A multiplexed real-time PCR assay for simultaneous
quantification of human immunodeficiency virus and Hepatitis
B virus for low-and-middle-income countries

Djeneba Bocar Fofanaa,*, Tenin Aminatou Coulibalyb, Mamoudou Maigac, Thuy Nguyend,
Joël Gozlane, Zoumana Diarraa, Amadou Konéb, Yacouba Cissokoa, Almoustapha Issiaka
Maigaa, Claudia A. Hawkinsc, Robert L. Murphyc, Laurence Morand-Jouberte, Mahamadou
Diakitéa, Jane L. Hollf, Sally M. McFallc

aFaculty of Medicine, University of Sciences, Techniques and Technologies of Bamako (USTTB),
Bamako BP 1805, Mali

bUniversity Clinical Research Center, International Centers for Excellence in Research (UCRC),
University of Sciences, Techniques and Technologies of Bamako, Bamako, Mali

cInstitute for Global Health, Northwestern University, Chicago, IL 60208, USA

dClinical Retrovirology Section, HIV Dynamics and Replication Program, National Cancer
Institute, Frederick, MD, USA

eSorbonne Université, INSERM, Institut Pierre Louis d’Epidémiologie et de Santé Publique
(iPLESP), for Department of Virology, Assistance Publique-Hôpitaux de Paris (AP-HP), Saint-
Antoine Hospital, Paris F-75012, France

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
*Correspondence to: USTTB, Bamako BP 1805, Mali., djesfof@gmail.com (D.B. Fofana).
Author statement
Hereby, I declare that the resubmitted manuscript has been carefully revised and I agree to proceed to the next step of the review.

CRediT authorship contribution statement
Djeneba Bocar Fofana: Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Validation, Writing
– original draft. Tenin Aminatou Coulibaly: Data curation, Formal analysis, Validation, Writing – review & editing. Mamoudou
Maiga: Conceptualization, Funding acquisition, Supervision, Writing – review & editing, Visualization. Thuy Nguyen: Formal
analysis, Resources, Writing – review & editing. Joe¨l Gozlan: Resources, Writing – review & editing. Zoumana Diarra: Resources,
Writing – review & editing. Amadou Koné: Resources, Writing – review & editing. Yacouba Cissoko: Resources, Writing –
review & editing. Almoustapha Issiaka Maiga: Resources, Funding acquisition, Writing – review & editing. Claudia A. Hawkins:
Resources, Writing – review & editing. Robert L. Murphy: Resources, Writing – review & editing. Laurence Morand-Joubert:
Resources, Writing – review & editing. Mahamadou Diakité: Resources, Writing – review & editing. Jane L. Holl: Resources,
Writing – review & editing. Sally M. McFall: Conceptualization, Formal analysis, Funding acquisition, Methodology, Validation,
Supervision, Writing – review & editing

Ethics approval
This study was approved by the ethics committee of the faculty of medicine and odonto-stomatology (FMOS) of the University of
sciences, techniques, and technologies of Bamako (USTTB), Mali under the number N°2021/ 175 / EC/USTTB.

Appendix A. Supporting information
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jviromet.2024.115026.

Data statement
All data and code used for producing the results are freely available for others upon request. Request can be sent to these emails:
http://sally@northwestern.edu and/or http://djesfof@gmail.com.

Declaration of Competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper.

HHS Public Access
Author manuscript
J Virol Methods. Author manuscript; available in PMC 2025 June 02.

Published in final edited form as:
J Virol Methods. 2024 December ; 330: 115026. doi:10.1016/j.jviromet.2024.115026.

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fBiological Sciences Division University of Chicago, IL 60637, USA

Abstract

Due to shared routes of transmission, including sexual contact and vertical transmission, HIV-

HBV co-infection is common, particularly in sub-Saharan Africa. Measurement of viral load

(VL), for both HIV and HBV, plays a critical role for determining their infectious phase and

monitoring response to antiviral therapy. Implementation of viral load testing in clinical settings

is a significant challenge in resource-limited countries, notably because of cost and availability

issues. We designed HIV and HBV primers for conserved regions of the HIV and HBV genomes

that were specifically adapted to viral strains circulating in West Africa that are HIV-1 subtype

CRF02AG and HBV genotype E. We first validated two monoplex qPCR assays for individual

quantification and, then developed a multiplex qPCR for simultaneous quantification of both

viruses. HIV RNA and HBV DNA amplification was performed in a single tube using a one-step

reverse transcription-PCR reaction with primers and probes targeting both viruses. Performance

characteristics such as the quantification range, sensitivity, and specificity of this multiplex qPCR

assay were compared to reference qPCR tests for both HIV and HBV viral load quantification.

The multiplex assay was validated using clinical samples from co- or mono-infected patients

and gave comparable viral load quantification to the HIV and HBV reference test respectively.

The multiplex qPCR demonstrated an overall sensitivity of 71.25 % [68.16–74.3] for HBV and

82 % [78.09–85.90] for HIV and an overall specificity of 100 % [94.95–100] for both viruses.

Although the overall sensitivities of the HIV and HBV assays were lower than the commercial

comparator assays, the sensitivity in the clinical decision range of >1000 copies/mL for HIV was

80 % [71.26–88.73] and >1000 IU/mL for HBV was 100 % [95.51–100] which indicates the test

results can be used to guide treatment decisions. This in-house developed multiplex qPCR assay

represents a useful diagnostic tool as it can be performed on affordable “open” real-time PCR

platforms currently used for HIV or SARS-Cov-2 infection surveillance in Mali.

Keywords

HBV DNA; Hepatitis B virus; Real-time PCR; Viral load

1. Introduction

Human immunodeficiency virus and acquired immuno-deficiency syndrome (HIV/AIDS) is

a leading cause of morbidity and mortality in sub-Saharan Africa (SSA), where 75 % of

AIDS related-deaths and 65 % of HIV new infections occur and where 71 % of people living

with HIV (PLHIV) reside (Full report, 2023, 2023). The Joint United Nations Program on

HIV/AIDS (UNAIDS) fast-track strategy has set diagnosis and treatment targets for 2020

and 2030, with the goal of markedly reducing both new infections and deaths by 2030 (Full

report, 2023, 2023). Despite these goals, a recent review concluded that the world is not

on track to end the HIV epidemic. Moreover, the 95–95–95 targets, in which 95 % of all

PLHIV should know their HIV status; 95 % of all diagnosed individuals receive sustained

antiretroviral therapy (ART); and 95 % of treated individual should have viral suppression,

are unlikely to be reached in SSA by the end of 2030, as planned (Full report, 2023,

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2023). This failure is in large part due to the lack of appropriate diagnostic and treatment

monitoring tools in the highest prevalent areas.

Chronic Hepatitis B infection, caused by the Hepatitis B virus (HBV), usually remains

asymptomatic for decades before leading to serious complications such as cirrhosis,

hepatocellular carcinoma (HCC), and death (Lemoine and Thursz, 2017). Due to shared

routes of transmission, including sexual contact, body fluid exposure, needle sharing, and

vertical transmission, HIV-HBV co-infection is common (Anon, 2023; Platt et al., 2020),

particularly in SSA with approximately 8 % of people living with HIV (PLWH) are also

living with HBV (Platt et al., 2020). Up to 25 % of people with chronic hepatitis B

(PCHB) die prematurely (Terrault et al., 2018) and those living with both viruses have

higher mortality and morbidity compared to those living with either HIV or HBV alone.

Knowledge of HBV status at initiation of HIV ART is important for selection of initial

treatment, as patients with co-infection should be treated with ARV combination containing

tenofovir disoproxil fumarate (TDF) +/− lamivudine (3TC) or emtricitabine (FTC), which

suppress both HIV and HBV replication (Singh et al., 2017).

Quantification of HIV and HBV viral loads (VL), is a key element to determine the stage of

HBV/HIV infection/disease, determining eligibility for and monitoring responses to antiviral

therapy. Real-time PCR assays are very sensitive, with as little as a few molecules of viral

DNA or RNA being detected. Several in-house PCR viral load assays have been developed

for HIV (Barnor et al., 2014) or for HBV (Lole and Arankalle, 2006).

Due to the high frequency of HIV and HBV co-infection in SSA, expanding access to

plasma viral genome testing to detect co-infection and to monitor response to antiviral

treatments is critical (Anon, 2023). The capacity for laboratory-based HIV viral load testing

has increased in low- and middle-income countries, but implementation of universal viral

load monitoring is still hindered by several barriers. In Africa, particularly in Mali, where

molecular diagnostics are often more expensive than the cost of a year of treatment of HBV,

affordable tests such as “homemade PCR” could be useful. Indeed, despite the availability of

conventional tests for decades, access to these tools remains limited due to their cost.

An accurate, low-cost, and easy to use assay for simultaneous quantification of both

HIV and HBV VLs to monitor patients’ response to treatment is therefore urgently

needed. However, the introduction of a new diagnostic or monitoring molecular tool in

low-resource settings can represent considerable challenges. Indeed, developers from high-

income countries (HICs) often struggle to consider the regional diversity of pathogens

when developing the molecular tools to quantify viral load (VL). The development of those

tools must also consider the developing pricing strategies, implementing, and supporting

products in healthcare systems with limited infrastructure. Thus, local design, development

and implementation of tools, such as PCR assays in the endemic countries is highly

advantageous. These technologies have the potential to expand viral load coverage and

improve viral suppression.

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In this paper, we report the development of an affordable, multiplex, real-time PCR assay for

simultaneous quantification of HIV and HBV, which can be widely used in resource-limited

countries.

2. Materials and methods

2.1. Assay standards

2.1.1. Standards for HIV quantification: 8E5-LAV cells, harboring a single copy of
the HIV-1 provirus per cell, were obtained from the Virology Quality Assurance Laboratory

(VQA; Rush Presbyterian/St. Luke’s Medical Center, Chicago, IL), as frozen cell pellets of

4000 cells/uL. The cell count was verified, as described previously (Jangam et al., 2009;

McFall et al., 2016). Cells were diluted in freezing medium (90 % fetal bovine serum,

10 % dimethyl sulfoxide), to concentrations ranging from 0.5 to 400 cells/μL and spiked

into the fresh HIV-1-negative EDTA-treated whole blood samples (Core Lab, North-Shore

University HealthSystems, Evanston, IL). HIV-DNA were extracted directly from a known

quantity of 8E5 cells reconstituted in whole blood and the eluates were diluted to create at

standard at 5–4000 HIV copies/uL of extraction. These HIV standards were used to validate

the primers/probes and testing of their effectiveness.

2.1.2. Standards for HBV quantification: The AccuSpan™ HBV DNA Linearity
Panel (genotype E) consisted of HBV-genotype E plasma samples (HBV DNA Linearity

Panel PHD802; SeraCare Life Sciences, Milford, MA). The panel includes 8 standards

(PHD802–01 to PHD802–07 and PHD802–09, WHO International Units (IU)) containing

1.1 × 108 U/mL (8.00 log IU/ mL), 8.6 × 107 IU/mL (6.90 log IU/mL), 8.6 × 106 IU/mL

(5.90 log IU/mL), 1.4 × 105 IU/mL (5.15 log IU/mL), 1.9 × 104 IU/mL (4.30 log IU/mL),

1.4 × 103 IU/mL (3.15 log IU/mL), and 1.5 × 102 IU/mL (2.20 log IU/mL) of HBV DNA

and no HBV DNA, respectively. These standards were used to validate HBV primers/probes,

to test their efficacy, and as a clinical standard for testing clinical samples of unknown viral

load.

2.1.3. Clinical samples panel: We validated the assay using archived serum samples
obtained from a prior Malian clinical cohort that included 80 samples from PCHB (viral

load range 7–100 million IU/mL, 1–8 logs measured by HBV Real-TM Quant Dx®, Sacace
Biotechnologie), 50 samples from PLWH (viral load range 40–10 million copies/mL, 1.60–
7logs measured by m2000 Abbott RealTime System), and 30 samples from HIV/HBV
uninfected people. CHB samples were collected from HBsAg positive individuals with

HBV genotype E and HIV samples were collected from naive or treated individuals. HIV

RNA and HBV DNA levels were measured with the commercial diagnostic tests described

above and these values were used to establish the multiplex assay’s clinical sensitivity and

specificity.

2.1.4. Internal control: All clinical samples were tested for human RNase P gene
(included in the Trioplex rRT-PCR kit, CDC) to assess the specimen quality and nucleic acid

extraction and amplification efficiency by spiking the RNase P primer and probe set into the

qPCR Master mix.

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2.1.5. Viral nucleic acid isolation: Total nucleic acids were isolated from 200 μL
of serum/plasma using the RNA QIAGEN KIT (Qiagen, Germany) and the Dynal Silane

Viral NA kit (Thermo Fisher Scientific, USA), respectively, according to the manufacturers’

instructions. We also used the QIAamp Circulating Nucleic Acid Kit (50) or QIAamp

DSP Virus Spin Kit for purification of viral nucleic acids following the manufacturer’s

instructions. Viral nucleic acids were eluted from the filter column with 100μL of nuclease-

free double distilled water. Different methods of nucleic acid isolation were used, due to the

temporary unavailability of different kits in Mali.

2.2. Selection of primers and probes for the HIV-1 and HBV qPCR assay

Sequences of HIV and HBV primers and probes from the literature were verified by

mapping them against sequences of HIV and HBV strains circulating in West Africa,

specifically HIV genotype CRF02_AG and HBV genotype E (Fofana et al., 2013; Traoré

et al., 2015) using Blast. Primer selection was based on conserved regions of viral

genomes to optimize the quantification of viruses of different genotypes. For the HIV

assay, we selected primers targeting long-terminal repeats (LTR) and gag genes (Barnor
et al., 2014). For the HBV assay, we selected primers targeting pre core/core genes, as

previously validated (Lole and Arankalle, 2006). Additionally, we excluded HBV primers/

probes located in regions known to contain drug-induced mutations, and/or premature stop

codons. As the primers and probes sets for HIV and HBV would be multiplexed, we

also checked the specificity of HIV and HBV primers and probes against HBV and HIV

genomes, respectively. We only selected primers and probes that targeted specifically HIV

or HBV and did not have any nonspecific binding with the other virus. Forward (Fv)

and reverse (Rv) primers with appropriate melting temperatures (Tm) and GC percentages

were selected using Primer Express 3.0 software (Applied Biosystems®). Eight primer

sets (4 for each virus), derived from the literature, were first adapted to the main

genotype (E) circulating in West Africa, then evaluated by monoplex end-point PCR

and qPCR assays (Table 1 supplementary data). The best primers sets were selected for

further validation in a multiplex assay (HIV-Fv5’--CAAGCAGCCATGCAAATGTTAA-3’,

HIV-Rv5’-AGTAGTTCCTGCTATGTCACTTCCC-3’ andHBV-

Fv5’TAGGAGGCTGTAGGCATAAATTGG-3′, HBV-Rv5’-
GCACAGCTTGGAGGCTTGA-3′). HIV and HBV fluorogenic probes were 5′-labelled
with FAM and VIC/HEX dyes, respectively and 3′-labelled with BKFQ dye (HIV-
probe 5’-/56-FAM/CAGTGCATG/ZEN/CAGGGCGTATTGCACCAG/3IABKFQ/−3’ and

HBV-probe 5’-/5HEX/TGACCTCTGCCTAATC/3MGBEC). These dyes are chosen to be

compatible with most real-time PCR instruments and for the assay applicability.

2.3. Development of a monoplex RT-qPCR assay for HIV and HBV

We evaluated primers and probes for the monoplex RT-qPCR and qPCR assays using

clinical standards (a serially diluted sample with known high HIV viral load) and technical

standards (HIV DNA extracted from 8E5-LAV cells and AccuSpan™ HBV DNA Linearity

Panel), as described in Sections 2.1.1 and 2.1.2.

Each monoplex qPCR reaction was composed of 20ul Master Mix, FastVirus 4X (Thermo

Fisher Scientic Balicus UAB V.A. Graciumo 8. LT02241 Vihnius. Lithuania) according to

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the manufacturer’s instructions, primers and probes were added at final concentrations of

200 and 250 nM, respectively, and 5uL of extracted DNA/RNA. The qPCR reaction was

performed on a Rotor-Gene Q MDx 5plex HRM (CA) (Qiagen technologies Canada) with

the cycling conditions including a reverse transcription (RT) step at 50°C for 5 minutes, at

95°C for 20 seconds with 40 cycles of annealing/extension at 95°C for 15 seconds, and then

at 55°C for 1 minute.

2.4. Multiplex qPCR assay development

The development of multiplex qPCR was performed using FastVirus 4X (Thermo Fisher

Scientific Balicus UAB V.A. Graciumo 8.LT- 02241 Vihnius. Lithuania) under the same

conditions as the monoplex qPCR experiment. The PCR reaction contained 12.5 μL of mix

with a final concentration of 200 nM primers and 250 nM probe each, nuclease-free water

adjusted to a final volume of 25 μL and 5 μL RNA/DNA (total nucleic acid) or molecular

grade water as negative control. The amplification program was RT step at 50°C for 5

minutes, for 95°C for 20 seconds with 40 cycles of at 95°C for DNA denaturation for 15

seconds and then annealing/extension at 55°C for 1 minute. The detection channels of the

probes used were the FAM and HEX for HIV and HBV, respectively. A housekeeping gene,

RNase P, was used as an internal control detected by Cy5. The multiplex qPCR experiments

were performed on the Applied Biosystem 7500 FAST and LightCycler 480 II (LC480)

instruments.

2.4.1. Validation of standard curves: We validated a clinical standard curve for
both HIV and HBV quantification in triplicates and in three independent experiments. We

constructed an HIV standard curve from a high titer sample obtained from an antiretroviral

treatment-naive patient. This sample was determined by commercial assay (m2000 Abbott
RealTime System) to contain until 1.21E7 copies of HIV RNA/mL of plasma. A 4-point
standard curve in triplicate was generated from tenfold serial dilutions of this sample which

was used to calculate the unknown viral loads of the clinical samples. For HBV, we used

AccuSpan™ HBV DNA Linearity Panel. Ct values, the cycle at which there is a statistically

significant increase in fluorescence, were plotted against the logarithm of the standard’s

concentrations. The slope, calculated from linear regression of samples’ concentrations

against Ct values, was considered as an indicator of amplification or PCR efficiency. Slopes

between −3.1 and −3.6, which indicated PCR efficiency between 90–110 % were considered

acceptable (Raymaekers et al., 2009). The log viral IU/mL for HBV or log copy number/mL

for HIV were calculated from the quantification cycle using the respective standard curve

equations (Raymaekers et al., 2009; Neto et al., 2017).

2.4.2. Determination of the limit of detection of the monoplex qPCR assays:
The detection limits of the monoplex assays were determined by testing limiting dilutions of

assay standards corresponding to 40 to 4 million copies/mL and 100 to 10e8 IU/mL plasma

for HIV RNA and HBV DNA, respectively.

2.4.3. Validation of assay repeatability and reproducibility: To assess precision
(intra-assay reproducibility), each sample from the Seracare® HBV DNA standard panel and

the developed HIV standard were tested in triplicate. To assess inter-assay reproducibility, a

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set of three HBV and HIV daily controls, including a negative control, a low-positive control

(Seracare LPC for HBV and a dilution of the standard for HIV), and a high-positive control

(Seracare HPC for HBV and 1.21E7 copies/mL for HIV) were tested on three different

days. Based on a linear regression, a conversion formula was calculated for the new qPCR

measurements (copies/mL) to the international standard units (IU/mL). This experiment was

repeated with clinical specimens.

The tests were conducted Applied Biosystems® 7500 Fast Dx Real-Time and Cepheid

SmartCycler Real-Time, as part of the existing infrastructure in our setting (SEREFO/

UCRC, Mali). They were also tested on other PCR instruments (qPCR machine,

RotorGene), currently available at the Center for Innovation in Global Health Technologies

(CIGHT) at Northwestern University in Evanston, Illinois USA and at other collaborative

laboratories from Mali and France (Laboratory of virology of Saint-Antoine Hospital) to

ensure the reproducibility and adaptability of the test.

2.5. Validation of the multiplex qPCR assay using clinical sample

We assessed the clinical specificity of both assays using 30 negative samples for HIV

and HBV and samples positive for other viruses HSV-1, HSV-2, HCV, and EBV that are

commonly found in blood specimens (Supplemental Data).

The analytic performance of the monoplex and multiplex qPCR assays were compared

to routine diagnostic assays of HIV, HBV, and HIV/HBV co-infection using the Abbott

RealTime HIV Viral Load Assay and HBV Real-TM Quant Dx®, by analyzing specimens at

various VLs.

2.6. Statistical analysis

Descriptive statistics are shown as the mean ± standard deviation (SD) or the median

and interquartile range, as appropriate, and provided by software. The limit of detection

was determined by means of Log analysis as the 95 % point estimate with a surrounding

95 % confidence interval, provided by the software which performs the quantification

by PCR. Sensitivity, specificity, and their 95 % confidence interval were calculated. We

used a Kruskal-Wallis test to determine whether or not there was a statistically significant

difference between the different experiments of repeatability.

3. Results

3.1. Development of a monoplex RT-qPCR assay for HIV and qPCR assay for HBV
quantification

We first tested different sets of primers by endpoint PCR and selected HIV Set 3 and HBV

Set 2 for the RT-qPCR assay based on the intensity of bands (suppl Data) and lack of

non-specific amplification products.

Quantification curves were constructed from linear regression analysis of 5- and 7-fold serial

dilutions for HIV and HBV, respectively. The efficiencies of the monoplex tests ranged from

0.98 to 1.05, corresponding to 98–105 % of PCR amplification efficiency (Fig. 1A, C).

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Statistical analysis of each standard curve revealed a high correlation coefficient (values R)

and a high PCR efficiency for both HIV-RNA and HBV-DNA amplification.

Regression coefficient for each standard curve is indicated (B, D). Amplification plots

generated with the Rotor Quantitative PCR System instrument from samples containing

HBV DNA (HEX signal) and HIV-1 RNA (FAM signal). The primers and probes were

found to be highly sensitive for VL above 1000 copies/mL or IU/mL, according to the

cycle threshold (Ct) values obtained with VL ranging from 40 to 4,000,000 copies/mL for

HIV and 100 to 100,000,000 IU/mL for HBV in accordance to HBV and HIV clinical

guidelines. The non-template control (NTC) showed no amplification. The experiment was

reproducible, as indicated by the low standard deviation among triplicates (Tables 1 and 2).

3.2. Specific detection of HIV and HBV by the multiplex qPCR assay

Additional experiments were performed to evaluate the impact of multiplexing on PCR

efficiency, such as the absence of cross reactivity of HIV and HBV primers/probes. For this

purpose, HBV primers/probe and/or HBV DNA (S5: 1,00E+03 IU/mL) were added at the

same concentration of HIV_RNA (S1: 4,00E+05 copies/mL) and HIV primers/probe mix.

We did a similar experiment where HIV primers/probe and/or HIV RNA (S3: 4,00E+03

copies/mL) were added at the same concentration of HBV_DNA (S1: 1,00E+07 IU/mL)

and HBV primers/probe mix. The experiments showed similar HIV Ct values despite the

addition of HBV primers/probe and HBV DNA (Table 3). Those results showed no cross

reactivity of HIV primers-probe with HBV template nor HBV primers-probe with HIV

template was detected even at high template concentrations.

3.3. The assays have high repeatability on clinical samples

To assess the repeatability of the assays, we performed three independent experiments in

triplicate on clinical samples from either mono or co-infected with HBV and HIV. The Ct

values obtained for both HIV and HBV quantification were nearly identical. Slope data,

efficiency and correlation between these serial dilutions were also similar (Supplementary

Table S1).

Three independent runs were performed on serial dilutions of clinical samples in triplicates.

The average Ct values between analyzes are shown. Results were identical according to

Friedman test with Dunn’s multiple comparisons test Fig. 2.

3.4. Clinical sensitivity and specificity of the in-house multiplex methods

Seventy-four HBV and fifty HIV clinical samples from infected patients were tested

separately using a reference method approved commercial assay (HBV Real-TM Quant Dx®

or Abbott HIV-1 M2000rt® with clinical cut-offs 12 UI/mL and 50 copies/mL respectively)

and our in-house multiplex qPCR. The results with our new qPCR were compared to VL

results obtained from reference methods. The results showed a very high correlation between

the different methods (Fig. 3). Nearly all clinical samples above 1000 copies/mL for HIV

and 1000 IU/mL of HBV were detected and quantified by our assay. Thirty persons, not

infected by HIV or HBV were also negative using our in-house. Our in-house methods failed

to detect samples with very low viral loads. Twenty-three of the 74 (31 %) samples tested

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had HBV VL <600 UI/mL and were not detected. Additionally, 9 of the 50 samples (18 %)

had HIV VL≤1000 copies/mL and were not detected. All infected patients with negative VL

(n = 11) were also undetectable using our HIV and HBV qPCR method (Table 5).

Only samples which had a detectable viral load for both the reference and multiplex assays

are included in the analysis.

4. Discussion

Implementation of viral load testing in clinical settings at a national level is a significant

challenge in resource-limited countries. Current commercial VL assays can only be

performed on closed real-time PCR systems, which are expensive and require proprietary

machines and reagents. Additionally, the cost of those assays is often higher than the cost

of treatment itself, which impedes their use in low-income countries. Several “in-house” VL

assays have been developed for HIV (Barnor et al., 2014; Zhou et al., 2015; Cobb et al.,

2011) or for HBV (Lole and Arankalle, 2006; Sun et al., 2011; Daniel et al., 2009), but

few methods have been developed to simultaneously quantify both HIV and HBV or been,

adapted to the circulating strains in West Africa. A multiplex assay to detect HBV, HCV and

HIV-1 has been previously reported, however the assay protocol contained separate steps for

amplification and detection, which was labor-intensive and time-consuming (Defoort et al.,

2000). Here, we describe a simple simultaneous quantification assay of HIV and HBV viral

loads that offers substantial benefits in reducing experimental and sampling costs, which

may be adapted for use at both national and regional levels. The assay provides significant

benefits for the clinical management of people living with HIV and HBV, which account for

a large number of individuals in SSA (Matthews et al., 2014).

In SSA, due to the high burden of HBV chronic infection and the limited resources allocated

to the elimination of this disease plus logistical constraints, lack of clear policies, and

the prevalence of home births, many countries have not successfully implemented HBV

vaccination at birth to prevent mother-to-child transmission (Candotti et al., 2004). Access

to VL testing to determine the need for antiviral therapy in pregnant women during the later

stages of pregnancy is essential to prevent mother-to-child transmission, especially where

access to newborn vaccinations is not available (Cheung et al., 2019). Hence, our assays are

clinically relevant both for screening for HBV and for identifying pregnant women requiring

treatment for HBV.

Despite extensive efforts to roll out HIV VL in Africa, there is still inequality among

countries and regions in access to testing and diagnostic reagents. To achieve the call

for UNAIDS 95/95/95 targets, access to VL testing must therefore be promoted. A

cost-effective VL test such as our multiplex qPCR (in-house multiplex assay) could be

tremendously beneficial for achieving this target. Furthermore, HIV VL is extremely

important to identify people living with HIV who are not adherent to ART or who develop

ART resistance. In SSA, ART adherence is the most common reason for therapeutic and

virological failure and is the most challenging issue in clinical management of PWH on ART

(Damulak et al., 2021). Hence, HIV viral load testing will be essential for early detection of

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this problem, which will in turn reduce the risk of drug resistance mutations development in

non-adherence individuals.

Our approach for simultaneous quantification of HBV and HIV VLs constitutes the first step

towards the development of a reliable tool for VL testing that is feasible for low-resource

settings. The advantages of our assay include (1) use of fewer reagents and consumables

and (2) less processing time and labor resulting in significant reduction of the assay cost

without compromising assay efficiency (Elnifro et al., 2000). Additionally, this assay was

first adapted targeting conserved HIV and HBV strains that were circulating in West Africa

particularly in Mali, such as HIV-1 subtype CRF02AG and HBV genotype E. Previous

studies have demonstrated the utility of identifying viral pathogens in various clinical and

epidemiological settings using multiplex PCR assays (Elnifro et al., 2000). This method

quickly became one of the most important methods for the detection of pathogens (Irshad

et al., 2016; Tombuloglu et al., 2021). Therefore, low-cost and efficient in-house qPCR

assays that can be used on open platforms are needed to support full implementation of VL

monitoring in such settings. In Africa, the unit cost of the HIV or HBV VL measurement

is ~$50-$100 for both tests including all steps from genome extraction. We estimate that

the cost of our multiplex qPCR is ~$30 for quantification of both viruses and ~$20 for a

single quantification. However, the effective introduction of this tool in clinical practice will

require other multi-center studies, involving a large number of samples to fully evaluate its

clinical performance and its clinical relevance, as well as, to assess the cost-benefits of the

tool in real-world settings.

A limitation to our in house multiplex assay is that compared to the commercial

comparators, our test showed lower analytical sensitivity when the viral load was lower

than 1000 copies/mL or 1000 IU/mL for HIV and HBV respectively. This may be because

we were restricted to using 200 μL of plasma samples for nucleic acid extraction compared

to 1000 μL used by commercial devices. Fig. 3 demonstrates high correlation between the

in-house multiplex assay and the commercial assays for both HIV and HBV quantification

which suggests that the inhouse assay accurately quantifies the viral targets across a broad

range of input concentrations. This is consistent with the lower assay sensitivities for HIV

and HBV detection being caused by the lower specimen volume used in the inhouse assay

compared to the commercial ones.

The HIV RNA amplification showed higher intra- and inter -assay variation than HBV

DNA amplification. This may be due to the long-term storage of clinical samples, which

could compromise the stability of HIV RNA more than HBV DNA, suggesting that the

RNA quality may be a source of variability across experiments. However, the assay was

still able to detect HIV RNA at a level of >1000 copies/mL which is above the threshold

for classification of virological failure on ART by WHO. The sensitivity of our assay will

certainly be improved by using fresh samples and larger volumes of samples (Matthews

et al., 2014). For HBV, the results show a high reproducibility as indicated by the low

standard deviation of cycle threshold (Ct) among replicates, from 0.01 for high VL to 0.83

delta Ct when the VL is very low. Therefore, we believe that the use of this assay could

be used in the clinical management of HIV and HBV infection. Importantly, we have also

demonstrated the robustness of the assay on different qPCR platforms, such as the ABI

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Applied Biosystems™ 7500 Real-Time PCR System, Roche Light Cycler 480 or Qiagen

Rotor-Gene Q, suggesting the feasibility of implementation of the assay at both national and

regional levels.

5. Conclusion

We developed and validated a specific and sensitive multiplex qPCR assay to simultaneously

monitor HIV and HBV VLs in people with HIV and HBV. This qPCR assay uses a mixture

of two pairs of primers and probes for HIV and HBV specifically designed for the regional

viral genetic diversity in West Africa. These assays can be performed on affordable, “open”

real-time PCR platforms such as what is currently used for monitoring HIV in Mali to also

perform low-cost molecular tests for HBV.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

The authors are grateful to Northwestern University’s Institute for Global Health program, Catalyzer project, the
Agence Nationale de la Recherche sur le SIDA et les Maladies Infectieuses Emergentes (ANRS-MIE). We also
thank the team of the University Clinical Research Center (UCRC) of the University of Sciences, techniques,
Technologies of Bamako (USTTB) Mali for their valuable scientific and technical assistance during the study.

Funding

This research was supported by Fogarty International Center, grant number: K43TW011957 for DBF, the National
Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Number
U54EB027049 and the Fogarty International Center of the National Institutes of Health under award number
Building the Next Generation of Researchers in TB/HIV Diagnostics in Mali (B-NextGen) Mali, D43TW010350,
Agence Nationale de la Recherche sur le SIDA et les Maladies Infectieuses Emergentes (ANRS MIE) ANRS-
MIE22295. The content is solely the responsibility of the authors and does not necessarily represent the official
views of the National Institutes of Health.

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Fig. 1.
Standard curves of HIV (A, B) and HBV (C, D). qPCR curves of serial dilutions of HIV and

HBV standards, respectively (A,C). The x-axis is the cycle number of the amplification; the

y-axis is the increase in fluorescence (dRn). Threshold cycle number (Ct) were plotted vs.

serial dilutions of RNA HIV-1 or DNA HBV (B, D).

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Fig. 2.
Repeatability and Reproducibility experiment of HIV qPCR (4 A) and HBV qPCR assay

using clinical samples.

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Fig. 3.
Correlation between two methods of virus quantification: A) HIV/HBV Multiplex Assay

(FAM signal) versus Abbott RealTime HIV Viral Load Copies/mL (y). R2 = 1.00; B)

HIV/HBV Multiplex Assay (HEX signal) versus HBV Real-TM Quant Dx® (y) IU/mL R2 =

0.99.

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Table 1
Threshold cycle values for serial dilutions of HIV standard.

Target Name Ct value Concentration (Copies/mL) Mean of Ct value Mean of Standard deviation Ct value

HIV_RNA-S 22,61 4×106 22,6 0,04

HIV_RNA-S 22,56 4×106

HIV_RNA-S 22,63 4×106

NTC

HIV_RNA-S1 25,68 4×105 25,7 0,04

HIV_RNA-S1 25,69 4×105

HIV_RNA-S1 25,75 4 ×105

NTC

HIV_RNA-S2 28,95 4×104 28,8 0,15

HIV_RNA-S2 28,81 4 ×104

HIV_RNA-S2 28,65 4 ×104

NTC

V_RNA-S3 31,98 4×103 32,26 0,38

HIV_RNA-S3 32,7 4 ×103

HIV_RNA-S3 32,1 4 ×103

NTC ND

HIV_RNA-S4 ND 4×102

HIV_RNA-S4 35,65 4 ×102

HIV_RNA-S4 34,98 4 ×102

NTC

HIV_RNA-S5 ND 40

HIV_RNA-S5 ND 40

HIV_RNA-S5 ND 40

S1, S2, S3, S4 and S5 represent ten-fold serial dilutions of HIV RNA Standard, which contains 4,000,000 copies/mL.

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Table 2
Threshold cycle values for serial dilutions of HBV standard.

Target Name Ct values Concentration (IU/mL) Mean of Ct value Mean of Standard deviation Ct value

HBV DNA-S 8,26 108 8,32 0,05

HBV DNA-S 8,37 108

HBV DNA-S 8,33 108

NTC

HBV DNA-S1 12,74 107 12,77 0,05

HBV DNA-S1 12,75 107

HBV DNA-S1 12,83 107

NTC

HBV DNA-S2 16,41 106 16,4 0,01

HBV DNA-S2 16,38 106

HBV DNA-S2 16,4 106

NTC

HBV DNA-S3 19,07 105 19,21 0,13

HBV DNA-S3 19,33 105

HBV DNA-S3 19,24 105

HBV DNA-S4 22,56 104 22,57 0,04

HBV DNA-S4 22,54 104

HBV DNA-S4 22,62 104

NTC

HBV DNA-S5 26,15 103 26,59 0,61

HBV DNA-S5 27,02 103

HBV DNA-S6 28,76 100 29,35 0,83

HBV DNA-S6 29,93 100

NTC

S1, S2, S3, S4, S5 and S6 represent different ten-fold serial dilutions of primary HBV DNA solution (DNA S). The results show an efficient primer
and probe sets with a high reproducibility as indicated by the small standard deviation among replicates, from 0.01 for high VL to 0.83 when the
VL is very low. The no template control (NTC) had no amplification.

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J Virol Methods. Author manuscript; available in PMC 2025 June 02.

A
uthor M

anuscript
A

uthor M
anuscript

A
uthor M

anuscript
A

uthor M
anuscript

Fofana et al. Page 21

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J Virol Methods. Author manuscript; available in PMC 2025 June 02.