ABSTRACT

Patients undergoing hemodialysis are at high risk of Hepatitis C virus (HCV) infection due to

frequent exposure to contaminated medical equipments.

This study was conducted to determine HCV viremia, genotype distribution and associated clinical
parameters with active HCV infection among hemodialysis patients in West Bengal.

A total of 310 HCV sero-reactive chronic kidney disease (CKD) individuals were enrolled in this
three-year study. Patients’ samples were analyzed for viremia, clinical parameters, sanger
sequencing and phylogenetic analysis.

Out of 310 HCV sero-reactive individuals, 157 (50.64%) were HCV RNA positive. The most
prevalent circulated HCV subtype was 1c (67.52%) followed by 1a, 4a, 3a, 1b and 3b in this study
population. CKD patients were infected with unique HCV genotypes 1c (67.52%) and 4a (7.64%)
which was unfamiliar in this region. Urea, creatinine, calcium and uric acid are significantly higher
(p-value:0.02, 0.03, 0.04 and 0.05) in HCV RNA positive CKD individuals than HCV RNA
negative CKD patients.

Calcium and uric acid level might be useful markers for management of CKD patients. Unusual
HCV subtypes may evolve in near future in India. Overall, this study indicates the alarming
situation of dialysis centers and Urgent need of infection control plan in Eastern India.

1
INTRODUCTION:

Hepatitis C virus is an enveloped, positive sense single-strand RNA virus with genome

size~9.6kb, belonging to the Flaviviridae family and Hepacivirus genus, responsible for liver

cirrhosis and hepatocellular carcinoma1. HCV is mainly transmitted via contaminated blood, body

fluids, sharing of intravenous drugs and contaminated medical equipment putting multi-

transfused β-thalassemia patients, patients with hemophilia, chronic kidney disease (CKD), HIV

infected patients and people who inject drugs (PWIDs) are at higher risk of acquiring this virus 2.

An estimated 177.5 million adults are infected with HCV3. In India, approximately 12-18 million

people are infected with HCV4. Due to high error rate of HCV RNA-dependent RNA polymerase

(RdRp), it has high genetic diversity with 8 genotypes and more than 86 subtypes 5. Recently, it

has been reported that non-liver related HCV infection also increases mortality and morbidity in

patients with extrahepatic diseases such as CKD6. Chronic kidney disease is defined as damage

of kidney which results from the presence of abnormal level of albumin in the urine (i.e

albuminuria) or decreased kidney function (i.e, glomerular filtration rate; GFR; <60 ml/min per

1.73 m2) for 3 months or more regardless of specific clinical manifestations7. The prevalence of

CKD ranges from 11-13% in high-income countries and 4.7- 41.9% in low-income countries8.

Diabetes, hypertension and oxidative stress have been attributed as some of the key factors

responsible for CKD9. At present, the prevalence of CKD in India is approximately 16.8%10.

HCV plays a key role in mortality and morbidity in CKD patients and the risk of infection in stage-

V hemodialysis patients is very high11. Most of the reports suggests that HCV genotype 1

(subtypes a and b) is the most prevalent genotype among HCV infected CKD patients

worldwide1213141516. There are few studies on the prevalence of HCV genotypes among CKD

patients in the other parts of India but this information is very scarce in the eastern part of India.

2
So, the present study was undertaken to identify HCV genomic diversity among CKD patients in

the eastern region of India over a period of three years and the impact of HCV infection on clinical

parameters like urea, creatinine, and calcium and uric acid etc. levels among CKD patients.

3
METHODS:

Ethical statement:

This study was conducted following the ethical guidelines of Helsinki declaration 1975, amended

in 2013, and was approved by the institutional ethical committee of the Indian Council of Medical

Research-National Institute of Cholera and Enteric Diseases, Kolkata. Written informed consent

was obtained from every individual patient before enrolled them in this study.

Study design:

A total of 310 CKD patients who had undergone at least 6 months of hemodialysis from August

2016 to July 2019, referred from various dialysis centers of West Bengal, India, (Figure 1a) were

included in this study. Whole blood was collected from patients in sterile clot vials. Patient’s

demographic and clinical data were collected from the dialysis unit at the time of enrollment. The

serum was separated and stored at - 80ºC for further study. Individuals above 14 years of age and

those who have undergone hemodialysis for more than 6 months were only included in this study.

HCV RNA positive patients were classified into six age groups, Group- I to VI. These were

categorized into Gr-I (14-24 years), Gr-II (25-34 years), Gr-III (35-44 years), Gr-IV (45-54 years),

Gr-V (55-64 years) and Gr-VI (65-74 years).

Measurement of clinical parameters:

Clinical parameters of HCV sero-reactive patients were evaluated by various methods such as

creatinine level was measured by Jaffe’s alkaline picrate method (Full auto Biochem Erba XL 600,

Germany), urea level was estimated by glutamate dehydrogenase (GLDH) kinetic method (Full

4
auto Biochem Erba XL 600, Germany) and in case of uric acid, it was determined by the reagent

based on Trinder’s reaction, enzymatic and colorimetric method (Full auto Biochem Erba XL 600,

Germany) respectively. Electrolytes such as sodium and potassium were quantified by ion-

selective electrodes (Roche electrolyte full auto analyzer, Switzerland) and minerals like calcium

and phosphorus levels were detected by Colorimetric endpoint method (Full auto Biochem Erba

XL 600, Germany) and Gomori’s method (Full auto Biochem Erba XL 600, Germany)

respectively.

Detection of HCV RNA:

Viral RNA was isolated from 140µl of anti-HCV reactive serum samples using QIAamp viral

RNA mini kit (Qiagen, Hilden, Germany) and eluted in 50µl elution buffer and stored at -80ºC for

further usage. 5′ UTR region of the HCV genome was amplified for viral RNA detection using

nested RT-PCR as described previously17. Briefly, first-round one tube RT-PCR was done in 20μl

total reaction volume containing 2μl isolated RNA and second-round nested PCR was performed

in 25μl total volume using 2μl of 1st round RT-PCR product using ABI9700 Thermal Cycler

(Applied Biosystems, Foster City, USA) and primers used as mentioned elsewhere18. The PCR

amplicons (256 bp) were visualized in a 1.5% agarose gel using a gel documentation system (Bio-

Rad, USA).

HCV viral load estimation:

To determine HCV viral RNA quantitatively, Quanti-Fast Pathogen RT-PCR+IC kit (Qiagen,

Germany) was used. The HCV primers and probe sequences were selected against the 5′ UTR of

5
HCV genome19. Viral load in serum of HCV RNA positive individuals were expressed as log10

international units per milliliter. The 4th WHO International Standard for HCV, NIBSC code

06/102, was used as a standard for viral load estimation in this study.

HCV genotyping and recombinant study:

Partial core and NS5B region of the HCV genome were amplified by nested RT-PCR for

genotyping and phylogenetic analysis and also to find the presence of any recombinant HCV strain.

PCR was performed as previously described for amplifying the core and NS5B region of HCV

genome17. The amplified product was electrophoresed in 1.5% agarose gel and stained with

ethidium bromide. Positive bands were visualized at 405 bp and 389 bp for the Core and NS5B

region respectively. The amplified PCR products were gel excised and purified using Wizard® SV

Gel and PCR Clean-Up System (Promega, Madison, USA) and used for sequencing using Big Dye

Terminator 3.1 kit (Applied Biosystem, Foster City, USA) in an automated DNA sequencer 3130

XL (ABI, USA). Sequences were aligned and edited using Bio-Edit tool. HCV genotypes and

subtypes were determined by NCBI genotyping tool

(www.ncbi.nlm.nih.gov/projects/genotyping/).

Phylogenetic analysis:

Phylogenetic analysis was performed using 82 representative partial core sequences of HCV RNA

positive samples. To investigate the evolutionary linkage among laboratory isolates and reference

strains, partial core sequences of the representative HCV laboratory were aligned with HCV

reference strains using Molecular Evolutionary Genetics Analysis (MEGA-X) tool20. The

evolutionary history was inferred by using the Maximum Likelihood method and Kimura 2-

parameter model21 (according to in-built model selection option in MEGA-X). The tree with the

6
highest log likelihood (-3110.59) is shown. Initial tree(s) for the heuristic search were obtained

automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances

estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the

topology with superior log likelihood value. A discrete Gamma distribution was used to model

evolutionary rate differences among sites (5 categories (+G, parameter = 0.4677)). All positions

containing gaps and missing data were eliminated (complete deletion option). There was a total of

297 positions in the final dataset.

Statistical analysis:

Clinical parameters viz., urea, creatinine, sodium, potassium etc. level was compared between

HCV positive and HCV negative group with Z-test. Categorical demographic data of patients were

compared with Chi-square test. P-value ≤ 0.05 was considered as statistically significant.

7
RESULTS:

Demographic data of HCV sero-reactive individuals:

A total of 310 CKD HCV sero-reactive individual cases were studied of which 50.64% (n=157)

were found to have an active HCV infection. HCV viremia of male patients (51.90%) were slightly

higher than that of female patients (48%) although no significant difference had been found(p-

value-0.52). However, a strong co-relation had been observed between HCV viremia and higher

age CKD patients, Gr-VI (65-75 years) patients were found to have more RNA positive status

(75%) than any other age groups (p-value-0.004). It was also observed that Patients who had

undergone higher frequency of dialysis per month, were at higher risk of HCV infection (p-value-

0.002). Moreover, it was also found that Patients who reside in sub-urban area had more active

HCV infection (59.15%) than patients reside in rural area or urban area (p-value-0.034) (Table 1).

Clinical Parameters:

Urea, creatinine, uric acid, electrolytes such as sodium, potassium, minerals like calcium and

phosphorus are considered as the important clinical parameters for the determination of renal

conditions among CKD patients. So, we tried to find any significant impact of HCV viral infection

on these clinical parameters among CKD patients. In this study, it was observed that significant

difference in levels of urea (p-value = 0.02), creatinine (p-value = 0.03), calcium (p-value = 0.04)

and uric acid (p-value = 0.05) in HCV RNA positive compared to HCV RNA negative CKD

patients (Table 2).

8
HCV genotype distribution:

Partial core gene (405 bp) of 157 HCV RNA positive samples was amplified for HCV genotyping

and the NCBI genotyping tool was used for genotype determination. Sequence alignment with

reference HCV strains revealed that the major circulating strain was genotype 1 (82.16%, n=129)

followed by genotype 3 (10.20%, n=16) and genotype 4 (7.64%, n=12) (Table 3). In this study,

six HCV subtypes were noticed and they were:1a, 1b, 1c, 3a, 3b and 4a. Among these subtypes,

genotype 1c (67.52%, n=106) was the predominant strains followed by genotype 1a (10.83%,

n=17), genotype 4a (7.64%, n=12), genotype 3a (6.37%; n=10), genotype 1b (3.82%, n=6) and

genotype 3b (3.82%, n=6) (Figure 1b). Comprehensive sequence analysis of paired partial HCV

core and NS5B genes showed no discordant HCV genotype and subtype, thus the presence of any

recombinant HCV strain or any dual HCV co-infection was ruled out.

Phylogenetic analysis:

To better understand the evolutionary relationship among the different HCV strains identified and

sequenced during this study, a phylogenetic tree was constructed by MEGA X software with 82

representative partial core gene sequences (405 bp) and submitted in the Gen Bank, i.e., 1c (n=61,

MN590016-MN590019, MN642002-MN642058), 1a (n=6, MN650197-MN650202), 1b (n=2,

MN889529-30), 4a (n=8, MN590020-MN590027), 3b (n=2, OK148442-43), 3a (n=3, MN889526-

MN889528). (Figure 1c).

9
Discussion and Conclusion:

Clinical and therapeutic management of HCV among CKD patients is challenging in developing

countries, including India. Individuals with renal disease are prone to other viral infections like

HBV, HIV due to frequent exposure to contaminated medical equipment or by nosocomial

transmission. Though, an international initiative by Kidney Disease: Improving Global Outcomes

(KDIGO) was able to reduce the prevalence of HCV infection in hemodialysis patients

worldwide22, but new challenges remain in preventing CKD patients from acquiring HCV

infection. HCV infection among chronic renal failure patients vary from 2% to 60% 23. Previous

studies revealed that HCV RNA positivity in CKD patients from multiple hemodialysis centers in

eastern India ranged between ~ 36%-63%, which is pretty high (Data not shown) and corroborate
16, 24
previous studies of high HCV infection among the hemodialysis population in India . This

also indicates the previously studied fact that lack of awareness of proper HCV screening

(collected during “window period”), mishandling of contaminated medical equipment during

dialysis or hospital-acquired nosocomial infection due to frequent visits to hospitals and dialysis

centers and exposure to the HCV infected individuals are some of the key factors contributing to

this high prevalence of HCV infection in CKD patients25. Additionally, every CKD patient who is

planning for hemodialysis should get tested for HCV infection through sensitive Nucleic Acid

Testing (NAT) based molecular screening along with Enzyme Immunoassay (EIA)26.

This study showed male CKD patients had a little higher risk of HCV infection (51.90%) than

female patients (48%) but significant differences (P-value:0.52) could not been observed between

two groups. It had also been reported that males are at a higher risk of acquiring HCV infection

than females 27 which is also reflected in this study. (Table 1)

10
This study also revealed that CKD patients belonging to age Gr-V and Gr-VI (55-64 years and 65-

74 years) had a significantly higher percentage (69.77% and 75%) of HCV RNA positivity than

other age groups. It indicates that CKD patients with older age are more prone to HCV infection(P-

value:0.004) (Table 1) which is familiar with previous study 28.

It can also be inferred by this study that risk of HCV infection is greater when frequency of dialysis

increases (P-value:0.002). Patients undergoes higher number of dialysis (8-12 times/month) per

month are more prone to HCV infection probably because patients were compelled to take dialysis

from different centers other than hospitals as number of dialysis equipments are insufficient to

serve over populated country like India. CKD patients reside in sub-urban area were more tend to

have active HCV infection (P-value: 0.034). (Table 1)

Nephrons are the basic unit of the kidney that helps to regulate electrolyte homeostasis and acid-

base balance. Urea and creatinine levels are good indicators of kidney health and a decrease in

estimated glomerular filtration rate (eGFR) leads to an increase in blood urea and creatinine levels

due to impaired kidney function. Hyper and hypokalemia, and hyper and hyponatremia are

associated with abnormalities of potassium ions (K+) and sodium ions (Na+) that leads to increased

mortality and morbidity in CKD patients293031. Additionally, minerals like calcium (ionized or total

corrected for albumin), phosphorus plays a key role in developing complexity in CKD patients,

termed as mineral and bone disorder (MBD)31. Several studies have also indicated that the

progression of CKD can be decreased by reduction of uric acid level in CKD patients 323334. It has

been previously reported that the mean urea and creatinine level in virus-infected CKD patients

was higher than uninfected individuals35. HCV infection also triggers calcium depletion in

endoplasmic reticulum and induce oxidative stress by inducing the uptake of calcium ion in

mitochondria36. Based on that information, a comparative analysis of the effects of HCV infection

11
on these clinical parameters is done on HCV RNA positive and HCV RNA negative individuals.

Result suggests that the levels of urea, creatinine, calcium and uric acid levels were significantly

higher (P-value: 0.02, 0.03, 0.04, and 0.05) in HCV RNA positive compared to HCV RNA

negative CKD patients (Table 2). Significantly, higher levels of calcium ions among HCV RNA-

positive CKD patients indicate that HCV infection might play a role in increasing calcium intake

in CKD patients which may lead to more complications in this high-risk group population. Uric

acid has a key role in developing end-stage renal disorder (ESRD). This study also tried to find

any significant impact of viral infection on uric acid levels among CKD patients and it also had

showed for the first time that HCV infection might lead to increase uric acid levels in CKD patients

compared to non-viremic (HCV RNA negative) patients (P-value:0.05). These finding suggests

Calcium and uric acid can be useful markers of HCV infection in dialysis patients. However, we

did not find any significant difference in the levels of Na+, K+ ions and phosphorus between HCV

RNA positive and negative CKD patients. (Table 2)

Genotypic distribution of HCV among CKD patients revealed that genotype 1 was the most

predominant genotype followed by genotype 3 and genotype 4. This finding is unusual as per the

HCV genotype distribution pattern in eastern India. The most prevalent genotype in the eastern

region of India is genotype 3, while genotype 1 is the dominant genotype in the south India37.

Previous studies have reported that HCV genotype 1a or 1b is the most predominant genotype

among CKD patients worldwide, in this study, it was observed for the first time that genotype 1c

is the most prevalent genotype (p-value:<0.001) (Table 3) among CKD patients in West Bengal

followed by 1a, 4a, 3a, 1b and 3b (Figure 1b), which indicates a unique HCV subtype distribution

among CKD patients in the eastern part of India. In short, 1c and 4a, these two subtypes are unique

subtypes among CKD patients in this region. Increasing of unusual genotype and subtype (gen-4a

12
and 1c) can be a future headache because new subtypes can recombine38 with older subtypes to

give rise a new type strain which might be a problem for clinical management of HCV infected

CKD patient.

In conclusion, this study was one of the largest analyses of HCV genotype prevalence study among

CKD patients enrolled from more than 18 dialysis Centers/Units in the West Bengal that provides

comprehensive knowledge about the most prevalent HCV strains circulating in CKD patients in

this part of India. In addition to urea and creatinine, for the first-time it has been shown that levels

of calcium and uric acid are also significantly increased in HCV RNA-positive CKD patients by

this study. This study also for the first-time reports that the predominant circulating HCV strains

is genotype 1c among the HCV-infected CKD patients in eastern India. Again, HCV genotype 4a

which is not common in eastern India, is still circulated among CKD patients. Till date, no study

has reported the circulation of these two subtypes among this study population. Overall, this study

indicates the alarming situation of dialysis centers and need immediate action of standard infection

control protocols to prevent HCV infection in dialysis centers.

Acknowledgment:
We would like to acknowledge other members of Dr. Sadhukhan’s laboratory, especially to Miss
Maya Halder. We would also like to thank the patients and their families for their kind co-operation
and participation in this study.

Conflict of interest:
The authors declare they have no conflict of interest.

Funding Statement: This work was supported by Department of Science and Technology, Govt.
of West Bengal; grant number- 758(sanc.)/ST/P/S&T/9G-8/2014, dated- 27.11.2014. All
instruments’ facilities were provided by Indian Council of Medical Research (ICMR-NICED);
grant number – N/A.

13
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18
Tables:

Table 1: Comparative analysis of viremia between various demographic patients’ groups by

Chi-square test.

Variables HCV (+) HCV (-) Viremia (%) p-Value

(n=157) (n=153) (50.64)

Gender Male 109 101 51.90476 0.52

Female 48 52 48

14-24 13 22 37.14286

25-34 29 55 34.52381
Age-group (year)

35-44 40 37 51.94805
<0.001#
45-54 42 25 62.68657

55-64 30 13 69.76744

65-74 3 1 75

2 times/month 12 29 29.26829
Dialysis interval

4 times /month 22 34 39.28571

0.002#
8 times/month 96 70 57.83133

12 times/month 27 20 57.44681

Rural 49 69 41.52542
Locality of
Sub-Urban 42 29 59.15493 0.034#
residence

Urban 66 55 54.54545

# Denotes statistically significant.

19
Table 2: Comparative analysis of clinical parameters between HCV RNA positive and RNA
negative CKD patients by Z-test
CLINICAL HCV RNA HCV RNA P-VALUE
PARAMETERS POSITIVE (N=156) NEGATIVE (N=154)
(MEAN±SD)
Urea (mg/dL) 117.06± 31.04 108.59± 33.67 0.02#
Creatinine (mg/dL) 9.00± 2.92 8.28± 2.95 0.03#
Sodium (mEq/L) 136.70± 3.15 136.73± 3.04 0.93

Potassium (mmol/L) 5.00± .59 4.91± .68 0.21

Calcium (mg/dL) 7.95 ± .77 7.78± .73 0.04#
Uric acid (mg/dL) 5.65± 1.38 5.33± 1.16 0.05#
Phosphorus (mg/dL) 5.35± 1.20 5.33± 1.16 0.88

mg/dL= milligram/deciliter
mEq/L= miliequivalents/liter
mmol/L=milimoles/liter

# Statistically significant

20
Table 3: HCV genotype Distribution and comparison between various demographic groups of CKD
patients by chi-square test.

HCV genotypes
Variables
Total
Genotype Genotype Genotype Genotype Genotype Genotype p-value
1a 1b 1c 3a 3b 4a (n=157)
(n=17) (n=6) (n=106) (n=10) (n=6) (n=12)
Male 12 4 72 8 5 8 109 <0.001#
Gender

Female 5 2 34 2 1 4 48 <0.001#

14-24 1 1 8 1 1 1 13 <0.001#

25-34 5 1 19 1 1 2 29 <0.001#

35-44 3 2 26 3 1 5 40 <0.001#
Age-group
(Year)

45-54 2 0 33 4 1 2 42 <0.001#

0.94
55-64 5 2 18 1 2 2 30 <0.001#

Insufficient
65-74 1 0 2 0 0 0 3 sample
number

2 times 2 1 5 1 1 2 12 <0.001#
(n Times/Month)
Dialysis Interval

4 times 4 1 13 1 1 2 22 <0.001#

0.32
8 times 6 2 75 5 2 6 96 <0.001#

12 times 5 2 13 3 2 2 27 <0.001#

Rural 6 2 31 4 1 5 49 <0.001#
Locality of
Residence

sub-
7 2 25 2 1 5 42 <0.001#

0.49
Urban

Urban 4 2 50 4 4 2 66 <0.001#

# Statistically significant

21
 Figure 1:

 Figure 1a: Map of West Bengal and relative position of West Bengal in India

 Figure 1b: A donut pie-chart shows Distribution of HCV genotypes in CKD

patients.

 Figure 1c: A phylogenetic tree constructed by MEGA-X software containing 82

representative core sequences with 26 reference sequences downloaded from

NCBI. Maximum likelihood method with Kimura-2+G parameter model is used

according to in-built model selection option of MEGA-X. The word “CKD” is used

to encode local sequences.

22
Table and figure legends:

 Table 1: Comparative analysis of viremia between various demographic patients’ groups

by Chi-square test.

 Table 2: Comparative analysis of clinical parameters between HCV RNA positive and

RNA negative CKD patients by Z-test.

 Table 3: HCV genotype Distribution and comparison between various demographic groups

of CKD patients by chi-square test.

 Figure 1:

 Figure 1a: Map of West Bengal and relative position of West Bengal in India

 Figure 1b: A donut pie-chart shows Distribution of HCV genotypes in CKD

patients.

 Figure 1c: A phylogenetic tree constructed by MEGA-X software containing 82

representative core sequences with 26 reference sequences downloaded from

NCBI. Maximum likelihood method with Kimura-2+G parameter model is used

according to in-built model selection option of MEGA-X. The word “CKD” is used

to encode local sequences.

23