Implementation of quantitative HBsAg testing in clinical microbiology laboratories in Spain: Results from a national GEHEP-SEIMC survey

Abstract

Introduction

Quantitative hepatitis B surface antigen (qHBsAg) is increasingly recognized as a key biomarker for chronic hepatitis B (CHB) management. This study aimed to assess the adoption, practices, and barriers to qHBsAg testing in Spanish clinical microbiology laboratories.

Methods

A cross-sectional survey was distributed through the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) Viral Hepatitis Study Group (GEHEP). Clinical microbiology laboratory professionals across Spain were invited to complete an anonymous online questionnaire. The survey collected information on qHBsAg implementation, technical practices, barriers, and patient-related data. Descriptive statistics were applied.

Results

Of 118 laboratories invited, 91 responded (77.1%); only 37.4% performed qHBsAg. Among non-performing centers, 86% did not refer samples, mainly due to perceived low clinical demand (57.1%), plus lack of equipment/resources. Implementation correlated with hospital size (p = 0.041): 0% (<200 beds), 23.3% (200–500), 44.9% (500–1000), 62.5% (≥1000). Among performing labs, 57.6% quantified after a positive qualitative test, 27.3% on request, and 15.2% as initial screening. Proactive reporting was 69.7%, and 81.8% diluted samples above limits. In the last year, 40.7% of results were <100 IU/mL and 27.1% >3000 IU/mL; over five years (≈14,500 tests), 29.5% >3000 IU/mL and 27.3% <100 IU/mL, supporting the clinical relevance of these thresholds.

Conclusions

qHBsAg testing is not uniformly implemented across Spanish clinical microbiology laboratories and workflows and reporting practices are heterogeneous. Given the descriptive design and potential selection bias, these results should be interpreted as a baseline assessment to inform harmonization efforts and future evaluations linking testing availability to clinical decision-making.

Resumen

Introducción

El antígeno de superficie de la hepatitis B cuantitativo (qHBsAg) es un biomarcador clave en el manejo de la hepatitis B crónica (HBC). Este estudio evaluó su implantación, prácticas y barreras en laboratorios de Microbiología Clínica en España.

Métodos

Se realizó una encuesta transversal distribuida por el Grupo de Estudio de Hepatitis Víricas (GEHEP) de la SEIMC. Profesionales de laboratorios de España completaron un cuestionario online sobre implantación del qHBsAg, prácticas técnicas, barreras y datos relacionados con los pacientes. Se realizó análisis descriptivo.

Resultados

De 118 laboratorios, respondieron 91 (77,1%); el 37,4% realizaba qHBsAg. Entre los que no lo realizaban, el 86% no derivaba muestras. Las principales barreras para su implementación fueron la baja demanda percibida (57,1%) y falta de recursos. La implantación se asoció al tamaño hospitalario (p = 0,041): 0% (<200 camas), 23,3% (200–500), 44,9% (500–1000) y 62,5% (≥1000). En los centros que lo realizaban, el 57,6% cuantificaba tras un resultado cualitativo positivo, el 27,3% a petición clínica y el 15,2% como cribado inicial. El 69,7% emitía informes proactivos y el 81,8% diluía muestras fuera de rango. En el último año, el 40,7% de los resultados fueron < 100 UI/mL y el 27,1% >3000 UI/mL; en cinco años (≈14.500 determinaciones), el 29,5% y el 27,3%, respectivamente.

Conclusiones

La determinación de qHBsAg no está uniformemente implantada y existen diferencias en prácticas y en la emisión de resultados. Estos datos constituyen una eva evaluación inicial para orientar la armonización y futuros estudios sobre su impacto en la toma de decisiones clínicas.

Introduction

Chronic hepatitis B virus (HBV) infection remains a major global health problem, responsible for substantial morbidity and mortality due to cirrhosis and hepatocellular carcinoma (HCC), despite the availability of effective vaccines and potent antiviral therapies.^1,2 Loss of hepatitis B surface antigen (HBsAg) is widely used as a surrogate endpoint for functional cure in chronic HBV infection. However, HBsAg loss does not necessarily indicate sterilizing cure, as low-level viral persistence (including occult infection) may remain,³ and interpretation can be influenced by the broader serologic context and assay-related limitations.

Traditionally, monitoring of HBV infection has relied on clinical, biochemical, and virological markers such as HBV DNA and HBeAg/anti-HBe serology.^4,5 However, these parameters have limitations in predicting disease progression, treatment response, or the safety of therapy discontinuation.⁶ In this context, quantitative HBsAg (qHBsAg) has emerged as a clinically valuable biomarker.⁷ Serum qHBsAg reflects, to varying degrees, transcriptional activity from intrahepatic cccDNA as well as expression from integrated HBV DNA, and therefore should be interpreted as an indirect—rather than exclusive—marker of viral transcriptional activity. Moreover, the relevance of qHBsAg is expected to further increase with the potential arrival of novel therapeutic agents aiming to achieve functional cure, where reliable biomarkers will be essential for treatment monitoring and decision-making.^9,10

The most recent European Association for the Study of the Liver (EASL) Clinical Practice Guidelines (2025) explicitly recommend annual quantification of HBsAg in HBsAg-positive patients, both in HBeAg-positive and HBeAg-negative phases, as part of the standard monitoring strategy.¹¹ Multiple studies have also highlighted its predictive role in clinical and therapeutic outcomes, with greater applicability than emerging biomarkers such as HBV RNA or HBcrAg, which remain limited by lack of standardization and clinical availability.^8,12

In Spain, healthcare delivery and laboratory organization are regionally structured, which may contribute to heterogeneity in access to specialized biomarkers and variability in diagnostic workflows. Establishing a national baseline on qHBsAg availability, implementation models, and barriers is therefore necessary to identify inequities, inform harmonization strategies, and align routine laboratory practice with international recommendations and evolving therapeutic needs.

To our knowledge, no prior nationwide survey has characterized how qHBsAg is implemented, requested, and reported across Spanish clinical microbiology laboratories. This limits the ability to identify gaps between guideline recommendations and real-world practice. Within this framework, the Spanish Group for the Study of Viral Hepatitis (GEHEP-SEIMC) has conducted a nationwide survey to assess the level of awareness, use, and barriers to the implementation of qHBsAg in Spanish clinical microbiology laboratories. The primary objective was to estimate the proportion of Spanish clinical microbiology laboratories implementing qHBsAg testing. Secondary objectives were to describe current testing workflows (reflex vs on-request vs screening), reporting practices, and perceived barriers to implementation. An exploratory objective was to assess associations between implementation and structural characteristics such as hospital size and geographic region.

Materials and methods

We conducted a cross-sectional, descriptive survey study aimed at evaluating the degree of implementation and awareness of quantitative hepatitis B surface antigen (qHBsAg) testing in clinical microbiology services across Spain.

Eligible participants were professionals from clinical microbiology laboratories in Spanish hospitals. All hospitals were eligible regardless of whether they performed qHBsAg testing, in order to capture a comprehensive overview of current practice. Participation was voluntary and anonymous. To ensure confidentiality, personal identifiers were collected only for clarification purposes and were not used in the analysis.

A structured online questionnaire was developed by the study coordinators to assess the current status of qHBsAg testing in Spanish clinical microbiology laboratories. Invitations were sent to 118 hospital-based clinical microbiology laboratories included in the GEHEP-SEIMC distribution list at the time of the study. The online questionnaire was distributed in March 2025 and remained open until May 2025. One reminder email was sent midway through the study period to maximize participation. The survey captured implementation and use patterns, not diagnostic performance or discordance resolution, and included both closed- and open-ended questions organized into thematic sections covering implementation, testing practices, patient statistics, and respondent feedback, divided into four main sections: (a) Current status of qHBsAg testing – whether testing is performed, reasons for not performing it, referral practices; (b) Testing practices – conditions under which qHBsAg is performed, reporting practices, dilution procedures, and techniques used; (c) Patient statistics – number of HBsAg-positive patients identified over the past five years and the previous year, number of those quantified, and distribution of qHBsAg values into predefined categories (>3000 IU/mL, 1000–3000 IU/mL, 250–1000 IU/mL, 100–250 IU/mL, < 100 IU/mL); and (d) Additional comments – open-ended questions allowing suggestions on qHBsAg implementation. Respondent information (hospital, position, and contact details) was also collected but was excluded from analysis to preserve anonymity.

Responses were automatically collected through Google Forms and exported into a database for analysis. To minimize duplicate entries, hospital identification fields were used to detect multiple submissions. Only one response per hospital was considered for analysis and no duplicates were identified. Data were collected through an online structured questionnaire. The full structure and content of the survey are provided in Table 1.

Table 1.

Structure and content of the national survey on qHBsAg implementation in Spanish clinical microbiology laboratories.

Section	Content
1. Current status of qHBsAg testing	• Whether qHBsAg testing is performed (Yes/No)• Reasons for not testing (resources, demand, equipment, other)• Referral to reference lab (Yes/No)
2. Testing practices	• Conditions under which qHBsAg is performed• Proactive reporting to clinicians (Yes/No)• Dilution practices for results above quantification limit• Technique used (commercial platform)
3. Patient statistics	• Number of HBsAg+ patients in past 5 years• Number quantified in past 5 years• Number of new HBsAg+ patients in past year• Number quantified in past year• Distribution across categories (>3000, 1000–3000, 250–1000, 100–250, <100 IU/mL)
4. Additional comments	• Open-ended comments and suggestions

Structure and content of the national survey on quantitative HBsAg (qHBsAg) implementation in Spanish clinical microbiology laboratories. The questionnaire was organized into four thematic sections plus respondent information. It included both closed-ended questions (Yes/No, multiple choice, numerical counts) and open-ended items for comments and suggestions.

Categorical variables were summarized as absolute numbers and percentages. The association between hospital size and implementation of qHBsAg testing was assessed using Pearson's Chi-square test. A two-sided p value < 0.05 was considered statistically significant.

Results

A total of 91 responses were received from 118 clinical microbiology laboratories invited to participate (response rate 77.1%). Among these, only 37.4% (34/91) reported performing quantitative HBsAg (qHBsAg) testing in their center.

Of the 57 centers not performing qHBsAg, 86.0% (49/57) reported not referring samples to an external laboratory for quantification. The most frequently cited reason was lack of clinical demand (57.1%), followed by lack of equipment and resources, often in combination. A smaller proportion of laboratories mentioned lack of necessity (1.8%). Table 2 shows the barriers most frequently reported by laboratories not performing qHBsAg testing.

Table 2.

Reported barriers to qHBsAg implementation.

Reason	Centers reporting (%)
Lack of demand	57.1
Lack of equipment	5.4
Lack of resources	3.6
Lack of demand + Lack of equipment	8.9
Lack of demand + Lack of resources	5.4
Lack of demand + not considered necessary	3.6
Lack of demand + Lack of equipment + Lack of resources	5.4
Lack of resources + Lack of demand + Lack of equipment + Not considered necessary	1.8
Not considered necessary	1.8
Others	7.1

The table summarizes reasons provided by centers that do not perform qHBsAg testing. Lack of demand was the most frequent reason, followed by lack of equipment and resources.

Others refers to respondents who indicated reasons such as awaiting the availability of reagents, noting that the assay was not included among the determinations available in their laboratory, or planning to implement it in the near future.

Hospital size, measured by the number of beds, was significantly associated with qHBsAg implementation (Pearson's χ² = 8.26; df = 3; p = 0.041). No hospital with <200 beds reported performing qHBsAg, compared to 23.3% in hospitals with 200–500 beds, 44.9% in hospitals with 500–1000 beds, and 62.5% in hospitals with ≥1000 beds. Post hoc pairwise comparisons did not remain statistically significant after Bonferroni correction, likely reflecting limited subgroup sample sizes. Table 3 details the proportion of centers performing qHBsAg testing by hospital size.

Table 3.

Implementation of qHBsAg testing by hospital size. Implementation rates increase significantly with hospital size, with highest adoption observed in hospitals with ≥1000 beds.

Hospital size (beds)	n	qHBsAg; n (%)
<200	4	0
200–500	30	7 (23.3)
500–1000	49	22 (44.9)
≥1000	8	5 (62.5)

Geographical distribution of responses encompassed all autonomous communities. Rates of qHBsAg implementation varied widely, from 0% in some regions (e.g., Canary Islands, Extremadura, La Rioja) to 100% in others (Asturias, Cantabria, Navarra, Balearic Islands), but no statistically significant differences were found across regions. Low sample size in several regions limited the power of this analysis.

Among centers that performed qHBsAg, the most common practice (57.6%) was to reflex quantification after a positive qualitative HBsAg screening result. A further 27.3% performed quantification only upon specific clinician request, while 15.2% used the quantitative assay as the initial screening test. Proactive reporting of qHBsAg results – defined as the active communication of quantitative results to clinicians trough their inclusion in the laboratory report – was reported by 69.7% of laboratories, and 81.8% reported diluting samples exceeding the assay's upper limit of quantification to obtain an exact value.

Commercial assays most frequently used were Abbott Alinity HBsAg Quantitative Assay (55.9%), Diasorin Liaison XL Murex HBsAg Quant (26.5%), Roche Elecsys HBsAg II Quant (11.8%), and Siemens ADVIA Centaur Quantitative HBsAg assay (5.8%). All were chemiluminescent immunoassays, differing mainly in platform and signal detection method.

Marked heterogeneity was observed across centers regarding the number of HBsAg-positive patients detected and quantified. Some hospitals reported >3000–4000 positive cases in the past five years, whereas others reported only a few dozen, reflecting differences in hospital size and the timing of qHBsAg implementation. Over the past five years, a total of 14,576 qHBsAg determinations were reported. The largest proportion corresponded to values > 3000 IU/mL, accounting for 29.5% (n = 4306), followed by values <100 IU/mL, which represented 27.3% (n = 3972). Intermediate categories were less frequent: 16.0% (n = 2339) fell within 1000–3000 IU/mL, 16.5% (n = 2410) within 250–1000 IU/mL, and 10.6% (n = 1549) within 100–250 IU/mL. Some centers were unable to provide the detailed distribution across all predefined ranges. One laboratory reported grouped data for determinations > 1000 IU/mL (n = 2165), while another reported 25 determinations grouped as <250 IU/mL. In the most recent year, 40.7% (n = 197) of quantified determinations were <100 IU/mL, 27.1% (n = 131) were >3000 IU/mL, 12.2% (n = 59) between 1000 and 3000 IU/mL, 11.6% (n = 56) between 250 and 1000 IU/mL, and 8.5% (n = 41) between 100 and 250 IU/mL. Fig. 1 summarizes the distribution of qHBsAg categories among quantified determinations in the last year and over the past five years.

Fig. 1.

Distribution of qHBsAg categories in the last year and over the past five years. Bar chart showing the distribution of unique patients across predefined qHBsAg categories (<100, 100–250, 250–1000, 1000–3000, >3000 IU/mL) over the past year (n = 484) and the previous five years (n = 14,576). The y-axis represents the percentage of patients within each category. Centers reporting grouped categories were included as provided, and missing values were not imputed. The largest proportion was observed in the <100 IU/mL and >3000 IU/mL.

Discussion

This nationwide survey provides the first comprehensive overview of the current status of quantitative hepatitis B surface antigen (qHBsAg) implementation in clinical microbiology laboratories in Spain, with relevance extending to other countries where implementation remains largely undocumented. Although qHBsAg has been incorporated into clinical algorithms in guidelines issued by the European Association for the Study of the Liver (EASL)¹¹ and the World Health Organization (WHO),¹⁵ published data describing its real-world laboratory implementation at a national level remain scarce. In this context, our study provides one of the first nationwide assessments of qHBsAg implementation in clinical microbiology laboratories. Our findings show that only one third of laboratories currently perform qHBsAg, highlighting the limited adoption of this biomarker in routine practice. Historically, qHBsAg gained interest during the interferon era^13,14 but later fell into relative disuse, which, together with its limited presence in some clinical practice guidelines, has likely contributed to its weak implementation. By systematically exploring barriers, practices, and patient-level data, the study fulfills its main objective of identifying the degree of implementation and the obstacles that need to be addressed to optimize the role of qHBsAg in chronic hepatitis B management. In addition to qHBsAg, other biomarkers such as hepatitis B core-related antigen (HBcrAg) and HBV RNA are increasingly recognized as potentially useful tools for monitoring chronic HBV infection. However, their use in routine clinical practice remains limited due to restricted availability, lack of standardized assays, and variability in laboratory implementation. In contrast, qHBsAg is more widely accessible in clinical microbiology laboratories and benefits from standardized quantification protocols, which may facilitate its integration into diagnostic and monitoring workflows.

The most striking finding is the limited uptake of qHBsAg testing, with fewer than 40% of participating laboratories performing this assay. Lack of clinical demand was the most frequently cited barrier, reflecting the insufficient integration of qHBsAg into hepatology and infectious disease care pathways. This suggests a need for stronger alignment between clinical specialties and microbiology services, as well as educational initiatives to increase awareness of the clinical value of qHBsAg. In this context, coordinated national educational activities involving hepatologists, infectious disease specialists, and clinical microbiologists may be warranted to disseminate updated evidence on the role of qHBsAg in patient stratification and monitoring. Professional societies could play a key role in promoting training programs, consensus documents, and practical guidance to facilitate broader and more standardized implementation across Spain. Importantly, most laboratories not performing the assay did not refer samples externally, which risks depriving patients of a biomarker now endorsed by major international guidelines for monitoring chronic HBV infection.^5,15 Furthermore, ensuring widespread availability of qHBsAg testing is critical not only for current clinical decision-making but also to prepare healthcare systems for the potential arrival of new therapeutic agents for hepatitis B, where reliable biomarkers will play a central role in evaluating treatment response and functional cure endpoints.^11,16,17

Hospital size was significantly associated with implementation, with larger hospitals more likely to perform qHBsAg. This reflects the greater availability of resources and technical capacity in larger institutions. To overcome this disparity, establishing referral networks or centralized testing models could ensure that patients from smaller hospitals also benefit from qHBsAg measurement. Additionally, incorporation of qHBsAg into standard diagnostic panels, supported by guideline-based recommendations, could help normalize its use across institutions of different sizes. We did not collect variables such as HBV patient volume, academic/tertiary referral status, or local/regional policies, which could confound the association between hospital size and qHBsAg implementation.

Among laboratories performing qHBsAg, notable heterogeneity in practices was observed. Most laboratories used quantification after a positive screening result, whereas a minority employed it as the initial test. While proactive reporting of results was common, nearly one third of laboratories did not communicate the results to clinicians unless specifically requested. Standardizing reporting practices and ensuring clinicians receive actionable information is crucial to maximize the clinical impact of this biomarker.

This study has several limitations. First, the voluntary nature of the survey implies a limited sample size relative to all microbiology laboratories in Spain, which may affect representativeness; in addition, the sampling frame reflects the GEHEP-SEIMC laboratory network and may not capture laboratories outside this distribution list; although this list might include more hepatitis-oriented laboratories, it is broadly representative of public hospital microbiology laboratories across Spain. Moreover, as a primarily descriptive survey-based study conducted within Spain, the findings should be interpreted with caution and may have limited generalizability beyond the Spanish healthcare setting. Second, implementation of qHBsAg is likely to vary according to local resources, and our results may not fully capture this heterogeneity. Third, as an online self-administered survey, responses may also be subject to recall and reporting biases, and the possibility of self-selection bias must be considered, as laboratories already performing qHBsAg may have been more likely to respond, potentially overestimating uptake. Fourth, the reliance on self-reported data introduces the risk of inaccuracies regarding testing volumes, practices, and barriers. In addition, the survey did not capture patient-level serologic panels (anti-HBs, anti-HBc), HBV DNA results, or confirmatory/neutralization testing practices in cases of discordant HBsAg reactivity. Therefore, we cannot estimate the frequency of atypical serologic patterns, potential variant-related non-reactivity, or the operational value of confirmatory testing. Future studies integrating laboratory and clinical datasets should address these questions. Despite these limitations, the survey provides valuable insights and establishes a foundation for future structured evaluations.

In conclusion, this nationwide survey indicates that qHBsAg testing is not uniformly implemented across Spanish clinical microbiology laboratories and that workflows and reporting practices are heterogeneous. Reported barriers—particularly perceived low clinical demand and resource constraints—highlight areas where targeted education and organizational approaches may improve access. Given the descriptive design and potential selection bias, these results should be interpreted as a baseline assessment to inform harmonization efforts and future evaluations linking testing availability to clinical decision-making.

Author contributions

All authors contributed to data collection, and some additionally to data analysis, manuscript drafting, and critical revision. All authors reviewed and approved the final version of the manuscript.

Ethical considerations

Specific ethics approval was obtained from Hospital Universitario San Cecilio's Ethics Committee.

Informed consent

Informed consent was not required for this study.

Use of artificial intelligence

Artificial intelligence tools were used exclusively for writing assistance and linguistic revision of the manuscript. They were not used for the generation of results, analysis, or conclusions. The author guarantees the integrity and accuracy of the content.

Funding statement

This work was supported by the GEHEP-SEIMC group.

Conflict of interest

None of the authors have a conflict of interest to disclose for this work.

Data availability

Data available to investigators upon reasonable request.