Seroprevalence and viremia of hepatitis C virus among HIV-infected patients on ART with unsuppressed HIV viral load in Ethiopia

Abstract

Background

Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV) share common transmission routes, resulting in a significant burden of co-infection. This study assessed the seroprevalence and viremia of HCV among people living with HIV (PLWH) on ART with unsuppressed viral load in Ethiopia.

Methods

A cross-sectional study was conducted between June 2022 and February 2023 among 900 PLWH with unsuppressed viral loads. Plasma samples were screened for HCV antibodies by ELISA, and positives were confirmed for HCV RNA using the Abbott m2000sp/rt PCR system. Data were analyzed using SPSS V 26, applying chi-square tests and logistic regression to assess associations.

Results

Among 900 participants, 59.3% were females. Overall HCV seroprevalence was 5.4%, with higher rates among females (5.8%) and those aged 40–49 years. Active HCV viremia was confirmed in 14.3% of ELISA-positive cases (0.8% overall). Non-DTG regimens showed higher HCV co-infection (9.7%) than DTG-based regimens (4.4%, p = 0.062). HCV RNA positivity was more frequent in males and participants with medium HIV viral loads.

Conclusion

A subset of PLWH with unsuppressed viral load showed evidence of HCV exposure and active infection. The result also indicates the exitance of active co-infection HIV/HCV. The data may also reflect spontaneous clearance of HCV or a high rate of past, resolved infections.

Introduction

Hepatitis C virus (HCV) is a single-stranded, positive-sense ribonucleic acid (RNA) virus belonging to the genus Hepacivirus. It is primarily transmitted through blood exposure, including blood transfusion, injecting drug use, unsafe medical procedures, tattooing, and sexual contact [1, 2]. Chronic HCV infection is a major cause of liver-related morbidity and mortality, leading to liver cirrhosis and hepatocellular carcinoma [3].

Human immunodeficiency virus (HIV) is a retrovirus of the genus Lentivirus that shares similar transmission routes with HCV [4]. As a result, people living with HIV (PLWH) are at increased risk of HCV co-infection, particularly because HCV is transmitted more efficiently than HIV through percutaneous exposure [5, 6].

This study focuses specifically on PLWH with unsuppressed HIV viral load, defined as HIV RNA > 1,000 copies/mL, in accordance with WHO and national treatment guidelines. Although antiretroviral therapy (ART) has no direct antiviral effect against HCV, uncontrolled HIV replication is associated with immune dysregulation, impaired CD4⁺ T-cell recovery, and reduced capacity for spontaneous clearance of HCV. In addition, individuals with unsuppressed viral load often experience treatment interruptions, suboptimal adherence, or delayed engagement in care, which may increase exposure to blood-borne infections. These biological and clinical factors may contribute to both increased HCV exposure and persistence in this vulnerable subgroup.

Clinically, HCV co-infection is a major contributor to non-AIDS–related morbidity and mortality among PLWH, accelerating liver disease progression and impairing immunologic recovery even after ART initiation [7]. The World Health Organization recommends a two-step diagnostic approach for HCV infection, consisting of serological screening followed by molecular confirmation of HCV RNA to identify active infection [8]. However, access to confirmatory molecular testing remains limited in resource-constrained settings due to cost and laboratory capacity challenges [9].

In Ethiopia, data on both HCV seroprevalence and active viremia among PLWH—particularly those with unsuppressed HIV viral load—are limited. This study aims to address this gap by assessing HCV exposure and active infection using both serological and molecular methods in PLWH receiving ART but with unsuppressed HIV viral load.

Material and method

A cross-sectional study was conducted between June 2022 and February 2023 among 900 people living with HIV (PLWH) with unsuppressed viral load in Ethiopia. Unsuppressed HIV viral load was defined as HIV RNA > 1000 copies/mL, in accordance with World Health Organization and Ethiopian national HIV treatment guidelines. All participants had been on antiretroviral therapy (ART) for at least nine months prior to viral load testing, ensuring sufficient time to achieve viral suppression. Unsuppressed status was determined based on the most recent routine viral load measurement.

Study participants were identified through the national viral load testing program. Plasma specimens were obtained from 14 viral load (VL) testing laboratories that collectively serve approximately 82% of the national ART population across multiple regions of Ethiopia. Specimen allocation from each laboratory was performed proportionally to the number of unsuppressed viral load tests reported, as recorded in the national VL/EID database maintained by the Ethiopian Public Health Institute (EPHI). Eligible specimens were selected randomly using a lottery method in Microsoft Excel from unsuppressed routine VL testing samples. The inclusion criterions were PLWHI, at least on ART for 9 months, have unsuppressed VL, at with 500Ul remnant samples, and complete data for Age, Sex, unique ART number, and drug regimen. Individuals with incomplete demographic or clinical data (age, sex, viral load result, ART regimen, or ART identification number) or insufficient sample volume were excluded from the study.

ART regimen information and HIV viral load results were extracted from the VL/EID database and linked using unique ART identification numbers. No individual-level behavioral, socioeconomic, or HIV transmission risk data were available, as the study relied on routine laboratory and programmatic data.

HCV testing followed a two-step diagnostic approach. Plasma samples were initially screened for anti-HCV antibodies using an enzyme-linked immunosorbent assay (ELISA). HCV antibody–positive samples were subsequently tested for HCV RNA using the Abbott RealTime HCV assay on the Abbott m2000sp/m2000rt platform. Nucleic acid extraction was performed automatically by the m2000sp system, with co-amplification of an internal control to ensure assay validity. Quantitative real-time reverse transcription PCR was conducted on the m2000rt system, where fluorescence detection cycles correlate with the initial HCV RNA concentration [10].

Data were analyzed using Statistical Package for the Social Sciences (SPSS) version 26. Descriptive statistics were used to summarize sex, viral load group, Region, age groups, and ART regimen. Means with standard deviations and proportions were reported as appropriate.

Bivariate logistics regression analysis was performed to assess the association between potential risk factors and HIV and HCV co-infections. Variables with P- value < 0.2 in the bivariate analysis were entered into the multivariable logistics regression model to control for potential confounders. Adjusted odds ratios (AORs) with 95% confidence intervals (CIs) were reported to indicate the strength of association. A P-values < 0.05 was considered statistically significant.

Descriptive statistics (frequencies, means, medians, and percentages) were used to summarize participant characteristics. Associations between categorical variables and HCV seropositivity were assessed using chi-square tests and logistic regression analysis. Crude and adjusted odds ratios (COR and AOR) with 95% confidence intervals (CI) were reported. A p-value < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS version 26.

Results

A total of 900 people living with HIV (PLWH) with unsuppressed HIV viral load were included in the analysis. The study population was predominantly female (59.3%), with a mean age of 33.7 years (SD ± 13.3). Participants were enrolled through public ART clinics across multiple regions of Ethiopia.

The overall HCV seroprevalence was 5.4% (49/900; 95% CI: 4.05–7.13). HCV seropositivity was observed in 5.8% (31/534) of females and 4.9% (18/366) of males, with no statistically significant difference by sex. The highest proportion of seropositive cases occurred in the 40–49year age group, accounting for 38.8% (19/49) of all HCV antibody–positive individuals.

In multivariable logistic regression analysis, overall variation in HCV seropositivity across age groups was statistically significant (global p = 0.042). However, compared with individuals aged < 18 years (reference group), those aged 40–49 years had borderline lower adjusted odds of HCV seropositivity (AOR = 0.44; 95% CI: 0.18–1.10; p = 0.077). After adjustment for covariates, participants receiving DTG-based ART regimens showed higher odds of HCV seropositivity compared with those on non-DTG regimens (AOR = 2.20; 95% CI: 1.01–4.75; p = 0.046) (Table 1).

Table 1.

Factors associated with HCV seropositivity among PLWH on ART with unsuppressed viral load in Ethiopia (N = 900)

Variable	Category	HCV serology		COR (95% CI)	P value	AOR (95% CI)	P value
		Positive N (%)	Negative N (%)
Sex	Female	31(5.8%)	503(94.2%)	0.84(0.46–1.52)	0.565
	Male	18(4.9%)	348(95.1%)	1
HIV Viral load result	Lower (< 10,000)	18 (4.4.%)	391(95.6%)	1
	Medium (10,000–100,000)	23(6.2%)	350(93.8%)	1.261(0.510–3.115)	0.615
	Higher (> 100,000)	8(6.8%)	110(93.2%)	0.708(0.324,1.545)	0.385
Drug regimens	DTG	40(5%)	767(95%)	0.487(0.228–1.038)	0.062	1
	NDTG	9(9.7%)	84(90.3%)	1		2.20(1.01–4.75)	0.046
Age Groups	< 18	7(5%)	133(95%)	1		1	0.042
	19–29	10(5.3%)	180(94.7%)	0.947(0.35–2.55)	0.915	0.85(0.31–2.32)	0.754
	30–39	10(3.7%)	255(96.3%)	1.3(0.500–3.61)	0.560	1.25(0.46–3.39)	0.660
	40–49	19(9.7%)	176(90.3%)	0.49(0.199–1.194)	0.116	0.44(0.18–1.10)	0.077
	> 49	3(2.7%)	107(97.3%)	1.877(0.474–7.434)	0.370	1.71(0.43–6.80)	0.449

COR: Crude Odds Ratio; AOR: Adjusted odds Ratio CI: Confidence Interval: *= statistically significant (p-value < 0.05)

Among the 49 individuals who tested positive for anti-HCV antibodies, 14.3% (7/49) were confirmed to be HCV RNA positive, corresponding to an overall active HCV infection prevalence of 0.8% (7/900). HCV RNA positivity was observed more frequently among males (57.1%) than females (42.9%) and was most commonly detected in individuals aged 40–49 years. Due to the small number of RNA-positive cases, analyses of factors associated with HCV viremia were descriptive, and no statistically significant associations were observed (Table 2). The low proportion of RNA positivity among seropositive individuals suggests that the majority of HCV antibody positivity reflects past or resolved infection rather than ongoing viral replication.

Among the 49 ELISA-positive samples, 14.3% (7/49) were confirmed HCV RNA positive, indicating active infection (0.8% overall). Viremia was more frequent in males (57.1%) than females (42.9%) and most common in the 40–49-year group. The 14.3% RNA positivity rate among antibody-positive samples strongly indicates that seropositivity largely reflects past, re solved infections rather than ongoing viral replication (Table 2).

Table 2.

HCV RNA prevalence among ELISA-positive PLWH samples on ART with unsuppressed viral load in Ethiopia (N = 900)

Variable	Category	HCV RNA		Chi-square test	P value
		Positive N (%)	Negative N (%)
Sex	Female	3(0.6%)	531(99.4%)	0.794	0.373
	Male	4(1.1%)	362(98.9%)
Viral load result group	< 10,000	2(0.5%)	407(99.5%)	2.245	0.523
	10,000–100,000	3(0.8%)	370(99.2%)
	> 100,000	2(1.7%)	116(98.3%)
Drug regimens	DTG	7(0.9%)	800(99.1%)	0.813	0.367
	NDTG	0(0.0%)	93(100%)
Age Group	< 18	0(0.0%)	140(100%)	3.321	0.506
	19–29	2(1. %)	188(99. 0%)
	30–39	1(0.4%)	264(99.6%)
	40–49	3(1.5%)	192(98.5%)
	> 49	1(0.9%)	109(99.1%)
Regions	AddisAbaba	4(1.7%)	235(98.3%)	7.518	0.377
	Somali	0(0.0%)	9(100%)
	Oromia	1(0.3%)	292(99.7%)
	Amhara	0(0.0%)	248(100%)
	SNNP	2(2.1%)	93(97.9%)
	Harari	0(0.0%)	12(100%)
	Benishangul	0(0.0%)	3(100%)
	Afar	0(0.0%)	1(100%)

The χ2 and p-values in this table were calculated based on HCV RNA-positive cases, *= statistically significant (p-value < 0.05)

Discussion

Viral hepatitis remains a major public health concern, particularly among people living with HIV (PLWH), where hepatitis C virus (HCV) co-infection substantially contributes to liver-related morbidity and mortality. HCV infection among PLWH accelerates progression to advanced liver disease, including cirrhosis and hepatocellular carcinoma, and increases non-AIDS-related mortality [11]. In this study, which applied both serological and molecular testing among 900 PLWH with unsuppressed HIV viral load in Ethiopia, the overall HCV seroprevalence was 5.4%.

This prevalence is comparable to findings reported from Dessie (5.2%), northern Ethiopia (6.6%), Nigeria (4.4%), and Rwanda (5.7%) [5, 12–15]. However, it is higher than reports from Western Ethiopia (0.23%), Afar (1.5%), and Addis Ababa (0.9–1.1%) [16–20]. Conversely, the prevalence observed in this study is lower than that reported from Europe (12.3%), Mekelle (8.6%), and Hawassa (10.5%) [21–23]. These variations likely reflect differences in study populations, underlying HCV endemicity, exposure risk profiles, diagnostic methods, and temporal factors across settings.

Although a higher proportion of HCV antibody–positive cases was observed among females and individuals aged 40–49 years, these differences were not statistically significant and should therefore be interpreted descriptively rather than as evidence of true demographic disparities. The higher concentration of seropositive cases in older age groups likely reflects cumulative lifetime exposure to HCV rather than ongoing transmission.

A key finding of this study is that only 14.3% (7/49) of HCV antibody–positive individuals had detectable HCV RNA, corresponding to an overall active HCV infection prevalence of 0.8%. This low proportion of viremia suggests that most HCV seropositivity in this population reflects past or resolved infection, consistent with spontaneous viral clearance reported in other studies [18]. Alternative explanations, including false-positive serological results or very low-level viremia below the assay detection threshold, cannot be excluded but are less likely given the use of a sensitive molecular platform.

Sometimes, HCV antibody tests can give false-positive results. This is more common in people living with HIV, especially when their viral load is high. The reason is that HIV can cause the body to produce many non-specific antibodies (a condition called hypergammaglobulinemia), which may confuse the test. In these cases, the ELISA test may react to other antibodies, leading to a positive antibody result even though the person does not actually have detectable HCV RNA [24].

All individuals with detectable HCV RNA had HIV viral loads exceeding 1,000 copies/mL and had been on antiretroviral therapy for more than nine months. While the small number of RNA-positive cases limits causal inference, this observation supports the biological plausibility that uncontrolled HIV replication may impair immune-mediated clearance of HCV and highlights the vulnerability of this subgroup.

Importantly, these findings emphasize the necessity of confirmatory molecular testing following HCV antibody screening to avoid overestimation of active infection based solely on serology [19]. Integrating routine HCV antibody screening with RNA confirmation into HIV care services particularly for PLWH with unsuppressed viral load may improve early detection of active co-infection and support national and global hepatitis elimination efforts.

Conclusion and recommendation

A subset of people living with HIV with unsuppressed viral load showed evidence of HCV exposure, and a smaller proportion had active HCV infection confirmed by RNA testing. Although the prevalence of active co-infection was low, these findings underscore the importance of routine HCV antibody screening with confirmatory molecular testing among PLHIV, particularly those with unsuppressed HIV viral load. Strengthening the integration of HIV and viral hepatitis services within ART programs may improve early identification and clinical management of HIV/HCV co-infection and contribute to improved patient outcomes.

Strength and limitation of the study

This study has several notable strengths: samples were obtained from across the entire country, the sample size is robust, and both serological and nucleic acid tests were employed to confirm the participants’ positive status. Since the study utilized samples originally collected for HIV drug resistance testing, there are certain limitations. These include the absence of individual-level socioeconomic data and comprehensive clinical histories, such as information on HCV transmission routes, HIV acquisition methods, time since diagnosis, precise ART duration, and previous HCV treatment history. These limitations suggesting that the ability to control for confounding factors related to socioeconomic status is limited. This also make difficult in interpret findings in the context of treatment-experienced versus treatment-naïve individuals. Thus, results should be interpreted with caution.

Author contributions

DA was the principal investigator who participated in the conceptualization of the study, drafted the manuscript, performed the data analysis, and participated in data collection and laboratory tests; KD, RD and JF were advised throughout the process and participated in editing and reviewing the manuscript; GG, AG, GG, TS, DL, HM and GT were participated in data collection, data analysis and interpretation. Each author reviewed and gave their approval to the final draft.

Funding

Financial support for the research was granted to the author(s) by Addis Ababa University, Destin project and Ethiopian public health institute.

Data availability

In order to maintain the confidentiality of participants, not all data will be provided. The datasets that were utilized and/or analyzed in the current study are accessible from the corresponding author upon a reasonable request via email **: destaayana185@gmail.com**.

Declarations

Ethics approval and consent to participate

Ethical clearance was obtained from the Department Research and Ethical Review Committee (DRERC) of Addis Ababa University. The study qualified as routine program monitoring; thus, a waiver of consent was granted by the EPHI IRBs. This was based on the fact that the study used remnant, stored samples from a large patient pool, making retrospective consent impractical. HCV test results were returned to patient clinics via ART numbers for clinical follow-up.

Consent for publication

All authors confirm that we have seen and approved the final version of this manuscript. We affirm that this work is original and has not been published elsewhere, nor is it under consideration for publication by any other journal. we also confirm that all co-authors (if any) have agreed to its content and submission for publication. We understand that the journal may reproduce this work in all forms and media.

Competing interests

The authors declare no competing interests.