Genetic Diversities and Spatiotemporal distribution of antimalarial drug

resistant gene mutations in a Plasmodium falciparum parasite population

in Ethiopia: A Drug surveillance epidemiological and TES study

Advisors: Fekadu Massebo (PhD, Associate Professor, Arba Minch University, Ethiopia)
Fitsum Girma (PhD, Assistant Professor, Armauer Hansen Research Institute)

February, 2022
ARBA MINCH, ETHIOPI
1 CHAPTER-1

Spatiotemporal distribution of antimalarial drug resistant gene mutations in a P. falciparum

parasite population in Ethiopia: Molecular Epidemiology and TES Study

SUMMARY
Background: Recent research has revealed that the R561H artemisinin-associated resistance
marker has made its first appearance in Africa, highlighting the significance of ongoing genetic
surveillance to assess the selection and spread of this and other drug resistance indicators in the
region. Spatiotemporal changes in Drug resistance Markers can aid policymakers in selecting
effective antimalarial drugs and slowing the spread of multidrug-resistant parasites. In Ethiopia,
little is identified about malaria drug resistance molecular markers. Part-I of the study will assess
the prevalence of Drug resistance Markers of anti-malarial drug resistance (PfK13, PfCRT,
PfMDR1 PfDHFR, PfDHPS, Plasmepsin2, Mitochondrial genome (PfCYT-B)) in samples archived
from previous therapeutic efficacy study and current cross-sectional samples. The study will also
be designed to investigate spatiotemporal distributions along with drug resistance loci genotypic
linkage. Part-II of the Study is intended and designed to i) assess gametocyte carriage in locally
recruited gametocyte doner patient as proxy markers of development of drug resistance. ii).
investigate different P. falciparum clones and antimalarial drug associated alleles in local
gametocyte carriers, and iii). measure the transmission success of different drug resistant/sensitive
genotypes in natural isolates of P. falciparum by their local mosquito vector or mosquito infectivity.

Methodology: PCR Amplicon Sequencing and digital PCR platforms will be used to examine
prevalence of mutation on P. falciparum drug resistance gene and their spatiotemporal distribution
in 500 samples (Cross-sectional N=? and TES sample, N=?) collected from current cross-sectional
study and archived TES DBS samples from previous AHRI Study. The genotypes and linkage
disequilibrium test will be performed to study the association between the SNPs of the drug
resistance markers genes. Nanoplate-based dPCR will be used to study CNV of drug resistance
related genes (pfMDR1 and pfplasmopepsin2) from study samples. For Part-II study: Gametocyte
doner (N=?) will be recruited from Arba Minch Zuria Area. Membrane feeding Experimental set up
will also be designed to measure the transmissibility to mosquitoes of resistant genotypes of
parasites and calculate the potential resistant-genotype transmissibility of malaria parasites on this
part of the dissertation study. qRT-PCR protocol will be used to quantify posttreatment mature
gametocytes in the study samples. The correlation study will be checked with in gametocyte
densities estimates and mosquito infection rates.

Study Implication: The study from TES and cross-sectional study expected to show trends over
time and space across the study sentinel site and give information about spatial heterogeneities in
molecular markers. This study's findings could provide frequency of drug resistance and
information’s on its spatial distribution which can be important input data for future P. falciparum
malaria therapy, management, and control.

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2 CHAPTER-II (METHODOLOGY)

Assay development and validation to measure of gene amplifications related to malaria drug
resistance in P. falciparum and PvDBP gene amplification related to host invasion in P. vivax
using nanoplate-based digital PCR system
SUMMARY
Background: dPCR enables absolute quantification of target nucleic acid molecules (DNA, RNA
and oligos). For quantification we use the QIAcuity system from QIAgen. It utilizes a Nanoplate,
where each well is divided into a large number of partitions (micro chambers of nl-size). One
sample is added per well, and the molecules in each sample are distributed randomly into the
partitions. In each of the partitions target end-point PCR amplification will take place, and
amplification is detected by measuring fluorescence. Some partitions will contain either one or a
few target molecules (positive count) while other partitions will be empty (negative count). Due to
the random distribution in the partitions, Poisson statistics can be used to calculate target
quantification without the use of a standard curve. dPCR needs no standard curve or reference
samples, and the technology excels in complex samples of low target abundancy and high wild-
type background. 5 detection channels allow for multiplexed quantification. dPCR could be applied
for rare mutation detection (e.g., circulating tumor DNA), Gene expression studies, Genotyping,
Copy number variation (CNV) detection, Gene editing detection (CRISPR/Cas9), Quantification
of NGS libraries & Pathogen detection. In this study, nanoplate-based dPCR will be used to
measure gene CNV in plasmodium genome.
Parasite genetic factor like copy number variations (CNV) have shown their role in drug resistance.
Most of the studies have focused on the role of SNPs and drug resistance in parasite. However, it
has also been shown that CNV is associated with adaptation and drug resistance in parasite. It may
be suggested that CNVs in plasmepsin genes are the major driver of piperaquine resistance.
Moreover, CNVs in pfcrt and pfmdr1genes appear to play important role in adaptation and hence
survival of the parasite. Targeting of CNV formation in the parasite could be beneficial for
breakdown of its adaption in response to drug pressure.
Objectives: To develop single-plex, duplex and Multiplex assay and optimize multiplex dPCR
assay for detection of gene amplification in the plasmodium genome. Developed assay result will
be validated in qPCR assay using Laboratory reference samples and patient samples from the field.
Method:
i. Development of the dPCR assays
Development and validation of the dPCR assays will be done according to previous published
methods for dRCR assay development and validation (Srisutham et al., 2021) with modification on
dPCR (QIAgen and QIAcuity® Probe PCR Kit).
ii. Validation of dPCR assays
The accuracy and precision of uniplex, duplex and multiplex dRCR assays will be validated by
qualification of GCN of PfMDR1 and pfplasmepsin2 genes in the two-fold serial diluted strain 3D7
P. falciparum (approximate 40,000 P. falciparum genome copies/ul based on the absolute
quantification of pfβtubulin gene). Two-fold serial dilutions of P. falciparum strain D6
(approximate 50,000 P. falciparum genome copies/ul based on the absolute quantification of
pfβtubulin gene) will be prepared. Laboratory reference Strains of P. falciparum; 3D7, 7G8, D6,
D10, Dd2, HB3, W2 (N=?) will be obtained and used to compare the CNVs obtained by the dPCR
and qPCR assays. The GCN will be determined by three independent dPCR runs.
iii. Application of developed & validated nanoplate-based dPCR CNV assay: developed assay
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e be used to measure CNV of P. falciparum from field samples.
3 CHAPTER-III

Mapping and spatiotemporal dynamics analysis of Pfmdr1 CNV and PfCRT resistance alleles
across settings with varying levels of P. falciparum & P. vivax co-endemicity after
Introduction ACT.

Summary
Background: CNV, SNPs in the Pfmdr1, and SNPs in Pfcrt, genes of P. falciparum may confer
resistance to a number of anti-malaria drugs. Increased pfmdr1 copy numbers (CN) in field isolates
were associated with higher inhibitory concentrations of MQ, QN, HF, and artemisinin in vitro (34,
46) and were linked to the failure of MQ monotherapy and mefloquine-artesunate combination
therapy (Fredrick et al., 2013). Furthermore, direct evidence of the role of pfmdr1 in the modulation
of parasite susceptibility came from a report where inactivation of 1 of 2 copies of pfmdr1 in the P.
falciparum FCB line led to moderate increases in susceptibilities to artemisinin and
arylaminoalcohol drugs. Pervious study evidenced that pfmdr1 gene amplification and expression
are required in some parasites to withstand high concentrations of AL (Chavchich et al., 2010).
Given the high co-endemicity of the two species and the continued availability of CQ for the
treatment of P. vivax, it is currently unclear whether switching from CQ to AL for P. falciparum
treatment has implications current Artemisinin Lumefantrine’s potential for p. falciparum
treatment. It is important to find out whether released CQ pressure in endemic setting decreases
pfmdr1 gene amplification and results chloroquine sensitive p. falciparum strains compare with Co-
endemic setting.

Objectives: This study aimed to analyze dynamic of pfmdr1 amplification and PfCrt point
mutation on samples collected through health facility based cross-sectional surveys from settings
with varying P. vivax and P. falciparum endemicity.

Method: For this study, plasmodium falciparum diagnosed blood samples will be collected from
regions of different degree endemicity of p. vivax. Each collected samples from different setting
will be subjected to DNA extraction and species confirmation. Then the relative frequencies of
amplification/CNV of Pfmdr1 alleles and point mutation on PfCrt alleles will be assessed. For
CNV study, Optimized protocol for CNV of pfmdr1 will be used to detection presence of gene
copy with nanoplate-based Digital PCR platform. Point mutation on pfCrt will be performed using
nested PCR and RFLP.

Study Implication: This study's findings could provide important information for future P.
falciparum malaria therapy, management, and control.

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4 CHAPTER-IV

Genetic diversity of Plasmodium falciparum population in the study area.

Summary
Background: Monitoring for shifts in antimalarial drug efficacy using genetic markers,
including PfCrt and pfmdr1 (for CQ, ASAQ, and AL), Pfdhps and Pfdhfr (for SP), and
pfk13 (for ART), is essential to properly guide treatment. P. falciparum genetic diversity
facilitates adaptation to environmental changes, including antimalarial drug pressure. In
regions with low transmission, such as SEA, P. falciparum populations have been
characterized by single-clone infections, low genetic variation, reduced rates of
recombination, and strong linkage disequilibrium (LD) (i.e., nonrandom associations
among loci). Given those aggressive strategies have been implemented to eliminate P.
falciparum from SEA and that inbreeding predominates, it is not surprising that multidrug
resistance to antimalarials with different modes of action have arisen and spread. Although
studies in Ghana have provided longitudinal data on the trends for polymorphisms in the
genes mediating resistance, they have not addressed (1) whether MDR haplotypes are
circulating and/or expanding in the P. falciparum population and/or (2) whether LD exists
between/among the alleles at these drug resistance loci.
Methodology
Identifying Genetic Diversity of Plasmodium falciparum Populations
To genotype P. falciparum positive samples, high-resolution msp2 genotyping will be
performed as previously developed protocol by University of Maryland School of
Medicine https://www.medschool.umaryland.edu/malaria/Protocols/); this method is
specific to P. falciparum therefore only DNA samples positive for P. falciparum yield PCR
products. Briefly, a nested multiplex PCR approach will be used to amplify 3D7 and/or
FC27 family alleles of the gene encoding the highly polymorphic antigen, msp2, using
family-specific primers labeled with a fluorescent dye. The PCR products will be analyzed
by 1.5% agarose gel electrophoresis with PCR-positive samples being selected for further
analysis. The allele family and fragment size will be then determined by capillary
electrophoresis using an ABI 3730×1 DNA Analyzer platform with the internal size
standard GSLIZ500.
Genotyping of P. falciparum isolates will be carried out by Nested PCR amplification of
the two highly polymorphic regions of msp-2 (block3) genes and fragment size will be then
determined by capillary electrophoresis as described previously. (University of Maryland
School of Medicine. Available:
https://www.medschool.umaryland.edu/malaria/Protocols/). Protocol for MSP-2
Genotyping (umaryland.edu).
Expected Outcomes of study: Genetic variability is determined by sequence analysis of
the polymorphic segments of the merozoite surface protein 2 (msp-2) in P. falciparum

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5 CHAPTER-5

To investigate post-treatment gametocyte carriage female gametocyte markers as proxy

markers of development of resistance.
To measure the transmissibility to mosquitoes of resistant genotypes of parasites and analyses
the potential resistant-genotype transmissibility of malaria parasites

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1.1. STUDY SITES

Ethiopia is organized into 12 administrative regions including Addis Ababa and Dire Dawa
Cities. For current malaria drug resistance surveillance study, regions on the basis risk factors
for drug resistance emergence and expansions; Sites sharing border with neighboring
countries, high transmission endemicity. For the current Malaria drug resistance surveillance,
8 district or cluster i.e., Metema, Dasenech, Erer, Gambella, Qafta Humera, Guba, Gonder
zuriya, Salamago have be selected purposely because of acquisition of risk factors for
emerging and expansion of malaria drug resistance. Surveillance also will be done on
previous year Therapuetic efficacy study from Arba Minch (2019), Benishangul (2020),
Tanqua Abergelle (2020), Adama (2018) and metehara (2020) will also be included.

Figure: Map of Drug resistance surveillance study site

Metemma:
Metemma is a town in northwestern Ethiopia, on the border with Sudan. Located in the
Semien Gondar Zone of the Amhara Region, Metemma has a latitude and longitude of
12°58′N 36°12′E with an elevation of 685 meters above sea level. Across the border is the
corresponding Sudanese village of Gallabat. Metemma is located at 897 km North of Addis
Ababa and 197 km from Gondar and it has a latitude and longitude of 12°58′N 36°12′E. The
district has a total Population of 123,235 (Lemma, 2020) and covers an area of 6,969.97 km2.
The district is located in the lowlands, its elevation ranges between 550 and 1600 meter

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above sea level. Metema district has a hot semi-arid climate, with the annual average
temperature ranges from 20°C to 37°C. The annual rainfall ranges between 450 and 650 mm,
The district is malarious with the prevalence of 17% was reported recently (Ferede et al.,
2013; Lemma, 2020).

1.2. Sampling design and Sample size calculation

This study is a cross-sectional molecular epidemiological malaria drug resistance study of P.

falciparum infection in populations of 8 district across study population and archived BDS
sample from previous study. For cross-sectional molecular epidemiological, purposive
selection of 8 malarious districts with different malaria transmission setting, high labour
attracting, border to neighbouring countries, at the main transport corridors.

Required sample size for this cross-sectional study was calculation using Epi Info™ 7.2.5.0
version software-based sample calculation for cluster sampling with an assumption 12.1%
mutation prevalence, 95% confidence interval, 5% marginal error, 2 deff and 8 clusters in the
study. Total sample size is known to be 328 and sample size proportion allocation on each
cluster or district is calculated to be 41.

From previous TES sample, 172 DBS (Tanqua Abergele N= 43, Arba Minch N=43,
Benishangul N=43, and Adama N=43) Day-0 sample will be retrieved randomly for drug
resistance servilience. All P. falciparum Microscopically positive randomly selected samples
from Day-0 will be used for this study. TES Study were done according to World Health
Organization Guideline (WHO, 2016b).

1.3. Study populations

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1.4. Sample collection
This Study is part of an ongoing bigger population-wide epidemiological Pfhrp2/3 gene
deletion survey project where finger-prick blood samples were obtained from study
participants in accordance with the inclusion criteria. Briefly, 328 blood samples were
collected and screened in the field for P. falciparum by microscopy and rapid diagnostic test.
Parasitemia levels of P. falciparum positive samples were determined and recorded for
archiving. In total, 500 DBS (N= 328 Dried Blood Spots from cross-sectional survey and N=
172 Archived DBS sample from previous TES study) from P. falciparum positive samples
will be prepared by absorbing approximately 50 µl of the collected blood onto Whatman filter
paper and air-drying. DBS will be stored at ambient temperature at in sealed zip-lock bags
with desiccant awaiting DNA extraction.

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 TOTAL STUDY SAMPLE N=500
Cross-sectional Study Samples, N= 328
TES DBS Samples, N= 172

Cross-sectional Study site (8 district) and Total Samples, N=328 Therapeutic Efficacy Study, randomly selected D0 samples, N=172
Metemma, N = 41 Arba Minch (2019), N = 43
Dasenech, N = 41 Benishangul (2020), N = 43
Erer, N = 41 Tanqua Abergelle (2020), N = 43
Gambella, N = 41 Adama (2018) = 43
Qafta Humera, N = 41 metehara (2020)
Guba, N = 41
Gonder zuriya, N = 41
Salamago, N = 41

Parasitic DNA extraction Using MagMax™ DNA Multi-Sample Kit and qPCR
18srRNA Real Time qPCR For Species Confirmation and Quantification

MSP2 genotyping by CE Multiplex amplicon sequencing

Parasites Genetic Diversity Study, N =? Assay Development & Validation Malaria Drug resistance gene genotyping
for pfMDR1 CNV study, N = (Pfk13, PfCRT, PfMDR1, PfDHFR, PfDHPS,
Plasmepsin2, Mitochondrial genome or pfcytb),
N = 500
Nanoplate-based dPCR platform

Assay development & pfMDR1 CNV SNPs & CNV analysis

analysis
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