Molecular and serological investigation of hepatitis B virus in patients with acute undifferentiated febrile illness at Tha Song Yang hospital, Tak Province, Thailand

Abstract

Background

Hepatitis B virus (HBV) infection is associated with hepatitis, often progressing to liver cirrhosis or cancer, posing a significant public health challenge, particularly in Thailand. Previous research revealed that viral causes account for 18.5% of acute undifferentiated febrile illness (AUFI) in Asian countries. This study examined the prevalence of HBV and its seroprevalence among patients with AUFI at Tha Song Yang Hospital (Tak Province, Thailand).

Methods

A cross-sectional study was conducted using residual serum samples collected between 2016 and 2017 from patients exhibiting symptoms of AUFI at Tha Song Yang Hospital. DNA was extracted and identified for HBV using real-time polymerase chain reaction (PCR). Positive samples were further characterized for the HBV genotype using semi-nested PCR targeting the pre-S gene. The serum samples were subjected to enzyme-linked immunosorbent assay to detect antibodies against hepatitis B core antigen (HBcAg) and hepatitis B surface antigen (HBsAg).

Results

Among AUFI patients, the prevalence of HBV infection was 6.45% (18 of 279). Genotyping revealed the presence of genotype C with subgenotype C1 (88.89%) and genotype B with subgenotype B3 (11.11%). The seroprevalence of HBcAb and HBsAb was observed in 40.58% (112 of 276) and 48.98% (120 of 245) of the cases, respectively.

Conclusions

The detection of HBV infection among AUFI patients (6.45%) underscores the spread of HBV within neighboring countries, such as Thailand and Myanmar. Pending confirmation of test results, physicians should maintain vigilance regarding potential HBV infection in AUFI cases. The overall seroprevalence showed 40.58% positivity for HBcAb and 48.98% for HBsAb. Therefore, individuals residing near the Thai border who did not receive the HBV vaccine at birth were recommended to complete a catch-up vaccination series of three doses to mitigate the HBV infection rate and enhance HBV antibody levels.

Background

Hepatitis B virus (HBV) is a virus that attacks the liver and can cause acute and chronic diseases. HBV is a serious issue for global health and infects > 296 million people with chronic HBV infection [1]. HBV is a member of the Hepadnaviridae family. The HBV genome is a 3.2 kb circular, partly double-stranded DNA classified into 10 genotypes from A to J. In Asia, genotypes B and C are common, and genotype C causes more severe liver disease than genotype B [2]. Four open reading frames in the genome encode the envelope (pre-S/S), core (pre-core/core), polymerase, and X proteins (X) [3, 4]. Hepatitis B virus can be transmitted in several ways, such as from mother to child, through sexual contact with an infected person, or by contact with the blood or bodily fluids of an infected person. There is a safe and effective vaccine that provides 98–100% protection against hepatitis B [1]. Since 1992, Thailand has started universal HBV vaccination as part of the Expanded Program on Immunization (EPI) at birth, and ~ 95% of children were vaccinated [5]. However, individuals residing near the Thailand-Myanmar border may not have received adequate vaccinations or may have been vaccinated late. Factors contributing to this situation include caregivers’ illiteracy in Thai, children’s birth order, and proximity to healthcare facilities [6].

Hepatitis B surface antibody (HBsAb) is the antibody to HBsAg, becoming detectable 6 to 8 weeks after infection and remaining for life. A positive HBsAb indicates a past or resolving infection. The Hepatitis B vaccine induces HBsAb, vaccinated individuals will also test positive. Hepatitis B core antibody (HBcAb) targets a core viral protein and can be measured as IgG, IgM, or total (both). IgM indicates the early immune response and is later replaced by IgG. A positive HBcAb IgM suggests early or chronic infection, while a positive HBcAb IgG indicates past or chronic infection [7]. An epidemiological study of HBV in the Thai population found that the overall prevalence of HBV surface antigen (HBsAg) was 2.2%. HBV DNA was also detected, with 93% of the cases identified as genotype C [8]. A cross-sectional study conducted on hill tribe adults in Chiang Rai Province, Thailand, found that the prevalence of HBV infection was 26.6%; 7.6% of people tested positive for HBsAg, 19.2% for anti-HBsAg (HBsAb) and 18.9% for anti-hepatitis B core antigen (HBcAb) [9]. Another study discovered that the prevalence of HBsAb was 10.2%, and the total HBcAb was 8.1% among hill tribal youths in northern Thailand [10].

Hepatitis B virus (HBV) infection is a common cause of undifferentiated febrile illness (AUFI). During the early stages of HBV infection, many individuals experience flu-like symptoms, including fever. In some cases, fever may be the only presenting symptom, especially in individuals with mild or asymptomatic infections. Other symptoms of HBV infection can include fatigue, loss of appetite, nausea, vomiting, and abdominal pain [11, 12]. A comprehensive literature review of acute undifferentiated febrile illness (AUFI) in Asian countries revealed that viral etiologies were the most prevalent at 18.5%, whereas bacterial etiologies were 12.9% [13]. The prevalence and seroprevalence of HBV among Thai patients with AUFI residing in the border area adjacent to Myanmar have not yet been explored. This study aimed to employ molecular techniques to detect and classify the genotype of the HBV-pre-S gene target. The objective was to assess the seroprevalence of HBsAb and HBcAb using serological techniques in AUFI patients at Tha Song Yang Hospital (Tak Province, Thailand). Moreover, this study elucidated the epidemiology of HBV infection in Tha Song Yang, Tak Province, a neighboring area of Myanmar, particularly among AUFI patients. Furthermore, the findings will contribute to health promotion and disease prevention by providing valuable epidemiological insights.

Methods

Study area and sample

This study is a cross-sectional investigation conducted in Tha Song Yang District, Tak Province, Thailand, from August 2016 to July 2017. This province is located in the western region of Thailand and is close to the Kayin State of Myanmar. Both countries are divided by the Moei River (Fig. 1). A total of 279 leftover serum samples were collected from patients of all ages who presented symptoms of AUFI as indicated by physicians for < 1 week at Tha Song Yang Hospital. The samples were stored at − 80 °C until processing. This study was conducted following the principles of the Declaration of Helsinki. Approval was obtained from the Ethics Committee of the Faculty of Tropical Medicine Ethics Committee, Mahidol University (MUTM: 2024-006-01). Given the retrospective nature of the study, the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University determined that informed consent was not required.

Fig. 1

The study area shown on the map is at Tha Song Yang District, Tak Province, western Thailand. The precise coordinates of the study area are 17°13′36″N, 98°13′30″E. This area is close to Kayin State, Myanmar, and is separated by the Moei River. The Tha Song Yang District is represented in red

Sample size calculation

Applying Cochran’s formula to determine the sample size in a cross-sectional design involved utilizing the following formula: n = Z² × p(1– p)/d² [14], where n is the sample size, Z is the 95% confidence interval (95% CI; 1.96), p is the prevalence of HBV infection based on a previous study in the northern region of Thailand (7.2% or 0.072) [15], and d is the margin of error (5% or 0.05). This calculation yielded a sample size of 103. Consequently, a minimum of 103 serum samples was deemed necessary for the investigation. Accordingly, 279 stored leftover serum samples, representing an age range of 2 months to 88 years (mean age: 25.48 years), were subjected to analysis, constituting the final sample size for the study.

DNA extraction

Two hundred microliters of 279 leftover serum samples were subjected to viral nucleic acid extraction utilizing the Nucleic Acid Extraction A 200 − 32 Kit and the EXM3000 Nucleic Acid Isolation System (Zybio, Chongqing, China), employing the magnetic bead methodology. Extraction procedures were conducted in accordance with the manufacturer’s protocol. The viral nucleic acid samples were preserved at − 80 °C until analysis.

Screening of HBV by real-time polymerase chain reaction (PCR)

The extracted nucleic acid samples underwent screening for HBV utilizing the HBV Real™ Qual Kit (Sacace Biotechnologies, Italy), a commercially available test kit. PCR analysis was conducted using the CFX96 Touch Real-time PCR Cycler System (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s instructions. Each reaction comprised a total volume of 25 µl, comprising 15 µl reaction mix and 10 µl extracted nucleic acid. HBV positivity was indicated based on fluorescence signals detected in the yellow fluorophore HEX channel. The clinical sample was considered positive for HBV DNA if the Ct value did not exceed the 35 Ct value. Then, all HBV-positive samples were chosen for further processing via semi-nested PCR.

Genotype classification of the HBV pre-S gene by semi-nested PCR

The pre-S region of the HBV sequence was amplified, spanning from the inception of pre-S1 to the culmination of pre-S2, through semi-nested PCR in 18 positive samples using real-time PCR. PCR amplification of HBV DNA was utilized with a recombinant Taq DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA). Oligonucleotide primer sequences employed in this study were sourced from previous research [2] and are documented in Table 1. To obtain the pre-S region sequence, the initial PCR round with P1 and S2-2 primers underwent 40 cycles (98 °C for 10 s, 50 °C for 20 s, and 72 °C for 25 min), followed by a 10 min extension at 72 °C. The second PCR round with P1 and S4R primers underwent 35 cycles (94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min), followed by a 7 min extension at 72 °C. The resultant amplified PCR product of 522 bp was visualized on a 1.5% agarose gel utilizing the Gel Doc XR + Gel Documentation System (Bio-Rad).

Table 1 PCR primers for the pre-S gene of HBV

Nucleotide sequencing and phylogenetic tree analysis

The semi-nested PCR products were excised from a 1.5% agarose gel and individually purified using the QIAquick Gel Extraction Kit (Qiagen). The purified PCR products were sent for DNA sequencing at Macrogen, Inc. (Seoul, Republic of Korea). The nucleotide sequences were assembled using the MEGA X program version 10.2.6 and aligned with the reference complete genome sequence of HBV utilizing Geneious Prime version 2024.0.5, employing Clustal Omega version 1.2.3 for multiple alignment. A phylogenetic tree of the specific pre-S gene was constructed using the neighbor-joining (NJ) algorithm by the Tamura-Nei model. To ensure the robustness of pairwise comparisons and phylogenetic tree analysis, bootstrap resampling was performed 1,000 times using Geneious Prime version 2024.0.5. Reference complete genome sequences representing various globally circulating genotypes and subgenotypes were obtained from the GenBank database for analysis of the specific pre-S gene, with the details provided in Table 2.

Table 2 List of reference complete genome sequences of HBV to analyze the specific pre-S gene in this study

Determination of HBsAb and HBcAb

Serum samples were subjected to a qualitative serological test for HBV antibodies targeting surface and core antigens using an HBsAb and HBcAb commercial test kit (Dia.Pro Diagnostic Bioprobes Srl, Milan, Italy). In the HBsAb test, microplates coated with highly purified HBsAg facilitated the capture of HBsAb during the initial incubation. The captured antibodies were identified using HBsAg labeled with horseradish peroxidase (HRP), generating an optical signal proportional to the HBsAb concentration by measurement at 450 nm wavelength using a spectrophotometer. Results were interpreted based on a 10 WHO mIU/ml cutoff value, with concentrations below considered negative and above considered positive.

The HBcAb test relied on a competition-based principle, where serum antibodies competed with a monoclonal antibody (mAb) for a fixed amount of purified recombinant HBcAg coated on microplates. Serum samples and an interference-blocking additive were added to the well. After washing, a mAb specific for HBcAg conjugated with HRP was added, binding to free HBcAg coated on the plastic. Subsequent incubation, washing, and substrate addition led to color development inversely proportional to the HBcAg antibody concentration. Results were interpreted using a cutoff value calculated with the formula: Cutoff = (NC + PC)/5, categorized as negative if < 0.9, equivocal if ≥ 0.9 to ≥ 1.1, and positive if > 1.1, based on the sample OD_{450 nm} or Co/S ratio. In the case of an equivocal result, the data will be excluded from data analysis.

Statistical analysis

Data were inputted into an Excel spreadsheet and thoroughly reviewed for accuracy before analysis. Statistical calculations regarding incidence and the corresponding 95% CI were conducted using MedCalc version 22.023 (available at https://www.medcalc.org/calc/rate_ci.php) for comprehensive analysis.

Results

HBV DNA detection by real-time PCR Serum samples from patients presenting with AUFI at Tha Song Yang Hospital underwent screening for HBV infection. Among these samples, 6.45% (18 of 279; 95% CI = 0.0382–0.1020) tested positive, whereas 93.50% (261 of 279; 95% CI = 0.8254–1.0561) were negative for HBV infection based on real-time PCR, indicating non-HBV infection status. The 6.45% positive samples underwent amplification of the HBV pre-S gene to determine their genotype via semi-nested PCR. Genotype identification was achieved in 3.25% (9 of 279), with the remaining 3.25% (9 of 279) non-identified. The non-identified samples cannot be genotyped due to insufficient sample volume for DNA amplification during sequencing analysis.

Distribution of HBV genotypes and subgenotypes

All sequences in this study have been deposited in the GenBank database with accession numbers OQ975884 and PP680764 to PP680771. Phylogenetic tree analysis was conducted on a fragment of the partial pre-S genomic region of HBV isolates from this study, alongside reference HBV complete genome sequences from the GenBank database. The phylogenetic tree classified HBV sequences into 10 genotypes (A–J), encompassing 12 subgenotypes (C1–C12) for HBV genotype C and 5 subgenotypes (B1–B5) for HBV genotype B. Among the identified HBV infections, 8 of 9 cases (88.89%), including OQ975884 and PP680765–PP680771, were assigned to genotype C with subgenotype C1, whereas 1 of 9 cases (11.11%; PP680764) was determined to be genotype B with subgenotype B3 based on the phylogenetic tree topology.

Most HBV sequences from genotype C (subgenotype C1) exhibited close similarity to reference strain prevalence in Thailand (AB074756). Furthermore, these sequences were categorized within clusters originating from neighboring countries of Thailand, specifically Cambodia (LC535950) and Myanmar (KT987426). Sequences from Vietnam (AB031265) were included in this cluster, with their geographical locations within 100 km of Thai territory. These clusters spanned the years 2005 to 2017. Moreover, a single nucleotide HBV sequence belonging to genotype B (subgenotype B3) demonstrated a close relationship with reference strains from the Philippines (AB219427) and Indonesia (EF473977), which were deposited in 2006 and 2008, respectively. The phylogenetic analysis of HBV is depicted in Fig. 2.

Fig. 2

A phylogenetic analysis of HBV was conducted using the NJ algorithm with 1,000 bootstrap replicates based on a partial nucleotide sequence of the pre-S gene, utilizing Geneious Prime version 2024.0.5. HBV strains were categorized into 10 genotypes (A–J). The nine sequences derived from patient serum samples in this study were indicated by dark blue circles and annotated with accession and patient numbers. Reference strain names were provided alongside accession numbers and the countries where HBV isolates originated. The Tha Song Yang HBV strains in this study were grouped into genotype C (subgenotype C1) and genotype B (subgenotype B3). Bootstrap support values were represented by numbers on branches

Seroprevalence of HBV

A seroprevalence study employing enzyme-linked immunosorbent assay (ELISA) was conducted to detect HBcAb and HBsAb in serum samples from AUFI patients. Of 276 serum samples analyzed, 40.58% (112 of 276; 95% CI = 0.3341–0.4883) tested positive for HBcAb, whereas 59.42% (164 of 276; 95% CI = 0.5067–0.6924) were negative for HBcAb. Similarly, among 245 serum samples assessed, 48.98% (120 of 245; 95% CI = 0.4061–0.5857) tested positive for HBsAb, whereas 51.02% (125 of 245; 95% CI = 0.4247–0.6079) tested negative for HBsAb. The corresponding pie chart depicting these findings is presented in Fig. 3. Considering the positive rates of HBcAb and HBsAb, they were ~ 1 in every 2 individuals.

Upon analyzing the individual data of 243 patients, 38 individuals tested positive for HBcAb alone, accounting for 15.64% (95% CI = 0.1107–0.2146), whereas 60 individuals tested positive for HBsAb alone, accounting for 24.69% (95% CI = 0.1884–0.3178). Fifty-nine patients (24.28%; 95% CI = 0.1848–0.3132) tested positive for HBcAb and HBsAb, whereas 86 patients (35.39%; 95% CI = 0.2831–0.4371) tested negative for HBcAb and HBsAb. The summarized prevalence data among AUFI patients are presented in Table 3. The ELISA test, limitations by a small amount of volume, was unable to evaluate all serum samples equally, leading to the identification of 276 serum samples for HBcAb and 245 for HBsAb. Moreover, while analysing the identical sample from the same patient, both HBcAb and HBsAb were identified in 243 individual data sets for evaluation.

Table 3 Summary of prevalence and seroprevalence of HBV infection among AUFI patients at Tha Song Yang Hospital

Fig. 3

Pie chart depicting the distribution of seropositive and seronegative for HBcAb and HBsAb among patients with AUFI patients at Tha Song Yang Hospital

Discussion

HBV is prevalent in Thailand, but data on its molecular and serological epidemiology are limited in specific regions, especially those bordering Myanmar. HBV infection is a common cause of undifferentiated febrile illness (AUFI). Early symptoms include fever, fatigue, loss of appetite, nausea, vomiting, and abdominal pain. In some cases, fever may be the only presenting symptom. Despite thorough laboratory investigations, infectious agents remained undetected in ~ 50% of patients presenting with AUFI and residing in the Thai border region adjacent to Myanmar, an area that remains unexplored. This study was the first comparative investigation into the molecular characterization and seroprevalence of HBV circulating in Tha Song Yang District, in close proximity to Myanmar. Its findings contributed essential data for assessing the efficacy of health program systems in Thailand and its neighboring countries. The study involved identifying viral genotypes and detecting HBcAb and HBsAb in a significant proportion of HBV carriers from this region. This combined approach is more sensitive and reliable for screening and predicting positive test results than detecting HBV DNA or antibodies separately, which may miss acute or past infections within the study population.

Findings indicated that the prevalence of HBV infection among individuals associated with AUFI was ~ 6.45%. The predominant HBV strains were classified as genotype C with subgenotype C1, accounting for 88.89% of the cases, followed by genotype B with subgenotype B3 at 11.11%. Most HBV sequences from genotype C (subgenotype C1) exhibited clustering with reference strains prevalent in Thailand and were grouped within clusters originating from neighboring countries of Thailand, such as Cambodia and Myanmar. Conversely, a single nucleotide sequence of HBV belonging to genotype B (subgenotype B3) was significantly associated with reference strains from the Philippines and Indonesia. Consistent with previous studies, viral infections have been identified as the predominant etiological factor contributing to AUFI in Southeast Asia, with prevalence rates ranging from 26.0 to 39.6%. Among the commonly implicated viral causes of AUFI are dengue, chikungunya, HIV-1, hepatitis A virus, cytomegalovirus, hepatitis C virus, and HBV. Specifically, HBV positivity was detected in 1.1% of the cases [13, 16, 17]. Several researchers highlighted the spreading of HBV genotypes and subgenotypes across Southeast Asian countries. For instance, Tangkijvanich et al. reported that HBV genotypes C and B were prevalent in Thailand, accounting for 73% and 21% of the cases, respectively [18]. Sirilert et al. observed HBV associated with pregnant women in northern Thailand, which was predominantly subgenotype C1 in 77.4% of the cases [19]. Kyaw et al. demonstrated HBV genotype C, particularly subgenotype C1, was widespread across all states and regions of Myanmar, with prevalence rates ranging from 61.7 to 91.7%. Among genotype C, subgenotypes C1, C5, and C7 were identified, with subgenotype C1 as the most prevalent at 90.2% [20]. Nwe Win et al. reported that among migrant workers traveling from Myanmar to Thailand, genotype C was the most common (97.5%), followed by genotypes B (1.25%) and D (1.25%) [21]. Furthermore, Sa-nguanmoo et al. reported a high prevalence of HBV among migrant workers from Cambodia, Laos, and Myanmar, with 86% classified as genotype C (99% subgenotype C1) and 11.6% as genotype B (subgenotypes B2, B3, and B4) [22]. This study identified a high prevalence of HBV infection (~ 6.5%) among AUFI patients from Tha Song Yang District, indicating the spread of HBV among residents of neighboring countries, particularly Thailand and Myanmar. Moreover, these data underscored that HBV subgenotype C1 was the predominant strain occurring naturally among Southeast Asian populations, particularly in Thailand.

There was a slightly elevated prevalence of HBV infection (~ 6.45%) among AUFI patients. Consequently, this investigation aimed to assess the seroprevalence of HBV antibodies, specifically against core and surface antigens, in this population. This serological analysis revealed a positive HBcAb seroprevalence of 40.58%, indicative of past or ongoing HBV infection, as immunity from HBV vaccination typically does not elicit HBcAb. The HBsAb positivity rate was 48.98%, suggesting the presence of HBsAb, associated with recovery or immunity from HBV, which can also develop after vaccination, albeit with a potential decline in levels over time [23]. Nevertheless, approximately one in two individuals exhibited positive serological markers for HBcAb and HBsAb. This research aligned closely with previous hepatitis serology investigations in Thailand, which reported widespread HBV vaccination. For instance, Chongsrisawat et al. (2004) reported 26.5% and 41.6% seropositivity rates across a population aged 6 months to 60 years for HBcAb and HBsAb, respectively [24]. Similarly, Posuwan et al. (2016) demonstrated overall seropositivity rates of 17.09% for HBcAb and 43.81% for HBsAb [25]. Although the seropositivity rate of HBcAb increased over time, that of HBsAb remained consistent with the findings reported in 2004 and 2016. However, it can be observed that this research found a relatively low number of molecular positives for HBV DNA, while a high prevalence of HBcAb was found among the individuals, which demonstrates a high level of contact with the virus. This is because HBV DNA in the bloodstream is a reliable marker of active HBV infection. It can be detected within a few days of infection. The amount of HBV DNA often climbs to its highest point during a severe episode of hepatitis, then gradually decreases and disappears if the infection heals naturally [26]. Therefore, the period during which HBV DNA can be detected is short, which is why the number of HBV DNA detections is much lower than the number of antibody detections.

Upon analyzing the individual data of 243 patients, 38 individuals tested positive solely for HBcAb, representing 15.64%, whereas 60 individuals tested positive solely for HBsAb, accounting for 24.69%. Furthermore, 59 patients (24.28%) tested positive for HBcAb and HBsAb, whereas 86 patients (35.39%) tested negative for both antibodies. Focusing on individuals with undetectable or low antibody titers underscores the significance of HBcAb and HBsAb. Despite initiating universal HBV vaccination in Thailand in 1992 as part of the EPI at birth, ~ 95% of children were vaccinated [27]. In response to this situation, individuals living near the Thai border who missed receiving HBV vaccination at birth were advised to undergo catch-up vaccination, aiming to ensure they complete the three vaccine doses, leading to increased HBV antibody levels.

Additionally, a 95% confidence interval (CI) for the mean, which is commonly employed, represents a range with upper and lower limits calculated from a sample [28]. In serological studies, the CI is essential for assessing the uncertainty surrounding an estimate, such as the prevalence of a specific antibody. For instance data from Table 3, a CI of 0.3341–0.4883 indicates a lower prevalence, whereas a CI of 0.8254–1.0000 suggests a substantially higher prevalence of antibodies. In conclusion, confidence intervals are crucial role in serological testing, as they aid in evaluating the reliability of test results and contribute to guiding and assessing the overall quality of the study.

This study has some limitations. First, there is a lack of data regarding patient demographics, such as age, gender, marital status, and occupation, and potential risk factors, including HBV vaccination history, blood transfusion history, and engagement in multiple sexual partnerships. The primary constraint is the insufficient data for analyzing the relationship between variables and HBV presence and the seropositivity rate and associated risk factors. Another limitation is the limited volume of patient serum samples collected from 2016 to 2017, which has restricted the number of times PCR can be repeated to amplify DNA for sequencing due to certain samples being unable to undergo the process multiple times. The serological test is affected by a low amount of serum samples, resulting in a difference in the total number of tests conducted in this study.

Conclusions

The overall seroprevalence showed 40.58% positivity for HBcAb and 48.98% for HBsAb. Therefore, individuals residing near the Thai border who did not receive the HBV vaccine at birth were recommended to complete a catch-up vaccination series of three doses to mitigate the HBV infection rate and enhance HBV antibody levels. The detection of HBV infection among AUFI patients (6.45%) underscores the spread of HBV within neighboring countries, such as Thailand and Myanmar. Pending confirmation of test results, physicians should maintain vigilance regarding potential HBV infection in AUFI cases.