Laboratory Evaluation of Hepatitis C Virus Infection in Patients Undergoing Hemodialysis from North East India

Methods

It was a cross-sectional survey, where blood samples were collected from subjects admitted into a dialysis unit at a tertiary care hospital from the North-Eastern part of India. Adult subjects (≥18 years) who are at least two months into the dialysis unit and tested negative for anti-HCV antibody, HBV (Hepatitis B Virus surface antigen i.e. HBsAg), and HIV (anti-HIV antibodies, as per NACO guidelines) at the initial screening or any subsequent screening were selected for the study.²⁶ As a part of a Department of Biotechnology/North East Region Biotechnology Programme Management Cell funding scheme [Grant sanction letter no BT/248/NE/TBP/2011] of the central Government, between 1 October 2015 and January 2017, sample collection was carried out, followed by data analysis for the next two months. Prior informed consent was obtained before their inclusion into the survey; ethical clearance from the Institutional Ethics Committee of the host institute was obtained [MC/2/2015/82 dt.13.5.15].

Subjects were primarily tested for HCV, and their patient records were used to collect information on age, sex, duration, and frequency of hemodialysis. Five (5) ml of clotted blood was collected, serum separated, and aliquoted for HCV testing along with HBsAg, as well as anti-HIV as per NACO guidelines. Other biochemical parameters such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), and so on were either collected from patient's records or tested at the biochemistry facility of the host hospital. HCV testing included anti-HCV antibody detection by immunochromatography (ICT)-based rapid detection test (RDT) of Tulip make; third-generation anti-HCV antibody ELISA of J Mitra's Microlisa make; HCV core Ag with fourth-generation anti-HCV antibody ELISA of Biorad's Monolisa™ make (HCV Ag-Ab ULTRA assay); and HCV RNA testing by real-time PCR (HCV kit and manual pure nucleic acid extraction kit in Roche CobasTaqman 48 cycler). The kit insert of these reagents was strictly followed during the tests.

For data analysis, Statistical Package for the Social Sciences (SPSS), version 22 (IBM, USA) was used. Categorical variables were expressed in percentage (%), whereas continuous variables were expressed in mean, standard deviation, range, median, upper quartile (UQ), and lower quartile (LQ) wherever applicable. All screening tests were compared against the gold standard HCV RNA assay with standard parameters such as sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy with 95% confidence intervals (95% CIs). For agreement between HCV RNA test and other screening tests, such as RDT, third-generation anti-HCV antibody ELISA, and HCV Ag-Ab ULTRA assay, Kappa statistic (k) was performed with 95% CI values.

A cost-effective analysis on anti-HCV third-generation ELISA and fourth-generation (antigen-antibody combined) ELISA was performed, and the result was documented in a tabulated form (Supplementary Tables 1–4).

Results

Table 1 and Figure 1 summarize the methods and results of our study.

The baseline characteristics of all 270 enrolled patients are shown in Table 1. Levels of AST, ALT, and GGT were lower than 40 IU/L, and this was irrespective of HCV infection status (data not shown).

The rate of HCV RNA detection was 31.9% (86/270), and 83 of 86 were positive in the HCV Ag-Ab ULTRA assay. None of the HCV RNA–negative patients tested positive in the HCV Ag-Ab ULTRA assay (Table 2). Three HCV RNA positive HCV but fourth generation ELISA (i.e. Ag-Ab ULTRA assay) negative cases had a low viral load, i.e. below 1000 IU/ul (refer Table 3 for break up)

As shown in Table 2, third-generation anti-HCV antibody ELISA negative rate was 74.8% (202/270), of whom 11.4% (23/202) were HCV RNA positive (Table 2). About 11.5% of 68 seropositive cases had HCV RNA negative result. Table 2 clearly shows higher sensitivity and specificity of HCV Ag-Ab ULTRA assay than that of third-generation anti-HCV antibody ELISA. Similarly better PPV, NPV, and accuracy of HCV Ag-Ab ULTRA assay than those of third-generation anti-HCV antibody ELISA test is depicted in Table 2. The agreement between HCV RNA and HCV Ag-Ab ULTRA assay tests was much better (k = 0.947) than that between HCV RNA and third-generation anti-HCV antibody ELISA (k = 0.947). Similar parameters of the anti-HCV rapid test (ICT-based) fared worse than those of other two tests compared with the reference standard HCV RNA test (Table 2).

Cost-effective analysis of fourth-generation HCV antigen–antibody kit with third-generation anti-HCV ELISA kit is detailed in Supplementary Table 1, Supplementary Table 2, and Supplementary Table 3.

Discussion

HCV infection is a very frequently encountered threat in the population undergoing chronic hemodialysis globally.^4,8 It considerably contributes to the risk of death in this group of people.^27,28 Early detection and prompt initiation of antiviral treatment is key to minimize the infection-related adverse outcomes.²⁹ Early detection also goes a long way in the prevention of the spread of infection, which is otherwise efficient in vulnerable patients undergoing hemodialysis with frequent and multiple invasive procedures.^7,30 Biochemical markers are not of much help in the identification of HCV infection in the early phase in this group of patients. The present study had 270 patients undergoing hemodialysis, and all had aminotransferase values less than 40 IU/L, including those with active HCV infection (HCV RNA–positive). This corroborated well with other studies.^7,30

About 7.3% antibody positive cases had no detectable HCV, whereas in 11.4% cases, seronegative cases were HCV RNA–positive. This findings was in concordance with previous other study results.¹⁰

The superiority of fourth-generation HCV (core Ag-antibody combined detection) assay–based studies over third-generation anti-HCV antibody detection is established in our research, as was found out by previous works.^13,31 Besides much better sensitivity and specificity, a 100% specificity vis-à-vis gold standard HCV RNA testing is noteworthy. But an NPV of 98.4% implies 1.6% anti-HCV antibody negative cases had positive HCV RNA result. Multiple Indian studies on core antigen-based HCV detection in subjects undergoing dialysis primarily used pure core antigen detection kits (in contrast to our antigen–antibody combined assay). One older study from South India³² concluded on a negative note about antigen detection kit due to poor outcome, although in the other earlier study, the same authors concluded usefulness of the kit especially during pre-seroconversion.²² This could be due to the failure of older assays to detect combined antigen (once the antibody is formed). Currently available core antigen assays can detect both free and combined antigen. A Delhi-based study opined in favor of using HCV core antigen ELISA in subjects undergoing dialysis as it effectively reduces the window period which is otherwise longer in dialysis cases.²¹ In a study by Chakravarti et al³³ from Delhi, core antigen–positive cases in 13 of 14 pre-seroconversion window period cases were found. They recommended it as alternative to HCV PCR in resource scanty areas. Jindal et al.³⁴ found that overall HCV in patients undergoing hemodialysis to be 32% – one very similar finding to our findings. They detected HCV RNA in 2.8% of core antigen–negative cases against our finding of 1.6% of cases (we used antigen–antibody combined assay). Mahajan et al.³⁵ on the other hand recommended both antibody-based and HCV RNA–based tests in dialysis cases (without mentioning core antigen kits). However, few recently concluded works outside India recommended core antigen–based assays for hemodialysis setups in resource scanty areas due to its excellent correlation to HCV RNA level in blood and 100% PPV.^36,37

Combined HCV antigen-antibody kit–based study in subjects undergoing dialysis is rare from India. We found few from abroad and one such French study concluded that combined kits detect HCV infection much earlier (21.6 days) than antibody detection assays.¹⁸ Similar conclusion was drawn by an Egyptian study too.²⁰ In a comparative study between Ag-Ab combined kit and pure antigen detection kit found that in patients undergoing hemodialysis, antigen–antibody combined detection ELISA was more sensitive than that of only core antigen detection.¹⁹

In the present study, 3 cases who were not detectable by fourth-generation (antigen–antibody ELISA kit) assay had viral loads less than 650 IU/ml (see footnote of Table 3). Our false negative rate was 1.6% (3/187). The cutoff viral load for pure core antigen–based assays yielding false negative results was reported higher at 4500 IU/ml by Chakravarti et al.³³ from India. Similar data for pure core antigen–based assays were reported by few other studies too.^3,14

Implication of low HCV viral load in subjects undergoing dialysis needs attention. Occult hepatitis infection (OCI) by HCV (i.e. detection of HCV RNA in kidney/peripheral mononuclear cells in serum HCV RNA negative cases) is documented but reportedly without any clinical implications/relevance.³⁸ In a similar vein, an infection with very low viral load too (undetectable by fourth-generation ELISA) may have less clinical implications, but the risk of transmission (within dialysis units) certainly remains high, especially if it goes undetected in initial screening.³⁹

Table 2 shows sensitivity issue of RDT kits with the possibility of missing nearly half of positive cases. Our findings corroborate well with similar previous works.¹⁰

Because genotype distribution can have a bearing on antibody-based test results, a kit covering (responding to) multiple genotypes is ideal for diagnostic purpose.^40,41 A Paris-based study found differing sensitivities of ELISA kits depending on HCV genotypes.⁴² Antigen-based kits can be a distinct advantage in this regard. In our present study, we checked genotypes by direct sequencing (protocol of Verma and Chakravarti⁴³) in selected isolates and all such selected strains (data not included in this manuscript) were found to be of 3h genotype [GenBank Accession no: KX646407-KX646427]. In addition, the influence of HBsAg (HBV) and HIV positivity on fourth-generation ELISA could not be studied as the number of cases was too less to do it (not included in this manuscript).

Cost-effective analysis (Supplementary Table 1, Supplementary Table 2, and Supplementary Table 3) clearly shows a Rs. 27.03 extra expense per 1% gain (up to 7.4% extra yield) in detection rate by using fourth-generation ELISA (Ag-Ab combined Ultra ELISA) against anti-HCV antibody ELISA. Supplementary data is showing average costs of commonly available HCV tests in India. Given the advantage of early detection (in window period) with near specificity as HCV RNA testing and without any extra equipment/expertise (in a typical ELISA lab), fourth-generation ELISA (Ag-Ab combined Ultra ELISA) test can be introduced in a low-resourced hemodialysis setting.³

Overall, to the best of our knowledge, the present study is the first work from our country with fourth-generation ELISA (Ag-Ab combined Ultra ELISA) in subjects undergoing hemodialysis. Clearly the fourth-generation kit can be a better alternative, given its advantage in sensitivity, specificity, PPV, and so on. Less chance of false negativity (lower limit of cutoff viral load) could be one advantage over pure core antigen–based kits, besides the cost factor (Supplementary data table). The fourth-generation kit, core antigen being an integral target, brings along advantages of pure core antigen–based assays – e.g. efficient correlation with HCV viremia (HCV RNA results).

Fourth-generation kits are more expensive than third-generation (antibody-based); ultimately yield wise (as shown in Supplementary Table 3 and Figure 1) former is more preferable and feasible, especially given the fact that no additional infrastructure is needed to develop (in addition to already exiting ELISA facility). Availability of fourth-generation kit in India could be another aspect to look into; as of now only one manufacturer markets this kit here. But with wide acceptance, more manufacturers will surely take it up and market.

The Government of India flagship programme “National Viral Hepatitis Control Programme” envisages development of a strong laboratory infrastructure (ELISA setup, PCR setup, and so on) including ELISA facility from a basic level i.e. district-level laboratory onward. Although antigen detection (or antigen–antibody detection) is not included and only antibody detection (ELISA and rapid test) and PCR are recommended for HCV establishment of ELISA, facility at the district level onward will surely facilitate antigen-based ELISA in future (given its advantage over antibody-based ELISA).

One systematic review on data from multiple countries shows 60–80% prevalence of HCV in persons who inject drug (PWID).⁴⁴ Similar finding was obtained later by other studies too.⁴⁵ In this context, one study from Saudi Arabia found distinct advantage of antigen-antibody combined ELISA in detecting HCV amongst PWID population with higher detection rate than antibody ELISA and comparable result with RNA detection method.⁴⁶ In PWIDs from resource-limited African setting, antigen detection method was found to be with comparable result with RNA detection in contrast to antibody detection.⁴⁷

Efficient HCV transmission in vulnerable groups such as in patients undergoing dialysis makes it very essential to go for early detection of infection, mainly, to prevent cross-infection in dialysis units and institute treatment in the acute stage. As a conclusion to this work, we propose routine screening should be carried out for patients undergoing hemodialysis using fourth-generation HCV (e.g., HCV Ag-Ab ULTRA assay) ELISA, instead of the common practice of anti-HCV antibody-based tests. The cost-effectiveness and reduced labor-intensive HCV Ag-Ab ULTRA assay make it suitable for even lower resourced settings.

CRediT authorship contribution statement

Deepjyoti Kalita: Conceptualization, Methodology, Software, Supervision, Writing – review & editing. Sangeeta Deka: Writing – original draft, Software, Validation, Data curation. Kailash Chamuah: Data curation, Visualization, Investigation. Giasuddin Ahmed: Writing – review & editing, Validation, Supervision.

Conflicts of interest

The authors have none to declare.

Acknowledgement

The authors are very much indebted to Dept. of Biotechnology (DBT, Government of India) for providing them with a research grant under the North East Region Biotechnology. Programme Management Cell (NERBPMC) initiative called “Twinning Project scheme” (Sanction order and Date: BT/248/NE/TBP/2011 and 27/11/2012). The authors acknowledge staff and administration of Gauhati Medical College and Hospital, especially professor and head (then) of Nephrology dept. Dr. Anup Kumar Barman for kindly allowing sample collection and all other help and support.

Funding

Dept. of Biotechnology (DBT, Government of India) provided research grants under the North East Region Biotechnology Programme Management Cell (NERBPMC) initiative called “Twinning Project scheme.” Sanction letter no. BT/248/NE/TBP/2011 dated 27/11/2012.

Appendix A. Supplementary data

The following is/are the Supplementary data to this article: