Introduction

Viral hepatitis, particularly that caused by hepatitis B (HBV) and hepatitis C (HCV) viruses, represents a life-threatening liver infection and, despite the progress made in recent decades, continues to be a major global health problem.1 The World Health Organization identifies these viruses as the leading causes of liver cirrhosis and hepatocellular carcinoma worldwide. Consequently, the Global Health Sector Strategy on Viral Hepatitis 2016–2021 was introduced in 2016 as part of efforts to eliminate viral hepatitis as a public health threat by 2030.2 The strategy outlines key actions, including vaccination, diagnostic testing, treatment, and national awareness campaigns.

Paraguay, a landlocked South American country bordering Brazil, Bolivia, and Argentina. It has a population of approximately 6.4 million and is administratively divided into the capital district of Asunción and 17 departments. Geographically, the country is characterized by two distinct regions separated by the Paraguay River, each with unique epidemiological profiles. The Eastern region is the most densely populated, home to 97.3% of the population, while the Western region (Chaco) accounts for just 2.7%, and features vast plains and lower population density. 3,4

In 2019, national health authorities approved the National Strategic Plan for the Response to HIV/AIDS and Other Sexually Transmitted Infections (STIs) 2019–2023. This plan adopts a comprehensive approach to key issues and guides the health sector’s response through an integrated, multi-sectoral strategy. Through cross-sector collaboration, it aims to build an effective, integrated, and well-structured public health framework.5.

The hepatitis B vaccine is currently administered to newborns within 24 h of birth, followed by additional doses at 2, 4, and 6 months as part of the pentavalent vaccine. While the 2-, 4-, and 6 month doses were incorporated into the vaccination schedule in 2003, the birth dose was introduced more recently in 2017 6. By 2023, coverage for the newborn dose had reached 96%, and 89% of infants had received all three doses by six months of age.7.

Assessing the prevalence and distribution of HBV and HCV infections is essential for planning preventive measures and, in the case of HBV, for monitoring vaccination programs.8 While sampling the general population is the most accurate method for determining seroprevalence, the typically low prevalence of these infections makes large-scale cohort studies impractical. Although studies on blood donors tend to underestimate seroprevalence in the general population, they remain the most widely used approach, offering valuable insights into long-term trends and the impact of health policies.

Furthermore, altruistic repeat donors in Paraguay are the exception, accounting for only about 14%, with most blood donations coming from first-time donors.9 This should be taken into account, since the prevalence of reactive serological tests for infectious diseases is usually higher among first-time donors than among recurrent volunteers, who undergo periodic testing.10.

Despite being notifiable infections, the burden of HBV and HCV in Paraguay remains poorly understood due to the absence of population-based studies. Consequently, available data are scarce, fragmented, and often outdated, primarily derived from studies on specific groups rather than the general population.

This study therefore aimed to establish a comprehensive and nationally representative epidemiological baseline of hepatitis B virus and hepatitis C virus seroprevalence among blood donors in Paraguay, thereby providing valuable insights into the prevalence and distribution of these infections.

Methods

Population

A prospective, cross-sectional study was carried out from 1 June 2024 to 31 July 2025, on the serological markers of HBV, and HCV. Serum samples from 85,811 blood donors who attended blood banks across the country’s 17 departments and the capital district of Asunción were analyzed. Donors completed a routine questionnaire to identify potential risks that could affect the safety and quality of donated blood and underwent a physical examination conducted by trained physicians. These assessments included measuring blood pressure, body temperature, pulse and respiratory rate.

Eligible participants were donors aged between 18 and 65 years, weighed at least 50 kg, and appeared to be in good general health, as established by the national regulatory framework. Pre-donation screening and epidemiological exclusion criteria were implemented in accordance with the standardized protocols of Resolution No. 563/2008. In compliance with these regulations, individuals under 18 years of age required parental consent to donate, while those over 65 were exceptionally allowed to donate if they were repeat donors. Data collected included only age, sex, and donation location to ensure anonymity. After obtaining consent, each donor completed the form and had a blood sample drawn in a plain (non-anticoagulated) tube for serological testing. The cohort was categorized into the following age groups: individuals under 20 years, 5-year intervals for those aged 20 to 50, and individuals over 50.

All analyses were performed using the number of repeatedly reactive tests, according to the flow chart determined by the Ministry of Health11(Fig. 1).

Fig. 1
Fig. 1
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Screening and validation algorithm for blood donation. Blood samples undergo initial screening; non-reactive results allow donor acceptance. Reactive samples are retested in duplicate. If both retests are non-reactive, the donation is accepted. Conversely, if either retest is reactive, the donation is discarded, and the donor is further evaluated with a second sample. Persistent reactivity leads to definitive donor exclusion, while a non-reactive second sample allows for donor acceptance, ensuring rigorous safety and screening accuracy.

Serological assays

Hepatitis B virus core antibody (anti-HBc), HBV surface antigen (HBsAg), and hepatitis C antibody (anti-HCV) testing was performed on the ARCHITECT i2000SR fully automated chemiluminescent microparticle immunoassay platform (Abbott Diagnostics, Abbott Park, IL), according to the manufacturer’s instructions. In particular, HBsAg was detected using the ARCHITECT HBsAg Qualitative II assay; per the manufacturer’s specifications, this assay demonstrates a diagnostic sensitivity and specificity exceeding 99.5%.

Nucleic acid amplification test (NAT) assays

All 85,811 samples were screened for HBV and HCV nucleic acids using Individual Donor Nucleic Acid Testing (ID-NAT). Testing was conducted on the fully automated Roche Cobas 6800 system using a multiplex polymerase chain reaction kit (Cobas MPX test), following the manufacturer’s instructions.

Statistical analysis

Data were compiled and analyzed using IBM SPSS Statistics version 23.0 for iOS (IBM Corp., Armonk, NY, USA). Categorical variables were expressed as frequencies and percentages. Comparisons between qualitative variables were performed using the Chi-square test or Fisher’s exact test, as appropriate. To assess the independent associations between demographic and geographic variables and serological markers of viral hepatitis, we conducted multivariable logistic regression analyses. Separate models were specified for anti-HBc, HBsAg, and anti-HCV as dependent outcomes. Age (continuous), sex (binary), and department of residence (categorical, with Asunción as the reference category) were included as explanatory variables. Adjusted odds ratios (ORs) with 95% confidence intervals (CIs) were estimated for each covariate. Forest plots were generated to facilitate visualization and comparative assessment of effect sizes across predictors. Model fit and assumptions were evaluated using standard diagnostic procedures. Moreover, age-standardized seroprevalence was calculated using the direct standardization method. Age-specific seroprevalence rates were first estimated for each defined age group within the study cohort. These rates were then weighted according to the age distribution of the general population, based on the 2022 Paraguayan Census, which served as the reference standard.3 Prevalence estimates were reported with their corresponding 95% confidence intervals (CI) to assess statistical precision. Ap value below 0.05 was considered statistically significant.

Results

The analyzed cohort included 85,811 blood donors (1.33% of the country’s population) from the 17 departments and Asunción. Mean age was 35.3 ± 10.6 years (range: 16.6–75.3; IQR1: 29.6 -IQR3: 42.4) and 33,171 (38.7%) were women, (Table 1).

Table 1 Reactive rates of HBV and HCV markers by sex and age (n = 85,811). IQR: Inter Quartile Range. * Significant differences between sex were observed in the frequency of HBsAg positivity (p < 0.001) and anti-HBc positivity (p = 0.003). Furthermore, significant differences were also observed by age group in the frequency of anti-HBc positivity (p < 0.001) and HBsAg positivity (p:0.006).
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Seroprevalence of HBV markers

The overall age-standardized prevalence of anti-HBc was 1.66% (1,159 out of 85,811; CI95%: 1.27–1.42%), ranging among the different departments from 0.70 to 3.86% (Fig. 2). Due to the limited number of samples from the Alto Paraguay and Boquerón departments, these were excluded from further analyses. A significant progressive increase in anti-HBc seroprevalence was observed, correlating strongly with donor age. This rate ranged from 0.26% in the < 20 age group to 3.02% in the over- 50 age group, p < 0.001 (Table 1). Furthermore, the prevalence was significantly lower in women than in men (1.06% vs. 1.53%, p < 0.001).

Fig. 2
Fig. 2
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Source Base map from Vecteezy (https://www.vecteezy.com), edited with Adobe Illustrator v26.0 (https://www.adobe.com/illustrator).

Seroprevalence of anti-HBc, HBsAg, and anti-HCV across Departments. Departments are color-coded based on seroprevalence relative to the interquartile range (IQR 3): Green (≤ IQR 3), yellow (within IQR 3), and red (> IQR 3). AMB: Amambay, APY Alto Paraguay, APA: Alto Paraná, ASU: Distrito Capital, BOQ: Boquerón, CAG: Caaguazú, CAZ: Caazapá, CAN: Canindeyú, CEN: Central, CON: Concepción, COR: Cordillera, GUA: Guairá, ITA: Itapúa, MIS: Misiones, NEE: Ñeembucú, PAR: Paraguarí, PHA: Presidente Hayes, SNP: San Pedro.

The age-standardized prevalence of HBsAg was 0.079% (74 out of 85,811; CI95%: 0.067–0.108%), distributed unevenly across the country’s different departments, from 0.0 to 0.38% positivity (Table 2) and did not strictly follow the anti-HBc rates in the different departments. Particularly, the seroprevalence of HBsAg in the 35–40 age group (0.16%) was significantly higher than the national average (p = 0.001). Conversely, donors under the age of 25 exhibited notably lower rates (p = 0.001) (Table 1). Similarly, seroprevalence was substantially lower in women compared to men (0.048% vs. 0.110%, p = 0.003).

Table 2 Reactive rates of anti-HBc, HBsAg and anti-HCV by departments (n = 85,811). Department data was available on 81,820 blood donors. * Due to the limited number of samples from the Alto Paraguay and Boquerón departments, they were excluded from the analyses. n/a: Department not available.
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Among the 1,172 individuals who tested reactive for at least one HBV serological marker (anti-HBc, HBsAg, or HBV-DNA), five distinct patterns were identified (Table 3). The most common pattern indicated past infections, characterized by reactive anti-HBc only (n: 1,066). Less frequently, ongoing infections, with all markers reactive (n:67) and occult B infections (OBI) -reactive anti-HBc with detectable HBV-DNA- were observed (n:24). Interestingly, eight cases presented with detectable HBV-DNA in the absence of HBsAg, while five exhibited both HBsAg and HBV-DNA, suggesting recent infections (Table 3).

Table 3 Hepatitis B Virus serological marker patterns. Among the 1,248 individuals who tested positive for at least one HBV serological marker (anti-HBc, HBsAg, or HBV-DNA), seven distinct patterns were identified. OBI: Occult hepatitis B infection, NR: No Reactive.
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The impact of the national vaccine schedule’s implementation on HBV marker seroprevalence was also assessed. The study cohort comprised 7,412 individuals (8.6%) born after the vaccine rollout and 78,399 (91.4%) born before its implementation. Anti-HBc seroprevalence was notably lower in the post-implementation birth group compared to the pre-implementation group (0.27% vs. 1.45%, p < 0.001). Furthermore, no HBsAg- reactive samples were identified in the post-implementation group, in contrast to the 74 reactive donor samples (0.094%) observed among those born prior to vaccine introduction (p = 0.008).

Anti-HCV seroprevalence

The nationwide age-standardized prevalence of anti-HCV was 0.261% (229 out of 85,811; CI95%: 0.205–0.304%). Seroprevalence varied across departments, ranging from 0.09% in Amambay to 0.46% in Concepción (Table 2). Importantly, no significant differences in anti-HCV seroprevalence were observed across age groups or sex. Among the 229 individuals who tested reactive for anti-HCV, only one was HCV-RNA detectable. Furthermore, four cases consistent with the viral window period were identified, characterized solely by the presence of HCV-RNA.

Finally, we performed a multivariate regression analysis. Across the three logistic regression models, distinct patterns of association were observed for anti-HBc, HBsAg, and anti-HCV. Age showed a positive association with anti-HBc, but no significant effect on HBsAg or anti-HCV. Likewise, sex was independently associated only with anti-HBc, with males presenting higher odds of positivity, while no significant differences were detected for the other markers. Geographic variation was most evident for anti-HBc, with several departments exhibiting significantly higher or lower odds compared to the reference category (Asunción). In contrast, only one department was associated with HBsAg positivity, and none reached statistical significance for anti-HCV (Fig. 3). Due to the limited number of samples from the Alto Paraguay and Boquerón departments, these were excluded from the multivariable analyses. In addition, donors without information on their department of origin were removed from the analysis.

Fig. 3
Fig. 3Fig. 3Fig. 3
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Forest plot of multivariable logistic regression analysis for factors associated with anti-HBc (a), HBsAg (b), and anti-HCV (c). Statistically significant variables are displayed with red diamonds and bold lettering, whereas non-significant variables are shown with grey diamonds. Image generated using R version 4.3.2 (R Core Team, 2023, https://www.R-project.org/).

Discussion

Seropositivity rates in blood donors are key indicators of blood safety and the effectiveness of donor selection and screening protocols. Highly sensitive diagnostic tests have revolutionized blood safety, minimizing the risk of parenterally transmitted infections like HBV and HCV12. However, maintaining and improving this security requires continuous monitoring and adaptation to territorial variations.

To our knowledge, this study represents the most comprehensive assessment of HBV and HCV prevalence among Paraguayan blood donors to date, offering crucial insights into the current epidemiological landscape. However, notable disparities across age groups, sex and geographic regions highlight the need for targeted interventions and region-specific awareness campaigns to effectively address the disease burden.

The limited availability of detailed epidemiological data from population-based studies on HBV and HCV in Paraguay has hindered public health planning. Previous reports indicate that HBsAg prevalence ranges from 0.4% among women of reproductive age to 4.5% in clinically suspected hepatitis cases,13,14 underscore the critical need for more representative and robust data. Furthermore, a recent investigation involving 278 blood donors, primarily from the Central Department and Asunción, reported HBV and HCV marker rates consistent with our findings.15 In response to this critical gap, our national-scale cohort study provides a significantly more comprehensive understanding of HBV and HCV seroprevalence. Additionally, the absence of HBV infection markers among donors vaccinated through Paraguay’s childhood immunization programs, launched in 2003, offers evidence of their effectiveness.

Another finding of our study is that 76 out of 150 HBsAg-reactive samples showed no reactivity for anti-HBc or HBV DNA. In low-prevalence cohorts, such as blood donors, this result is consistent with false reactivity. This issue has been documented in other studies and represents a challenge that cannot be ignored.16 Similarly, HBV DNA was detected in 24 of the 1,090 donors (2.2%) who were anti-HBc reactive but HBsAg negative, indicating occult HBV infection.17 The identification of occult hepatitis B infection (OBI) is a critical concern for blood banks, as the absence of detectable HBsAg may conceal a transmissible viral load. This underscores the necessity of implementing advanced nucleic acid testing (NAT) and anti-HBc screening protocols to safeguard against post-transfusion hepatitis. While reported OBI rates in blood donors vary widely18-largely influenced by the overall prevalence of the infection in the population- several studies have reported findings similar to those presented here.19,20.

In addition, the decreasing prevalence of anti-HBc across age groups, from older to younger individuals, indicates a decline in HBV circulation over time. This trend has been widely documented in regions where preventive public health measures have been effectively implemented to control the infection.21,22,23.

In a regional context, Paraguay’s epidemiological profile aligns with a trend of intermediate-to-low endemicity observed across Latin America, where countries like Brazil (1.25%) and Mexico (0.21%) show varying HBsAg rates, yet al.l face the common regional challenge of managing a high prevalence of resolved or occult infections among first-time and ineligible donors24,25.

The anti-HCV seroprevalence rates observed in our study are substantially lower than those reported in neighboring countries. 26,27 Notably, only two other studies -one conducted in indigenous communities in the eastern Paraguayan Chaco in 1998 and another among individuals attending the Institute of Health Sciences Research in 2009- reported significantly higher anti-HCV prevalence (14.9% and 6.6%, respectively). 14,28 Interestingly, in contrast to several studies where older individuals usually have significantly higher seroprevalence rates than younger individuals,29,30 this cohort showed no age-related differences in anti-HCV rates. Furthermore, unlike what was observed for HBV, no differences in anti-HCV seroprevalence were found between men and women. This contrasts with findings from other regions, where men, who are more likely to engage in high-risk behaviors such as intravenous drug use, exhibit higher rates.31,32 These results suggest that the impact of risk behavior and exposure factors varies across different contexts.

HCV-RNA was detected solely in one of the 229 anti-HCV reactive samples (0.44%). Although it is well established that 20% to 50% of HCV infections resolve spontaneously, which may partly account for this finding, 33 the high sensitivity of screening assays can sometimes compromise specificity. This limitation is particularly relevant for anti-HCV tests, which have been extensively scrutinized.34,35 The challenge becomes even more pronounced in populations with low infection prevalence, such as blood donors, where a low signal-to-cutoff (S/Co) ratio further complicates interpretation.34.

Globally, most new cases of HCV infection are currently restricted to transmission among people who inject drugs.36 In Paraguay, the prevalence of injecting drug use is very low, and, importantly, even within this small population of people who inject drugs, the rate of anti HCV positivity is among the lowest reported in Latin America36. This combination of limited exposure to parenteral risk behaviors and low HCV seroprevalence among the few existing people who inject drugs provides a plausible explanation for the comparatively low burden of HCV infection observed in our donor population. These findings support the notion that differences in risk behaviors, particularly the rarity of injection drug use, are central to understanding Paraguay’s distinct epidemiological profile.

The implementation of universal nucleic acid testing (NAT) within the Paraguayan public blood bank system, completed in 2024, represents a landmark shift in the national strategy to ensure blood safety.37 By identifying potential HBV and HCV cases within the early “window period”—infections that traditional serology might have missed—this study underscores that while Paraguay maintains a lower seroprevalence than many of its neighbors, the risk of transfusion-transmitted infections remains a persistent threat.

From a public health perspective, these findings provide the robust, nationwide data required to move beyond generalized screening toward the identification of “epidemiological hotspots”. By leveraging this information to refine targeted interventions, Paraguay can more effectively align its national health policies with the WHO’s 2030 goal of eliminating viral hepatitis.

Some limitations of this study warrant consideration. First, the lack of confirmatory testing, using neutralization assays, 38 to validate the HBsAg reactive and non-reactive samples for anti-HBc and HBV-DNA, specifically to rule out false positives. Additionally, because the NAT assay employed is qualitative, viral load data for HBV-DNA and HCV-RNA reactive cases were unavailable. Regarding demographic constraints, the data were limited to age and gender; consequently, key variables associated with seropositivity, such as education level, housing conditions, and socioeconomic status, could not be evaluated. Moreover, while blood donors represent a select, typically healthier and younger demographic that does not fully mirror the general population, their data remains vital. These results provide essential estimates for the prevalence of transfusion-transmissible infections and serve as a primary indicator of broader epidemiological trends. Although infection rates among donors are generally lower due to rigorous selection criteria, they reveal specific risk profiles that are instrumental in refining epidemiological models.

In conclusion, HBV and HCV seroprevalence among Paraguayan blood donors remains low. However, achieving the WHO’s goal of eliminating viral hepatitis by 2030 requires extensive large-scale studies and targeted research on high-risk groups. As the WHO emphasizes, robust strategic data is essential for guiding effective public health interventions. In this context, the strength of this study lies in the fact that it was based on a nationwide representative cohort of over 85,000 donors, encompassing data from all departments across the country. Identifying “epidemiological hotspots” enables the efficient allocation of resources and enhances health policy development. Furthermore, understanding HBV and HCV infections across regions and age groups is keystone for refining public health strategies, optimizing prevention efforts, and addressing disparities in disease burden.