at the enrolment. Patients with HBV, HIV infections and those on antiviral treatment on HCV were excluded from the study. All necessary clinical details were obtained through proper questionnaires from each infected case and kept confidential. Written informed consent document was obtained from each patient.
Hepatitis C virus RNA quantitation
For viral load, blood was collected in ethylenediaminetetraacetic acid-containing tubes, and plasma was separated within 3 hours. Aliquots were stored at −80°C, until further testing within 15 days. Viral RNA was extracted using operating manual of Mylab Lifesolutions Pathodetect™ (Pune, India). HCV quantitative polymerase chain reaction (PCR) was done by Mylab Lifesolutions Pathodetect™ (Pune, India), an in vitro diagnostic test based on real-time PCR technology utilising reverse transcriptase (RT) reaction to convert RNA into cDNA, PCR for the amplification of specific target sequences and target specific probes for the detection. The test can quantitate HCV RNA in the linear range of 40 IU/ml to 7 × 107 IU/ml.
Genotyping of hepatitis C virus
In HCV RNA-positive samples, genotypes were determined by performing RT PCR using Mylab Lifesolutions Pathodetect™ HCV genotyping kit (Pune, India) as per the manufacturer's protocol. This method allows for the determination of genotypes 1a, 1b, 2, 3, 4, 5 and 6.
Statistical analysis of the data was performed using SPSS for Windows, version 16 (SPSS Inc., Chicago, Illinois, USA). The data are presented as the mean and standard deviation (SD) and percentage. Differences in the genotypes and viral load were analysed using t-test. In all tests, the values of P < 0.05 were considered as statistically significant.
| ~ Results|| |
Of 120 HCV carriers, 52 (43.3%) were females and 68 (56.7%) were males. The overall male-to-female ratio was 1.31. The median age of male HCV carriers was 42 years, and the median age of female HCV carriers was 48.5 years.
HCV-infected patients were categorised into four age groups. Analysis of associations between patients' age group and HCV positivity revealed that the maximum number of cases belonged to the age group of 41–50 years (n = 37; 30.8%) and minimum in the age group >60 years (n = 10, 8.3%) as shown in [Figure 1].
|Figure 1: Hepatitis C virus-infected cases (%) across different age groups|
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Analysis of mode of transmission revealed majority of cases, i.e., 62 individuals (51.67%) had a history of blood transfusion, 26 (21.7%) cases were on dialysis and 11 (9.17%) had a history of surgery. Four individuals (3.33%) were haemophiliacs, and one case (0.83) was of injection drug user. Eight (6.67%) cases were incidentally detected HCV seropositive during blood donation or medical evaluation while applying for a job abroad having no particular history of any blood transfusion, surgery or tattoo. Eight (6.67%) were admitted cases with no previous medical records.
Liver enzyme levels
Analysis of liver function tests revealed higher enzyme levels. The mean ± SD levels of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase in HCV cases were 70.48 ± 51.64, 53.57 ± 48.23 and 191.75 ± 127.60, respectively.
Detected hepatitis C virus genotypes
[Figure 2] shows the distribution pattern of HCV genotypes in the studied population. Genotype analysis revealed the presence of genotypes 1, 3 and 4 with subtypes 1a, 1b and mixed genotypes 1b + 3. The most predominant genotype was 3 (n = 70; 58.3%) across all age groups. The second most dominant viral genotype was 1b in 29 individuals (24.2%), followed by 1a and 4 in 14 (11.7%) and 2 (1.7%) cases, respectively. A total of three samples (2.5%) were found with untypeable genotype, whereas the infection of mixed genotype was observed in two individuals (1.7%) [Figure 2]. The overall number of male HCV carriers was greater than females.
Correlation between viral load and hepatitis C virus genotypes
The mean viral load of the patients infected with different genotypes is given in [Table 1]. In this study, genotypes 1a, 4 and mixed genotypes (1b + 3) were associated with significantly higher viral load when compared with untypeable genotype.
|Table 1: Comparison of mean age, gender ratio and mean log value of viral load levels across hepatitis C virus genotypes|
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Distribution of hepatitis C virus genotypes according to gender
[Figure 3] shows the frequency of HCV genotypes distribution according to gender. Among all genotypes, genotype 3 was predominant in both males and females. The frequency of HCV genotypes 1a and 1b was found to be higher in males than females. In addition, the frequency of HCV genotype 4 and mixed genotypes (1b + 3) was found to be equivalent in male and female patients.
|Figure 3: Distribution of hepatitis C virus genotypes according to gender|
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| ~ Discussion|| |
The distribution of HCV genotypes and subgenotypes varies substantially in different parts of the world. Knowledge of genotypes is crucial because it has a role in determining therapeutic prognosis and clinical status of HCV-infected cases. Genotypes 1 and 3 account for the majority of HCV infections worldwide. In Africa and the Middle East, the most prevalent genotype is 4. Genotypes 5 and 6 are most common in South Africa and Asia. Genotype 1a is most prevalent in the United States and Northern Europe, while 1b is the most common genotype worldwide. In Europe and Japan, genotypes 2a and 2b are most prevalent, whereas 2c subtype is most frequent in northern Italy. Genotype 3a is the most common in Pakistan in comparison to 3b and 1a.
The diversity of genotypes also varies considerably across countries. The highest diversity is observed in China, Southeast Asia, Western Europe and Australia while in countries such as Egypt and Mongolia, almost all HCV infections are due to single genotype. The current study revealed that genotype 3 is the most frequent and common, followed by 1b in HCV-infected patients. Several earlier studies have also reported that genotype 3 is the most prevalent in our country.,, Studies from northern, eastern and western parts of India have uniformly shown predominance of genotype 3; however, in southern India, both genotypes 1 and 3 HCV are prevalent.,
Studies have shown that genotype 3 is associated with higher rates of liver cancer, fibrosis and mortality. Genotype 3 may also have a higher risk of severe steatosis or fat accumulation leading to swelling and scarring of the liver.
In this study, we found two HCV cases infected with genotype 4. Genotype 4 – HCV is also reported in some cases from southern India. In another study of 75 isolates from the northeastern part of India with predominant tribal population, genotype 4 is reported to be the most common genotype (31%) followed by genotype 6 accounting for 13.6% of the cases.
Genotypes 5 and 6 were not detected in our study. Few studies from different parts of the country have reported the presence of genotype 5a HCV cases., Genotype 6 – HCV infection is reported from eastern and northeastern parts of India.
The current study has also detected rare coinfection 1b +3 which is less frequent and less reported. Mixed genotypes in a single patient may affect the antiviral therapy response and disease succession. No other coinfection combinations were detected in this study. In our study, an untypeable HCV genotype was also detected. We believe coinfection and untypeable HCV genotype is reported first time from India. In this study, genotypes 1a, 4 and mixed genotypes (1b + 3) were associated with significantly higher viral load when compared with untypeable genotype.
When comparing the different age groups, we found that the highest prevalence of HCV infection occurred in the age group 41–50 years. In HCV studies, age has been argued to be an important factor being more predominant in older people.
Determination of HCV genotype in different regions is essential, being an essential clinical parameter for molecular epidemiology, therapeutic implications and development of vaccine. This communication describes our small effort to provide up-to-date, general information regarding HCV genotype distribution and viral load in treatment naïve HCV-infected cases in a tertiary care hospital, Mumbai.
Genotype 3 is associated with higher rates of liver cancer, fibrosis and mortality. Because of this, it is essential to determine which HCV variation the patient carries as soon as possible. This allows to kick-start the necessary treatment needed, potentially limiting the damage done to your liver and other serious side effects. The longer the diagnosis and treatment is put off, the more difficult, it is to get treated having poorer overall outlook.
HCC is the most common form of liver cancer, often occurring in people with chronic hepatitis B or C. Stringent blood banking laws need to be implemented, sterilisation and reuse of needles discouraged. All these are not possible without increased public awareness of the magnitude.
| ~ Conclusion|| |
In this study, HCV 3 was the most prevalent genotype seen in chronic HC patients admitted to our hospital, followed by 1b. Genotype 3 is responsible for the rapid progression of liver disease and a higher risk for hepatocellular cancer. HCV infection still remains a burden on the health-care system worldwide as the disease may be underreported in most of the HCV-infected cases being unaware of their clinical status. Determination of HCV variation is important to kick-start the treatment thereby potentially limiting the damage to the liver. The longer the diagnosis of HCV and its treatment is delayed; it is more difficult to treat it. The limitation of the study includes consideration of cases admitted only in a tertiary care hospital, Mumbai.
To the best of our knowledge, this study is the first one to address the prevalence of mixed genotypes and untypeable genotype from India. Further studies with more sample size need to be conducted to investigate if there is the possibility of shift in genotypes. Expanding the current study will allow us to follow changes HCV genotype distribution, to identify recombinants or even new subtypes or genotypes. The World Health Organisation aims to eradicate HCV by 2030 (World Hepatitis Summit 2017). We feel our results contribute to the comprehensive body of data in the fields of HCV epidemiological research. The high variation of the HCV genome, being an RNA virus, in particular, may produce unexpected problems, which will make a new strategy to overcome HCV quasispecies or recombinant strains.
The authors would like to thank intramural funds of the Indian Council of Medical Research (ICMR). We would like to thank all laboratory staff of Transfusion Transmitted Diseases Department, NIIH for their direct and indirect help.
Financial support and sponsorship
This study was financially supported by the intramural funds of Indian Council of Medical Research (ICMR).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]