Invest Clin 66(2): 166 - 174, 2025 https://doi.org/10.54817/IC.v66n2a04

Corresponding author: Flor Helene Pujol. Laboratorio de Virología Molecular, CMBC, Instituto Venezolano de

Investigaciones Científicas (IVIC), Caracas, Venezuela. E-mail: fhpujol@gmail.com

Optimization of a real-time PCR assay

for hepatitis B virus load determination

in infected patients.

María Zulay Sulbarán, Yoneira Fabiola Sulbarán, Carmen Luisa Loureiro,

Héctor Rafael Rangel, Rossana Celeste Jaspe and Flor Helene Pujol

Laboratorio de Virología Molecular, CMBC, Instituto Venezolano de Investigaciones

Científicas (IVIC), Caracas, Venezuela.

Keywords: hepatitis B virus; viral load; optimization; qPCR; genotype.

Abstract. Hepatitis B virus (HBV) infection is a significant health problem

in the world, with around 294 million chronic carriers. Determination of viral

load is crucial for the monitoring and treatment follow-up of HBV-infected pa-

tients. In-house methods are economical alternatives for those settings where

HBV viral load determination might not be widely available commercially. Mo-

lecular diagnostic techniques need to take into account the variability of this

virus. The aim of this study was the evaluation and optimization of a real-time

PCR method for the determination of HBV load. After optimization, the in-

house assay evaluated in this study showed acceptable sensitivity and specific-

ity, allowing its use for monitoring patients in low-income settings.

Optimization of a real-time PCR assay for hepatitis B 167

Vol. 66(2): 166 - 174, 2025

Optimización de un ensayo de PCR en tiempo real para la

determinación de carga viral del virus de hepatitis B en

pacientes infectados.

Invest Clin 2025; 66 (2): 166 – 174

Palabras clave: virus de hepatitis B; carga viral; qPCR; optimización; genotipo.

Resumen. La infección por el virus de la hepatitis B (VHB) es un gran pro-

blema de salud en el mundo, con alrededor de 294 millones de portadores cró-

nicos. La determinación de la carga viral es de importancia crucial para el mo-

nitoreo y el seguimiento del tratamiento de los pacientes infectados por el VHB.

Los métodos propios son alternativas económicas para aquellos países donde la

determinación de la carga viral del VHB podría no estar ampliamente disponible

comercialmente. Las técnicas de diagnóstico molecular deben tener en cuenta la

variabilidad de este virus. El objetivo de este estudio fue la evaluación y optimiza-

ción de un método de PCR en tiempo real para la determinación de la carga del

VHB. La prueba propia analizada en este estudio mostró, después de la optimiza-

ción, una sensibilidad y especificidad satisfactorias, lo que permitió su uso para

el seguimiento de pacientes en comunidades de bajos ingresos.

Received: 10-03-2025 Accepted: 09-05-2025

INTRODUCTION

Around 294 million persons are chroni-

cally infected with the hepatitis B virus

(HBV) in the world 1. HBV chronic infec-

tion frequently leads to cirrhosis and hepa-

tocellular carcinoma (HCC), with at least

800,000 deaths each year 1. This infection is

highly endemic in Sub-Saharan Africa, Asia,

and Indigenous populations in the Americas

and Oceania 2,3.

HBV belongs to the Hepadnaviridae

family. It is a partially double-stranded DNA

virus of around 3200 bp. In addition to the

virus, viral-like particles also circulate, com-

posed exclusively of the HBV surface antigen

(HBsAg) within a non-infectious envelope 4.

Up to ten genotypes and several subgeno-

types have been described for HBV, with the

American genotypes being the most diver-

gent. The tenth HBV genotype J seems to be

a recombinant one, and only one sequence

is available 5.

A recombinant HBV vaccine has been

developed, based on the HBsAg 6. After more

than 30 years of worldwide vaccination, viral

infection and HCC incidence reduction have

been widely demonstrated 6,7. These mea-

sures have led the World Health Organization

(WHO) to propose a plan for hepatitis elimi-

nation by 2030 8. However, nearly 300 million

people remain chronically infected, and no ef-

fective treatment is currently available.

The functional cure of chronic hepatitis

B is defined as HBsAg loss after therapy, which

is rarely achieved with the current therapy.

Novel agents in development and preexisting

ones will probably bring the tools to approach

this goal 9. In the last WHO guidelines of

March 2024, HBV treatment is recommended

for all adults and adolescents (over 12 years

old) with chronic hepatitis B if:

168 Sulbarán et al.

Investigación Clínica 66(2): 2025

1. Evidence of significant fibrosis

2. HBV DNA viral loads above 2000 IU/mL

and ALT levels exceeding the upper li-

mit of the reference range

3. Or presence of coinfections, such as

HIV, hepatitis C, hepatitis D, or other

comorbidities 10.

The determination of the viral load is

then of crucial importance for confirma-

tion of viral replication and the monitoring

of chronically infected hepatitis B patients.

Several commercial assays are available to

assess this biomarker, based on real-time

PCR or transcription-mediated amplifica-

tion (TMA), in most of the laboratories from

high-income countries. However, this might

not be the case in some low-income coun-

tries 11.

In-house methods are economical al-

ternatives for those settings where HBV vi-

ral load determination might not be widely

available commercially. Molecular diagnos-

tic techniques need to take into account the

variability of this virus. Portilho et al.12 de-

veloped a real-time PCR to determine HBV

load. This study aimed to evaluate and op-

timize this real-time PCR method for deter-

mining HBV load.

MATERIALS AND METHODS

Sera from HBV-infected patients

This study was approved by the Bioethi-

cal Committee of IVIC (“Biología molecular

de virus de hepatitis y el Virus de la Inmuno-

deficiencia adquirida (VIH) en Venezuela”,

December 5, 2024). The sera of patients di-

agnosed with HBV infection, and who gave

informed consent, were kept at -70°C until

use. DNA was extracted from sera using the

QIAamp Viral DNA Mini Kit (Hilden, Germa-

ny) and used in all the other analyses.

HBV PCR and sequencing

Nested PCR was carried out using previ-

ously reported primers 58P-1100N and S6-

S3as 13. PCR-purified fragments were sent to

Macrogen Sequencing Service (Macrogen,

Korea) for sequencing. The phylogenetic

analysis was performed with MEGA11 soft-

ware 14.

Viral load determination

According to the manufacturer’s in-

structions, HBV viral load was determined

with Bosphore® Ultra HBV Quantitation/

Detection (Anatolia Geneworks®, Istanbul,

Turkey). This test has been used previously

by several groups 15-17.

In-house real-time PCR determination

Real-time PCR for HBV was performed

according to the protocol suggested by

Portilho et al. 12. Additionally, an anti-

sense modified primer was designed for

an optimized assay: 5’-GGCCAAAATTCG-

CAGTCCCCAACC-3’. Real-time PCR was run

in a final volume of 15 µL, containing 6 µL

of DNA, 1X PCR buffer, 1 mM MgCl2, 0.4mM

dNTP mixture, 0.23 µM of each primer and

probe, 1.75 U Platinum® Taq DNA Poly-

merase (Thermofisher, USA). The final con-

ditions for the optimized assay were: 95°C

for 10 minutes, then 40 cycles at 97°C for

30 seconds, 54°C for 90 seconds, and a hold

stage of 32°C for 60 seconds.

Statistical analysis

The correlation between the values of

HBV viral load determined by the commer-

cial assay and the in-house method was eval-

uated with the Pearson´s coefficient 18.

RESULTS

Serum samples from HBV-infected pa-

tients were analyzed by a commercial kit and

the in-house real-time PCR to evaluate the

performance of an in-house real-time PCR. A

total of nine sera (and dilutions of them for a

total of 14 samples tested) were tested, with

variable HBV loads: five sera were classified as

HBV genotype F3 (one not shown in the tree,

since the sequence was shorter, but it could

be classified as F3), and one each as genotype

A2, C2, F2, and F4 (Fig. 1).

Optimization of a real-time PCR assay for hepatitis B 169

Vol. 66(2): 166 - 174, 2025

A good agreement was found between

the commercial assay and the in-house

method for 9/14 samples, but with an over-

all low correlation of R2=0.47 (Fig. 2A). The

samples exhibiting the discrepant results be-

tween the two tests were derived from two

samples, corresponding to HBV genotype C2

and F3 isolates. When these samples were ex-

cluded, the correlation coefficient increased

to R2=0.98 (Fig. 2B).

Fig. 1. Phylogenetic tree of the HBV isolates tested in this study. The S gene is analyzed (559 nt). The evolu-

tionary history was inferred using the Maximum Likelihood method and the General Time Reversible

model14. The tree with the highest log likelihood (-2602.78) is shown. The percentage of trees in which

the associated taxa clustered is shown next to the branches. Initial tree(s) for the heuristic search were

obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the

Maximum Composite Likelihood (MCL) approach. A discrete Gamma distribution was used to model

evolutionary rate differences among sites (4 categories (+G, parameter = 0.6597)). The rate variation

model allowed some sites to be evolutionarily invariable ([+I], 62.58% sites). The tree is drawn to scale,

with branch lengths measured in the number of substitutions per site. This analysis involved 45 nucleo-

tide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions with less than

80% site coverage were eliminated, i.e., fewer than 20% alignment gaps, missing data, and ambiguous

bases were allowed at any position (partial deletion option). Bootstrap values are shown in the branches

of the tree. The isolates are named by their genotype and GenBank accession number, except for the

ones from this study (n=8), which are shown in purple. An additional HBV isolate (B02023) was not

included in the tree since the shorter sequence could also be classified as genotype F3.

170 Sulbarán et al.

Investigación Clínica 66(2): 2025

An alignment of the sequences of the

HBV samples for which discrepant results

were observed is shown in Fig. 3. The align-

ment reveals multiple mismatches between

the antisense primer and several HBV iso-

lates, absent in the sequences without dis-

crepant results. The mismatches were pres-

ent, particularly in genotype C isolates.

A new antisense primer for an opti-

mized real-time PCR was designed (Fig. 3).

The complementarity of the three primers

(sense, the new antisense and the probe)

was evaluated in a total of 16516 sequences

from the HBV database 19, as shown in Table

1. The three primers showed high sequence

identity with the analyzed HBV sequences

from all the genotypes analyzed: for some

sequences, one or two internal mismatch-

es were observed, frequently at the 5´end.

These few mismatches per sequence (not at

the 3´ end of the primer) may not hamper

the adequate performance of the real-time

PCR. The probe also demonstrated satisfac-

tory performance. In general, the sequence

of this probe was well conserved among all

the isolates. The only exceptions might be

for the recombinant forms of HBV, for which

the mismatches were frequent, particularly

with the probe, and for some HBV genotype

C sequences.

Fig. 4 shows the correlation of the opti-

mized in-house real-time PCR with the com-

mercial assay. A strong correlation was ob-

served across all the tested samples, using

the new antisense primer, including discrep-

ant samples and their dilutions (R2=0.99,

Fig. 4).

Fig. 5 shows the performance of the in-

house test with HBV-positive and negative

samples. Most negative samples did not ex-

hibit any signal during the PCR process (40

cycles), although 1/20 negative samples had

a cycle threshold (Ct) value of 39.01. The de-

tection limit was fixed at 50 UI/mL (Ct value

around 35), which is acceptable for manag-

ing HBV-infected patients.

DISCUSSION

Hepatitis B is still a significant health

problem in Venezuela, even if vaccination

campaigns may have reduced the burden of

this disease, particularly in some indigenous

populations, where HBV infection remains

highly endemic 20. The most common HBV

genotype in Venezuela is F (F3, followed by

F2, and less frequently F1 and F4), followed

by HBV genotypes with worldwide distribu-

tion (A and D) and infrequently the HBV

Asian B and C, particularly these last ones in

immigrants 13,20. The samples from this study

included different subgenotypes circulating

in the country, and this diversity was impor-

tant to assess the performance, particularly

in terms of sensitivity, of the real-time PCR

test analyzed.

Fig. 2. Correlation between viral load determination with a commercial assay and the in-house method of Portilho et

al. 12. 2A. All the samples were tested. Green and open circles represent the dilution of a sample, as shown

in the filled green. Orange open circles represent dilution of a synthetic control (amplicon from sample

B4810, which is shown in filled orange). 2B. Correlation for samples without discordant results (orange and

purple in Fig. 2A, derived from B4810 and B5415, respectively) were omitted.

Optimization of a real-time PCR assay for hepatitis B 171

Vol. 66(2): 166 - 174, 2025

Fig. 3. Sequence alignment of the HBV preS region amplified by the in-house real-time test. Reference se-

quences are shown with their genotypes. The sequence of the samples used in this study is also inclu-

ded, except for B02023, since the sequence was shorter and does not cover the region. The primers

from Portilho et al. 12 for the in-house test are shown in blue. The new modified reverse primer is

shown in red. The reverse complement sequences are shown for the reverse primers.

Fig. 4. Correlation between viral load determination

with a commercial assay and the optimized

in-house method. Dilutions of the sample

B4810 (10–1, 10–3, and 10–4) are shown in

open orange circles, while sample 5379 and

its dilutions (10–1 and 10–2 open circles) are

shown in green.

Fig. 5. Performance of the optimized in-house re-

al-time PCR with sera from HBV-infected

patients or non-infected with this virus. Ct

values of positive (left, 38 samples) and ne-

gative (right, 20 samples) HBV samples. The

HBV negative samples were samples from six

HCV positive, three dengue virus positive,

two HIV-1 positive, 1 HAV positive, 1 HEV po-

sitive, and six with any known infecting virus.

172 Sulbarán et al.

Investigación Clínica 66(2): 2025

A low correlation was observed between

the commercial assay and the original in-

house primers proposed by Portilho et al. 12.

This finding contrasts with their analysis of

40 serum samples, where a good correlation

with the commercial assay was observed 12.

However, the authors did not mention the

genotype of their samples.

Although HBV is a DNA virus, it displays

a level of variability intermediate between

RNA and DNA viruses. The dependence of

a reverse transcriptase for its replication,

which lacks proof-reading activity, increases

the mutation rate of this virus. In contrast,

the highly compact genome (one of the

smallest animal DNA genomes), with a high

degree of overlap in the different open read-

ing frames, reduces the viability of some of

these mutations. The result is an intermedi-

ate mutation rate, compared to a DNA and

an RNA virus 20. The variability exhibited by

different HBV genotypes leads to differential

pathogenesis and variable resistance to IFN

treatment 21. In addition, the accuracy of

primers in PCR reactions is always limited.

In this study, analyzing more than

16,500 sequences to evaluate the suitability

of the new proposed primers suggests that

we can be confident in using those modified

primers. The only exception might be the re-

combinant isolates (and some genotype C iso-

lates), although we could not test any recom-

binant isolates. Recombination is a common

phenomenon in HBV 22,23. Infection with some

HBV genotypes has been associated with a

more severe disease and more rapid progres-

sion to HCC, for example, infection with HBV

genotype C: the relationship between HBV

recombinant genotypes and pathogenesis is

unknown at present 20,21. The recombinant

forms, in any case, may affect the sensitivity

of molecular diagnostic techniques.

Table 1. Complementarity of the primers used in the modified in-house method with HBV

sequences available in the HBV database.

HBV genotype N of sequences Sequences with divergence1

preSf preSr2 preSprobe

A 2359 104 48 64

B23534 246 64 68

C35088 386 1639 160

D43220 399 123 45

E5894 28 124 17

F5357 39 4 5

G687 2 0 0

H748 4 3 0

RF8929 237 134 287

Total (%) 16516 8.85% 12.95% 2.34%

1 The complementarity of the primers tested in the new in-house method were analyzed in the 16516 HBV sequen-

ces of the HBV database 19. Most of the observed divergences in the complementarity with the primers correspond

to only one or two internal mismatches. 2 For preSf and PreSr2, most of the sequences exhibited one mismatch at

the 5´end, so this first nt of the primer was not considered in the analysis. 3 Some mismatches were found in the

3´end of the primers: 38 sequences exhibited this mismatch for primer preSf, only 3 for preSr2. 4 One sequence

with a mismatch at the 3´end. 5 For preSf primer, most of the sequences exhibited one mismatch at the 5,7´end, so

this first nt was not considered in the analysis. 6 All the genotype G isolates have a T instead of an A in the fifth po-

sition of the primer preSr2, from 3´to 5´end, so this position was not considered in the analysis. 8 RF: recombinant

forms. Many sequences exhibited one or more mismatches, internal or at the 5´end.

Optimization of a real-time PCR assay for hepatitis B 173

Vol. 66(2): 166 - 174, 2025

In conclusion, modifying the HBVpreS

reverse primer significantly improved the

correlation of the viral load determination

by the in-house method, with the commer-

cial assay, increasing then the performance

of this test. The new version of the in-house

test displayed a satisfactory sensitivity and

specificity, allowing its use for monitoring

patients in low-income settings. A limitation

of this study is the relatively low number of

samples evaluated by the commercial as-

say and the in-house test. The in-house test

tested a higher number of samples (n=38

positive samples, and 20 negative, the last

ones without any signal). In addition, the in

silico validation of the assay with more than

16,000 sequences makes us confident in the

performance of the optimized test.

ACKNOWLEDGMENTS

To the health personnel who effective-

ly attended to the cases of hepatitis in this

study.

Funding

The Ministerio del Poder Popular para

Ciencia y Tecnología de Venezuela (MinCyT)

financed this project, Proyecto Mujer 2024.

Declaration of conflicts of interest

The authors declare that they have no

conflict of interest.

Number ORCID of authors

• María Zulay Sulbarán (MZS):

0009-0008-9798-5593

• Yoneira Sulbaran (YS):

0000-0002-3170-353X

• Rossana C Jaspe (RCJ):

0000-0002-4816-1378

• Carmen L. Loureiro (CLL):

0000-0003-3665-1107

• Héctor R Rangel (HRR):

0000-0001-5937-9690

• Flor H. Pujol (FHP):

0000-0001-6086-6883

Participation of authors

MZS, YS, RJC, CL, FHP: Substantial

contribution to the conception and design

of the study; data collection or their analysis

and interpretation, critical review of the ar-

ticle and approval of the final versión to be

published. HRR: critical review of the article

and approval of the final versión to be pub-

lished.

REFERENCES

1. Jeng WJ, Papatheodoridis GV, Lok

ASF. Hepatitis B. Lancet 2022: S0140-

6736(22)01468-4. https://doi.org/10.10

16/S0140-6736(22)01468-4.

2. GBD 2019 Hepatitis B Collaborators.

Global, regional, and national burden of

hepatitis B, 1990–2019: a systematic anal-

ysis for the Global Burden of Disease Study

2019. Lancet Gastroenterol Hepatol 2022;

7: 796-829. https://doi.org/10.1016/S24

68-1253(22)00124-8.

3. MacLachlan JH, Cowie BC. Hepatitis B

virus epidemiology. Cold Spring Harb Per-

spect Med 2015; 5: a021410. https://doi.

org/10.1101/cshperspect.a021410.

4. Gerlich WH. Medical virology of hepa-

titis B: how it began and where we are

now. Virol J 2013; 10: 239. https://doi.

org/10.1186/1743-422X-10-239.

5. McNaughton AL, Revill PA, Littlejohn M,

Matthews PC, Ansari MA. Analysis of ge-

nomic-length HBV sequences to determine

genotype and subgenotype reference se-

quences. J Gen Virol 2020; 101: 271-283.

https://doi.org/10.1099/jgv.0.001387.

6. Pujol FH, Toyé RM, Loureiro CL, Jaspe

RC, Chemin I. Hepatitis B eradication:

vaccine as a key player. Am J Transl Res.

2023; 15: 4971-4983.

174 Sulbarán et al.

Investigación Clínica 66(2): 2025

7. Huzair F, Sturdy S. Biotechnology and the

transformation of vaccine innovation: The

case of the hepatitis B vaccines 1968-2000.

Stud Hist Philos Biol Biomed Sci 2017;

64: 11-21. https://doi.org/10.1016/j.shp-

sc.2017.05.004.

8. World Health Organization. Combat-

ing Hepatitis B and C to Reach Elimina-

tion by 2030. Available from: https://

apps.who.int/iris/bitstream/han-

dle/10665/206453/WHO_HIV_2016.04_

eng.pdf. Accessed on December 22, 2024

9. Feld JJ, Lok AS, Zoulim F. New Perspec-

tives on Development of Curative Strategies

for Chronic Hepatitis B. Clin Gastroenter-

ol Hepatol. 2023; 21: 2040-2050. https://

doi.org/10.1016/j.cgh.2023.02.032.

10. WHO. Guidelines for the prevention, di-

agnosis, care and treatment for people

with chronic hepatitis B infection. Geneva:

World Health Organization; 2024. Licence:

CC BY-NC-SA 3.0 IGO.

11. Pawlotsky JM. Virological markers for

clinical trials in chronic viral hepatitis.

Virological markers for clinical trials in

chronic viral hepatitis. JHEP Rep. 2024;

6: 101214. https://doi.org/10.1016/j.

jhepr.2024.101214.

12. Portilho MM, Mendonça ACDF, Bezerra

CS, do Espirito-Santo MP, de Paula VS,

Nabuco LC, et al. Usefulness of in-house

real time PCR for HBV DNA quantification

in serum and oral fluid samples. J Virol

Methods. 2018; 256: 100-106. https://doi.

org/10.1016/j.jviromet.2018.03.001.

13. Devesa M, Loureiro CL, Rivas Y, Monsalve

F, Cardona N, Duarte MC, et al.. Subgeno-

type diversity of hepatitis B virus American

genotype F in Amerindians from Venezuela

and the general population of Colombia. J

Med Virol. 2008; 80: 20-26. doi: 10.1002/

jmv.21024. https://doi.org/

14. Tamura K, Stecher G, and Kumar S.

MEGA11: Molecular Evolutionary Genetics

Analysis version 11. Mol Biol Evol. 2011;

38: 3022-3027. https://doi.org/10.1093/

molbev/msab120.

15. Uzunoğlu E, Şahin AM, Avci E, Kutlu H,

Güntepe G. Can HBsAg Be Used as a Viral

Replication Marker in Chronic Hepatitis B

Patients? Viral Hepa J. 2017; 23: 55-59.

16. Aynalia A, Ciftcia E, Aridogana BC,

Cetina ES, Kayab S, Carsancaklia SA,

Ozturka T. Evaluation of viral load distri-

bution of HBV DNA positive patients at Su-

leyman Demirel University Hospital. J Exp

Clin Med. 2015; 32: 147-150. https://doi.

org/10.5835/jecm.omu.32.04.002.

17. Guney M, Bakir A, Erdal H, Gunal A,

Yildiz F, Sig AK, Kurkcu MF, Yavuz MT,

Gulsen M. The Correlation between Histo-

pathological Stages and Viral Markers of

Chronic Hepatitis B infection in Ankara,

Turkey. Int J Innov Sci Res Technol. 2021;

6: 1066-1070.

18. Akoglu H. User’s guide to correlation co-

efficients. Turk J Emerg Med. 2018; 18:

91-93. https://doi.org/10.1016/j.tjem.20

18.08.001.

19. HBVdb. The Hepatitis B Virus database.

https://hbvdb.lyon.inserm.fr/HBVdb/

HBVdbIndex. Accessed on December 27,

2024.

20. Pujol F, Jaspe RC, Loureiro CL, Chemin

I. Hepatitis B virus American genotypes:

Pathogenic variants? Clin Res Hepatol Gas-

troenterol. 2020; 44: 825-835. https://doi.

org/10.1016/j.clinre.2020.04.018.

21. Chen J, Li L, Yin Q, Shen T. A review of ep-

idemiology and clinical relevance of Hepa-

titis B virus genotypes and subgenotypes.

Clin Res Hepatol Gastroenterol. 2023;

47: 102180. https://doi.org/10.1016/j.

clinre.2023.102180.

22. Araujo NM. Hepatitis B virus intergeno-

typic recombinants worldwide: An

overview. Infect Genet Evol. 2015; 36:

500-510. https://doi.org/10.1016/j.

meegid.2015.08.024.

23. Locarnini SA, Littlejohn M, Yuen LKW.

Origins and Evolution of the Primate

Hepatitis B Virus. Front Microbiol. 2021;

12: 653684. https://doi.org/10.3389/

fmicb.2021.653684.