Molecular epidemiology of malaria and detection of anti-malarial drug resistanceassociated markers (Pfcrt, pfmdr-1, pfdhfr and pfdhps) in Hadhramout governorate, Yemen / Omar Abdullah Ali Bamaga
Malaria, especially Plasmodium falciparum malaria is one of the main causes of mortality and morbidity worldwide. Yemen is an Eastern Mediterranean country where 68% of its population is at risk of malaria. In 2013, it was estimated that there were 150,000 cases recorded in Yemen with 55 malarial...
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2017
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| Online Access: | http://studentsrepo.um.edu.my/10359/4/omar.pdf http://studentsrepo.um.edu.my/10359/ |
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| Summary: | Malaria, especially Plasmodium falciparum malaria is one of the main causes of
mortality and morbidity worldwide. Yemen is an Eastern Mediterranean country where
68% of its population is at risk of malaria. In 2013, it was estimated that there were
150,000 cases recorded in Yemen with 55 malarial deaths, compared to 900,000 cases
in 2000. The anti-malarial treatment policy in Yemen was changed from chloroquine
(CQ) to artemisinin combination therapy (ACT) in 2005.The present study is the first in
Hadhramout, Yemen which aimed to assess the epidemiology of malaria parasites and
to determine the frequency of mutant alleles and genotypes associated with antimalarial
drug resistance in Plasmodium falciparum isolates. Blood specimens were collected
from seven villages in two different districts of the Hadhramout governorate by houseto-house visits from July 2011 to May 2012. A total of 735 individuals aged 1 to 75
years with a median of 16 years and 22 interquartile range participated in the study. A
pre-tested questionnaire was used to gather demographic, socioeconomic and
environmental data. Plasmodium species were first identified by microscopy
examination and subsequently genomic DNA was extracted from dried archive blood
spots of P. falciparum isolates and analyzed using nested PCR. Mutation-specific nested
polymerase chain reaction (MS-PCR) and restriction fragment length polymorphism
(PCR–RFLP) methods were used to investigate the mutations in the Pfmdr1 (codons 86
and 1246) and Pfcrt (codons 76, 271, 326, 356 and 371) genes. DNA was also amplified
using nested PCR and subsequently sequenced for Pfdhfr and Pfdhps genes. Sequences
were analyzed for mutations in Pfdhfr at codons 51, 59, 108, and 164 and in Pfdhps at
codons 436, 437, and 540. Results of the overall prevalence of malaria parasites in
Hadhramout governorate, Yemen via microscopy was 18.8% (138 of 735) with
Plasmodium falciparum being the predominant species (99.3%; 137 of 138), followed
by Plasmodium vivax (0.7%; 1). Nested PCR detected P. falciparum in four samples
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that were previously negative using microscopy. The combination of microscopy and
nested PCR detection resulted in three samples being identified as mixed infections of
P. falciparum and P. vivax. The infection rate was higher in Al-Raydah-Qusyer district
(21.8%) compared to Hajer district (11.8%). Fifty two percent of those positive for
Plasmodium were asymptomatic with low parasite density. The adults had a higher
infection rate as compared to children. Univariate analysis identified those whose
household’s heads are fishermen (OR = 11.3, 95% CI: 3.13–40.5) and farmers (OR =
4.84, 95% CI: 1.73–13.6) as high-risk groups. A higher number of positive rates were
observed in people living in houses with uncemented brick walls (OR = 2.1, 95% CI:
1.32–3.30), without access to toilets (OR = 1.6, 95% CI: 1.05–2.32), without a fridge
(OR = 1. 6, 95% CI: 1.05–2.30), or without TV (OR = 1. 6, (95% CI: 1.05–2.30).
People living in houses with water collection points located less than 200 meters away
were also at higher risk of acquiring malaria (OR = 1.6, 95% CI:1.05–2.30). Knowledge
about the importance of using insecticide-treated mosquito nets (ITNs) and indoor
residual spraying (IRS) for prevention of malaria was 7% and 2%, respectively. The
prevalence of Pfcrt mutations at codons 76, 271, 326 and 371 were 50.4%, 58.7%,
54.3% and 44.9%, respectively. All isolates had wild-type Pfcrt 356 allele. The majority
of Pfmdr1 86 alleles (83.3%) and all Pfmdr1 1246 (100%) alleles were also wild type.
There was no association between Pfcrt mutations and symptomatology, gender and age
groups. For Pfdhfr/Pfdhps mutations, each Pfdhfr mutant allele (I51 and N108) in P.
falciparum isolate had a frequency of 84%. Pfdhfr R59 mutant allele was detected in one
isolate. Pfdhps at codon G437 mutant allele was detected in 44.7% of Plasmodium
falciparum malaria isolates. Frequencies of Pfdhfr double mutant genotype
(I51C59N108I164) and Pfdhfr/Pfdhps triple mutant genotype (I51C59N108I164-S436G437K540)
were 82.8% and 40.6%, respectively. It is important to note that there was one isolate
each which harbored Pfdhfr triple mutant genotype (I51, R59, N108, I164) and
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Pfdhfr/Pfdhps quadruple mutant genotype (I51R59N108I164-S436G437K540). In conclusion,
several environmental, socioeconomic and behavioral issues were discovered to be the
contributing factors to the high prevalence of malaria in this southeast Yemen
governorate. High frequencies of point mutations in codons 76, 271, 326 and 371 of P.
falciparum, suggested a sustained high CQ resistance even after 6 years of shifting to
ACTs. High frequencies of Pfdhfr and Pfdhps mutant alleles and genotypes in P.
falciparum isolates from Hadhramout, Yemen, highlight the risk of decreasing efficacy
of sulfadoxine pyrimethamine antimalarial drugs. Novel strategies adapted to local
situations need to be established in order to improve the effectiveness of malaria
control. The current study findings necessitate continuous monitoring of the efficacy of
malaria treatment. |
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