Comparative Evaluation of Diagnostic Performance Among Three Commercially Available Rapid HCV

Antibody Tests Using Clinical and Blood Donor Samples

Nuzhat Salamat, Abdul Quddus, Zumara

Abstract

Background: Accurate diagnosis is crucial for hepatitis C virus (HCV) management and global elimination
efforts. Rapid diagnostic tests (RDTs) offer advantages for expanding testing access, particularly in
resource-constrained settings. This study aimed to compare the diagnostic performance of three
commercially available HCV rapid tests using well-characterized samples.

Methods: We evaluated three most commonly used , DRAP registerd anti- HCV rapid tests (LDS,
Healgen, and Abbott) using 400 samples (200 HCV-positive, 200 HCV-negative ) previously characterized
by enzyme immunoassay and nucleic acid testing. Only Abbott rapid test is WHO prequalified as well.
We calculated sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and
accuracy with 95% confidence intervals. Inter-test agreement was assessed using Cohen's kappa, and
McNemar's test was used for pairwise comparison of sensitivity and specificity.

Results: All three tests demonstrated high diagnostic performance with sensitivities from 97.01% to
98.00% and specificities of 97.00%. The LDS test showed the highest sensitivity (98.00%, 95% CI: 95.02%-
99.29%) and accuracy (97.50%, 95% CI: 95.44%-98.69%), while all three tests exhibited identical
specificity (97.00%, 95% CI: 93.58%-98.71%). Inter-test agreement was nearly perfect (Cohen's kappa
>0.95 for all pairs). No statistically significant differences were found in diagnostic performance among
the three kits.

Conclusions: All three rapid tests demonstrated excellent diagnostic performance for HCV antibody
detection, with high sensitivity, specificity, and near-perfect inter-test agreement. These findings
suggest that all three tests are reliable tools for HCV screening in various settings, including resource-
limited environments where laboratory-based testing may be challenging. The high sensitivity is most
desirable for blood donor screening where context justifies.

Keywords: Hepatitis C, rapid diagnostic test, sensitivity, specificity, diagnostic accuracy, screening

1. Introduction

Hepatitis C virus (HCV) infection represents a significant global public health concern, affecting
approximately 58 million people worldwide with chronic infection and causing an estimated 290,000
deaths annually, primarily due to cirrhosis and hepatocellular carcinoma [1]. Despite the availability of
highly effective direct-acting antivirals (DAAs) that can cure >95% of cases, the majority of HCV-infected
individuals remain undiagnosed, presenting a major barrier to treatment access and global elimination
efforts [2].

The current diagnostic algorithm, at many places typically involves initial screening with an enzyme
immunoassay (EIA) for anti-HCV antibodies, followed by nucleic acid testing (NAT) for HCV RNA to
confirm active infection [3]. While these laboratory-based methods offer excellent sensitivity and
specificity, they require sophisticated infrastructure, trained personnel, and have relatively long
turnaround times, limiting their utility in resource-constrained settings and for rapid screening programs
[4].
Rapid diagnostic tests (RDTs) for HCV antibody detection have emerged as valuable tools to expand
testing access, offering advantages including minimal infrastructure requirements, ease of use, rapid
turnaround times (typically <30 minutes), and potential for point-of-care implementation [5]. These
characteristics make RDTs particularly valuable for reaching populations with limited access to
conventional laboratory services and facilitating same-day testing and linkage to care [6].

The World Health Organization (WHO) has endorsed the use of quality-assured RDTs in their guidelines
for HCV testing, particularly in settings with limited laboratory infrastructure or for reaching key affected
populations [7]. However, the performance of commercially available HCV rapid tests varies
considerably, with reported sensitivities ranging from 78% to 99% and specificities from 80% to 100%
across different studies and populations [8,9].

Most blood banks operating at district and sub-district levels in low- and middle-income countries, in
public, private, or non-governmental sectors, use rapid devices for screening blood donors for
transfusion-transmitted infections including HCV. Therefore, it is critical to evaluate the diagnostic
accuracy of commonly available rapid tests to enable informed decision-making by users and regulators.
This is particularly important in countries with high prevalence of transfusion-transmitted infections,
where users often lack the capacity to scientifically verify the performance characteristics of tests they
intend to use.

Despite the growing market availability of HCV rapid tests, comprehensive comparative evaluations
using standardized methodologies and well-characterized sample panels are limited. Most published
studies evaluate a single test or involve different methodologies, making direct comparisons challenging.
Additionally, performance data across different sample types (clinical vs. blood donor), viral loads, and
genotypes remain incomplete for many commercially available tests [10,11].

This study addresses these critical knowledge gaps by conducting a direct head-to-head comparison of
three widely available HCV rapid tests (LDS, Healgen, and Abbott) using a consistent methodology and
well-characterized, anonymized clinical and blood donor samples. The findings provide valuable
information to guide test selection based on setting-specific requirements and help optimize HCV
screening strategies, particularly in resource-limited settings where appropriate test selection is crucial
for maximizing case detection within budgetary constraints.

2. Materials and Methods

2.1 Study Design

This research employed a retrospective diagnostic accuracy study design using banked biological
samples with known HCV status. All samples had been previously tested with reference standard
methods (Band had established HCV status prior to evaluation with the index tests (rapid HCV tests).
This design aligned with the Standards for Reporting of Diagnostic Accuracy Studies (STARD) 2015
guidelines for diagnostic accuracy studies [12] and recommendations for evaluating HCV rapid tests [7].

2.2 Sample Selection and Characterization

We utilized well-characterized archived samples from two distinct sources: clinical diagnostic samples
collected from patients with suspected HCV infection during routine clinical care, and blood donor
samples collected during blood donation screening. All samples had been previously tested with
reference standards and stored according to standard laboratory protocols.

Sample inclusion criteria were: adequate sample volume (minimum 500 μL), complete documentation of
previous testing results, proper storage conditions maintained throughout storage period, confirmed
HCV positive or negative status by both antibody and nucleic acid testing, storage date within the past
years, and maintained sample integrity (no visible hemolysis, lipemia, or microbial contamination).

Samples with incomplete reference testing documentation, evidence of improper storage or multiple
freeze-thaw cycles (>3), insufficient volume, hemolysis or lipemia that could interfere with testing, or
indeterminate or conflicting reference test results were excluded.

In total, 400 samples were included in the study, comprising 200 HCV-positive and 200 HCV-negative
samples. Sample types included serum in aliquot, serum in gel tube, EDTA whole blood, and serum in
clot activator tube.

2.3 Reference Standard

The reference standard for determining true HCV status was a composite algorithm following
international guidelines [3,7] consisting of a third-generation enzyme immunoassay (BioRad EIA) for HCV
antibody detection AND confirmatory nucleic acid test (NAT) for HCV RNA detection. Samples were
classified as true positive (positive by both EIA and NAT) or true negative (negative by EIA and NAT).

2.4 Index Tests (Rapid HCV Tests)

Three commercially available, commonly used, DRAP registered, rapid HCV antibody tests were
evaluated:

1. LDS HCV Rapid Test: A lateral flow immunochromatographic assay

2. Healgen HCV Rapid Test: A lateral flow immunochromatographic assay

3. Abbott Diagnostic HCV Rapid Test: A lateral flow immunochromatographic assay

All three tests detect anti-HCV antibodies in human serum, plasma, or whole blood samples. Tests were
selected based on their widespread commercial availability and use in local healthcare settings.

2.5 Testing Procedures

Testing was conducted in a standardized laboratory environment with controlled temperature (20-25°C)
and humidity (30-70%), adequate lighting for result interpretation (300-750 lux), calibrated timers for
precise readout timing, and standard operating procedures (SOPs) for each test.

Samples were thawed once and brought to room temperature, with time between sample thawing and
testing standardized and recorded. Pre-analytical processing followed manufacturers' instructions. All
tests were performed according to manufacturers' instructions by trained laboratory technicians who
were blinded to the reference test results. Positive and negative controls were included in each testing
batch.
Test results were read at the exact time specified by manufacturers and documented using standardized
recording forms with photographic documentation of test results maintained. For tests with time-
dependent results, readings were taken at the earliest and latest recommended times.

2.6 Blinding Procedures

To minimize bias, samples were relabeled with study-specific codes. Laboratory technicians performing
the index tests were blinded to reference standard results, patient information, other index test results,
and other readers' interpretations. Data analysts were blinded to the identity of the three test kits (using
codes A, B, C) until primary analyses were completed.

2.7 Quality Control

Internal quality controls (included in test kits) were performed daily, and external quality controls
(characterized samples) were run at the beginning of each test batch. Tests from at least two different
lot numbers were evaluated. Invalid test results were repeated according to manufacturers'
recommendations, and 10% of all samples were retested to assess reproducibility.

2.8 Data Analysis

For each rapid test, the following parameters were calculated:

Sensitivity: proportion of true positives correctly identified

Specificity: proportion of true negatives correctly identified

Positive predictive value (PPV): probability that subjects with a positive test truly have HCV

Negative predictive value (NPV): probability that subjects with a negative test truly do not have
HCV

Diagnostic accuracy: proportion of correctly classified samples

All parameters were reported with 95% confidence intervals calculated using the Wilson score method
for proportions [13]. McNemar's test was used to compare the sensitivity and specificity between tests
[14]. Cohen's kappa coefficient was calculated to assess inter-test agreement. A p-value <0.05 was
considered statistically significant for all analyses.

2.9 Ethical Considerations

The study protocol was approved by the Institutional Review Board. Since the study utilized previously
collected and de-identified archived samples, a waiver of informed consent was granted according to
relevant regulations. All samples were de-identified before testing, with only anonymous coding used
during the study.

3. Results

3.1 Sample Characteristics

A total of 400 samples were included in the analysis, comprising 200 HCV-positive samples and 200 HCV-
negative samples from clinical diagnostic and blood donor sources.
3.2 Diagnostic Performance of Rapid Tests

Table 1 summarizes the diagnostic performance of the three rapid HCV tests. All tests demonstrated
high sensitivity and specificity. The LDS test showed the highest sensitivity at 98.00% (95% CI: 95.02%-
99.29%), followed by Healgen at 97.50% (95% CI: 94.26%-98.95%) and Abbott at 97.01% (95% CI:
93.58%-98.71%). All three tests exhibited identical specificity of 97.00% (95% CI: 93.58%-98.71%).

Table 1: Diagnostic Performance of the Three Rapid HCV Tests

Sensitivity (95% Specificity (95%

Test PPV (95% CI) NPV (95% CI) Accuracy (95% CI)
CI) CI)

98.00% (95.02- 97.00% (93.58- 97.03% (93.64- 97.98% (94.93- 97.50% (95.44-
LDS
99.29) 98.71) 98.73) 99.28) 98.69)

97.50% (94.26- 97.00% (93.58- 97.01% (93.61- 97.49% (94.25- 97.25% (95.12-
Healgen
98.95) 98.71) 98.72) 98.95) 98.53)

97.01% (93.58- 97.00% (93.58- 97.01% (93.58- 97.00% (93.58- 97.01% (94.79-
Abbott
98.71) 98.71) 98.71) 98.71) 98.34)

The positive predictive values (PPV) ranged from 97.01% to 97.03%, and negative predictive values
(NPV) ranged from 97.00% to 97.98%. Overall diagnostic accuracy was high for all three tests, ranging
from 97.01% to 97.50%. No invalid results were reported for any of the three tests, resulting in an
invalid rate of 0% for all tests.

3.3 Contingency Tables

Table 2 presents the contingency tables for each rapid test. The LDS test had 196 true positives (TP), 4
false negatives (FN), 194 true negatives (TN), and 6 false positives (FP). The Healgen test had 195 TP, 5
FN, 194 TN, and 6 FP. The Abbott test had 195 TP, 6 FN, 194 TN, and 6 FP.

Table 2: Contingency Tables for the Three Rapid HCV Tests

Test True Positive False Negative True Negative False Positive

LDS 196 4 194 6

Healgen 195 5 194 6

Abbott 195 6 194 6

3.4 Pairwise Comparison of Test Performance

McNemar's test was used to compare the sensitivity and specificity between test pairs. No statistically
significant differences were found in sensitivity or specificity among the three tests (p>0.05 for all
comparisons), indicating that the three tests performed similarly in terms of diagnostic accuracy.

3.5 Inter-test Agreement

All three tests showed very high agreement with each other. Cohen's kappa coefficients for inter-test
agreement were all above 0.95, indicating almost perfect agreement (Table 3). The observed agreement
between test pairs ranged from 97.50% to 99.25%.

Table 3: Inter-test Agreement (Cohen's Kappa)

Test Pair Kappa Observed Agreement Expected Agreement

LDS vs Healgen 0.9722 99.25% 50.00%

LDS vs Abbott 0.9501 97.50% 50.00%

Healgen vs Abbott 0.9722 99.25% 50.00%

3.6 Overall Agreement Among All Tests

Among the 400 samples tested, 394 (98.50%) showed consistent results across all three tests, while only
6 (1.50%) had at least one discordant result. This high level of agreement further confirms the reliability
and consistency of the three rapid tests.

4. Discussion

This comparative evaluation of three commercially available rapid HCV antibody tests using well-
characterized archived samples demonstrated excellent diagnostic performance for all three tests, with
high sensitivity, specificity, and overall accuracy. The LDS test showed marginally better sensitivity
(98.00%) compared to Healgen (97.50%) and Abbott (97.01%), but these differences were not
statistically significant. All three tests exhibited identical specificity (97.00%), indicating similar
performance in correctly identifying HCV-negative samples.

The findings from this study are consistent with previous evaluations of HCV rapid tests. A systematic
review and meta-analysis by Tang et al. [9] reported pooled sensitivity and specificity of 98% (95% CI:
97-98%) and 100% (95% CI: 100-100%) for HCV rapid tests, which is comparable to our results. Similarly,
Shivkumar et al. [5] reported pooled sensitivity and specificity of 98% (95% CI: 95-99%) and 99% (95% CI:
98-100%) for rapid immunoassays.

The high level of agreement among the three tests (Cohen's kappa >0.95 for all pairs) suggests that
these tests perform consistently and reliably. This is particularly important in resource-limited settings
where confirmatory testing might not be readily available, and decisions need to be made based on the
results of a single rapid test.

The excellent diagnostic performance of these rapid tests makes them valuable tools for HCV screening
in various settings, including resource-limited environments where laboratory-based testing may be
challenging. They can facilitate expanded access to HCV testing, particularly in hard-to-reach
populations, and support global efforts toward HCV elimination. The WHO has set ambitious targets for
HCV elimination, including 90% diagnosis of all HCV cases by 2030 [15], and rapid tests can play a crucial
role in achieving these targets.

In blood banking settings, particularly at district and sub-district levels in low- and middle-income
countries, rapid tests are often the primary screening tool for transfusion-transmitted infections,
including HCV. Our findings provide reassurance that the three tested kits offer reliable performance for
this critical application. However, it is important to note that rapid tests detect antibodies, not the virus
itself, and therefore cannot distinguish between current and resolved infections. In blood banking, this
limitation is acceptable since the goal is to exclude all potentially infectious donations, regardless of
whether the infection is current or resolved.

In the context of Pakistan, where HCV prevalence is estimated at 6.2% according to the PC-1 national
program [16], understanding the real-world performance of these tests is particularly important. When
adjusting the positive and negative predictive values to reflect this 6.2% prevalence, the PPV for all three
tests would be approximately 68%, while the NPV would remain around 99.8%. This means that in
Pakistan's context, about 32% of positive results could be false positives, while negative results remain
highly reliable. This underscores the importance of confirmatory nucleic acid testing following a positive
rapid test result, particularly when making treatment decisions.

A strength of this study is the use of well-characterized archived samples with established HCV status
determined by reference standard methods. This allowed for a direct head-to-head comparison of the
three rapid tests under identical conditions. Additionally, the study included a substantial and balanced
number of samples (200 positive and 200 negative), providing adequate statistical power.

4.1 Limitations

This study has several limitations that should be considered when interpreting the results. First, the
retrospective design using archived samples may not fully reflect the performance of the tests in real-
world settings where factors such as operator variability, field conditions, and diverse patient
populations can influence test performance. Second, the study did not evaluate the performance of the
tests across different HCV genotypes or viral loads, which can potentially affect sensitivity [17]. Third,
the study did not include samples from patients with resolved HCV infection (antibody-positive, RNA-
negative), which would have provided insights into the tests' ability to detect past infections. Finally, the
study did not assess the tests' performance in the presence of potential interfering factors such as
rheumatoid factor, high bilirubin levels, or coinfection with other blood-borne viruses [18].

4.2 Implications for Practice

The findings of this study have several implications for practice:

1. All three rapid tests (LDS, Healgen, and Abbott) demonstrated excellent diagnostic performance
and can be reliably used for HCV screening in various settings, including resource-limited
environments.

2. The high inter-test agreement suggests that any of these tests can be used interchangeably,
which is valuable information for procurement decisions, particularly when availability or cost
considerations come into play.

3. The absence of invalid results indicates robust test performance, which is important for field
implementation where repeat testing due to invalid results can be challenging.

4. For blood banks and transfusion services, particularly those in resource-constrained settings, all
three tests offer reliable performance for donor screening, contributing to blood safety [19].
 5. In the context of Pakistan's 6.2% HCV prevalence, these tests provide extremely reliable
negative results but a notable proportion of false positives, highlighting the need for
confirmatory testing of positive results before initiating treatment.

5. Conclusions

In this comparative evaluation, all three rapid HCV antibody tests (LDS, Healgen, and Abbott)
demonstrated excellent diagnostic performance with high sensitivity, specificity, and inter-test
agreement. These findings suggest that all three tests are reliable tools for HCV screening in various
settings, including resource-limited environments where laboratory-based testing may be challenging.
The selection among these tests can therefore be guided by factors such as cost, availability, and
operational characteristics rather than diagnostic performance. Future studies should focus on
evaluating these tests in prospective field settings, assessing their performance across different HCV
genotypes and viral loads, and in the presence of potential interfering factors.

Acknowledgments

We thank the laboratory staff for their technical assistance and the institutional review board for their
ethical guidance.

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