THE UNIVERSITY OF ZAMBIA

SCHOOL OF VETERINARY MEDICINE

ONE HEALTH LABORATORY DIAGNOSTIC

PREVALENCE OF ACQUIRED HUMAN IMMUNO DEFICIENCY DRUG

RESISTANCE TRENDS AMONG ADULTS IN THYOLO MALAWI

FELISTUS KANJIRA HUSSEIN

VET 2200029
INTRODUCTION

The coming in of Highly active antiretroviral therapy (HAART) that allows combination of at
least three antiretroviral drugs in 1996 has had a great impact on the prognosis of people affected
with the Human Immunodeficiency Virus (HIV) worldwide (Rodger et al., 2013). HIV
prevention and monitoring boost first line antiretroviral therapy (ART)’s long term effectiveness,
thereby improving the patient outcomes and reduce the spread of HIV drug resistant and in a
way guarantee the sustainability of ART programs (World Health Organization, 2015). Increase
in the use of antiretroviral drugs has however, also led to emergence of acquired HIV drug
resistant strains due to the mutation of the virus (WHO, 2021). Acquired drug resistance (ADR)
is as a result of resistance of the HIV virus to the drug which might come about due to poor
adherence of ART, incorrect dosing, drug to drug interactions and also absorption problems.
Effects of this has led to inadequate viral load suppression and increase in ongoing transmission.
(World Health Organization. , 2021). Both HIV1 and HIV2 have shown to develop resistance
against the antiretroviral drugs(Abana et al., 2019)

The Human immunodeficiency virus (HIV) is one of the major burden of public health and a
deadliest pandemic globally (Zaki et al., 2020) . Acquired Immune- deficiency syndrome (AIDS)
is the most advanced stage of HIV when the virus is not properly managed (Zaki et al., 2020).
Since the start of the HIV epidemic, 79.3 million people have become infected with the virus,
and in 2020 alone they were over 37.7 million people living with HIV worldwide including
10.2 million who were not on treatment, 1.5 million were new HIV infections and 650,000
AIDS related death (World Health Organization, 2015).

The sub-Saharan Africa is one of the mostly affected regions worldwide with two thirds (67%)
of people living with HIV (Update, 2021). Malawi has about 990,000 people living with HIV,
with 12,000 AIDS- related deaths annually although 86% of the people living with HIV are on
antiretroviral treatment (Frontline AIDS, 2020).

In Malawi the standard first-line ART regimens for adults are tenofovir disoproxil fumarate,
lamivudine, and dolutegravir (TLD) and this was introduced in 2018 according to WHO
recommendations, and is recommended as high genetic barrier to drug resistance(Schramm et
al., 2022).
The status of acquired drug resistance for first line treatment is yet unknown, limited access to
high technological instruments, few infrastructures to conduct drug resistance testing and the
high cost of these technologies has led to priotization of only viral loading test being conducted
annually (Gupta-Wright et al., 2020). The limitation of access to drug resistance testing has
major consequence with individuals who have been on the first line for the antiretroviral
treatment especially with high viral load. Consequences that result from unmonitored high viral
loads has implication in mutations accumulation resulting into resistance (Boender et al., 2016).
High levels of viral resistance circulating within the population will have impact on treatment
programs by increasing the chances of transmission of resistant strains and treatment failure

Monitoring the treatment is critical to ensure antiretroviral therapy given to individuals is

effective and viral load suppression is being achieved, however sequencing is critical for
studying persistent viral measures in patients with low or suppressed viral loads to determine
resistance and this has shown to be one of the effective predictor of future virological failure
(Taiwo et al., 2011). Previous studies that were conducted in Malawi have been more focused on
transmitted HIV drug resistance (Wadonda-Kabondo, Banda, et al., 2012). There is not much
data on acquired HIV drug resistance studies within the country. This study aims to investigate
HIV drug resistance among young individuals accessing services at Thyolo hospital for acquired
HIVDR and Viral load re-suppression who have received first-line ART for at least 6 months
and who have failed to re-suppress their viral load (≥1000 copies/ml.)

RESEARCH PROBLEM

HIV is one of the major global burdens that has been around for decades and the emerging in of
HIV drug resistance is a major concern to the scaling up of the combination antiretroviral
therapy(cART) in developing countries which is high in Africa (UNAIDS, 2021).Treatment
failure in HIV may result from antiretroviral drug resistance and if these are not detected at an
early stage may result into AIDs. Transmission rate also gains due to viremic individuals who are
infectious and mortality levels are increased as a result in increase of opportunistic infectious
microorganisms(Engla and Journal, 2017).
The data for acquired HIV drug resistance in Malawi is limited with only few articles of HIV
drug resistance more focused on transmitted HIV drug resistance that have been produced and
most of them were published a couple of years ago(Wadonda-Kabondo, Hedt, et al., 2012).
There is a need for new studies and with coming in of Covid 19 pandemic also had negative
impact on access to viral load and testing in the country (Ministry of Health and Population,
2022). It is however vital to conduct drug resistance test to those individuals with viral load test
> 1000 copies/mL on first line regimen, as this can aid on timely change and monitor of the
therapeutic drugs that they are taking in. It is vital to study in this subject to gain more insight on
the status of drug resistance pattern in the country especially in the districts with high prevalence
of HIV like Thyolo.

Major Objective

The main objective of the study is to determine the prevalence levels of acquired HIV drug
resistance in adults living with HIV in Thyolo.

Specific objectives

1. Detect high levels of viral load >1000 copies/mL in individuals who are on first line
antiretroviral treatment.
2. Detect drug resistance prevalence among individuals with viral load > 1000 copies /mL
3. Phylogenetic relationship of the mutation among individuals with acquired HIV drug
resistance.

METHODOLOGY

Study site

Malawi is located at the southern part of Africa and Thyolo is one of the districts that is found in
the southern part of Malawi. It is one of the districts with high HIV prevalence among the top
five high prevalent zones in the southern part of Malawi (Jerry John Nutor, 2020). Thyolo has
about 56 632 people accessing its ART services (Thyolo ART records). Malawi is among the
world's least developed countries and is one of the countries affected with HIV epidemic in
Africa.

Sampling Method

Analytical cross-sectional study design will be used were HIV-infected adults (≥15 years old to
40 years) who have been on first-line ART for more than six months and willing to participate in
the study will be sequentially and anonymously enrolled, Those with viral load result of ≥ 1000
copies/Ml samples. This involves dividing the population into subpopulations that will allow to
draw more precise conclusions by ensuring that every sub group is properly represented
(Taherdoost, 2020). The gender selection criteria will also be considered.

Sample analysis

Whole Blood samples (dried blood spot) will be quantified using Abbott Real Time HIV-1 assay
(Abbott Molecular, Des Plaines, IL, USA) with a quantification threshold of less than 839 copies
per mL and those samples with a viral load of >1000 copies/mL will be subjected for drug
resistance for the first line regime. Extraction will be achieved by using Easy Mag and the
samples will be amplified using the Verriti thermos cycler and for sequencing, the samples to be
tested for HIV drug resistance will be analyzed using an Applied Bio Systems Genetic Analyzer
model 3500/3500XL (Sanger sequencing). This will be done at the Malawi reference laboratory
unit in Lilongwe.

Sample size

The sample size refers to the number of participants to be included in the study. For overall
prevalence estimates, the expected prevalence of acquired HIV drug resistance varies widely
depending on the context and regimens in use in the country. For this reason, the expected
acquired HIV drug resistance prevalence is set at 50%, the point of maximum variability. This
means that the resulting required sample size, if the expected prevalence is set at 50%, will yield
a confidence interval no wider than ±6%, regardless of the actual observed prevalence. Using the
formulas presented by WHO results in a required overall sample size of 267 eligible case
specimens. Therefore, the required sample size for estimating the prevalence of overall acquired
HIV drug resistance is 267. Since not all specimens will successfully amplify, the required
sample sizes must be inflated to account for the genotyping failure rate. WHO recommends that
an anticipated genotyping failure rate of 30% be used in this calculation (WHO Team, 2021).

Data analysis

The data collected from records will be manually checked for missing, irrelevant and inaccurate
information. Data will be cleaned and checked for completeness before analyzing for the
detection levels of viral load and drug resistance pattern in the dried blood spots samples.

Results will be originally entered into SPSS database for further data cleaning, and statistical
analyses. The data will include both continuous and categorical variables such as age, will be
described using means and standard deviations, and medians with value ranges and interquartile
ranges (IQR’s). Categorical variables, such as virological failure and mutation, will be described
using frequency and percentage distributions. The results of the HIV drug resistance tests will be
reported using the Stanford HIV drug resistance database at http://hivdb.stanford.edu/.
According to (WHO) 2009 Surveillance Drug Resistance Mutation list, HIVDR was defined as
the existence of at least one drug resistance-associated mutation (DRM), using the Stanford
Calibrated Population Resistance analysis tool (version 4.1 beta, accessible at
http://hivdb.stanford.edu/).To check for acquired of drug resistance mutations, phylogenetic trees
will be reconstructed using Cluster for alignment and Mega software for phylogenetic
relationship. Confidence levels in the groupings in the phylogeny will be assessed using 1000
bootstrap replicates as part of the phylogeny reconstruction.

Expected outcomes

The study aims investigate HIV drug resistance in detectable viral load results and the outcome
can be used in any opportunity to create awareness on the importance of HIV drug resistance
detection in our health sector and also how to manage them to policy makers and the responsible
stakeholders in order to reach global 95% viral load suppression.

Timeline
Tas Description Progress Au Sep Oct No Dec Ja Feb Mar Apr May Jun Jul Au Sep Oct
k g v n g
No
1 Topic finding
1.1 Preliminary
research
1.2 Amend topic
focus based
on supervisor
feedback
1.3 Concept
approval
2 Research
proposal
2.1 Write up
research
objectives
2.2 Understanding
the need for
research
2.3 Studying
previous
research
3.0 Summarizing
previous
literature
3.1 Understanding
present
knowledge of
the research
area
3.2 Understanding
the knowledge
gap
3,3 Define the
scope and
limitations
4 Development
of
methodology
5 Identify the
resource and
budget
6 Ethical
clearance
Data
Collection
Data analysis
Results
interpretation
Results
validation and
verification
Presentation
Compiling the
research thesis

Budget

EXPENSES Description of Activities Duration Amount Requested

Personnel collecting samples and analysis 3 months K3500.00
Supplies for sample collection K2000.00
Transport From collection site to K3500.00
reference laboratory for
sequencing
Communication Airtime K1000.00
Materials and Reagents Analysis 2 months K15000.00
Stationery Write up , consent K2000.00
Dissemination cost K2000.00
Total cost K29000.00
References

Abana, C.Z. et al. (2019) ‘Drug resistance mutations and viral load in human immunodeficiency
virus type 2 and dual HIV-1/HIV-2 infected patients in Ghana’, Medicine (United States), 98(6).
Available at: https://doi.org/10.1097/MD.0000000000014313.

Boender, T.S. et al. (2016) ‘Accumulation of HIV-1 drug resistance after continued virological
failure on first-line ART in adults and children in sub-Saharan Africa’, Journal of Antimicrobial
Chemotherapy, 71(10), pp. 2918–2927. Available at: https://doi.org/10.1093/jac/dkw218.

Engla, N.E.W. and Journal, N.D. (2017) ‘New engla nd journal’, pp. 1605–1607.

Frontline AIDS (2020) ‘Malawi Hiv Prevention Shadow Report 2020 Hiv Prevention 10-Point
Plan’.

Gupta-Wright, A. et al. (2020) ‘Virological failure, HIV-1 drug resistance, and early mortality in
adults admitted to hospital in Malawi: an observational cohort study’, The Lancet HIV, 7(9), pp.
e620–e628. Available at: https://doi.org/10.1016/S2352-3018(20)30172-7.

Rodger, A.J. et al. (2013) ‘Mortality in well controlled HIV in the continuous antiretroviral
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40 years of AIDS’, Global Aids Update, pp. 1–386.

Wadonda-Kabondo, N., Hedt, B.L., et al. (2012) ‘A retrospective survey of HIV drug resistance
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Wadonda-Kabondo, N., Banda, R., et al. (2012) ‘Prevalence of transmitted HIV drug resistance
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https://doi.org/10.1093/cid/cir993.

WHO (2021) Technical report HIV drug resistance 2021, Geneva, Switzerland: World Health
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World Health Organization (2015) ‘Guideline on When To Start Antiretroviral Therapy and on
Pre-Exposure Prophylaxis for HIV’, (September), pp. 1–76.

Zaki, E.A. et al. (2020) ‘Genotyping and antiretroviral drug resistance of human
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