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High prevalence of pfcrt-CVIET haplotype in isolates from asymptomatic and symptomatic patients in south-central Oromia, Ethiopia
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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2014 (English)In: Malaria Journal, ISSN 1475-2875, Vol. 13, 120- p.Article in journal (Refereed) Published
Abstract [en]

Background: As a result of extensive chloroquine resistance (CQR) in Plasmodium falciparum in late 1990s, Ethiopia replaced CQ with sulphadoxine-pyrimethamine (SP) as first-line drug, which in turn was replaced by artemisinin combination therapy in 2004. Plasmodium falciparum resistance to CQ is determined by the mutation at K76T of the P. falciparum chloroquine resistance transporter (pfcrt) gene. Understanding diversity in the P. falciparum genome is crucial since it has the potential to influence important phenotypes of the parasite such as drug resistance. Limited data is available regarding the type of pfcrt mutant allelic type, the effect of CQ withdrawal and diversity of the parasite population in south-central Oromia, Ethiopia. Methods: Finger-pricked blood spotted on Whatman 3MM filter papers were collected from falciparum malaria patients. Parasite DNA was extracted from individual blood spots on the filter papers. The presence of K76T mutations was determined using nested PCR for all isolates. Complete sequencing of mutations in pfcrt 72-76 was done for a set of randomly selected resistant isolates. Four microsatellite (MS) markers were analysed to determine the heterozygosity. Results: Although CQ was withdrawn for more than a decade, 100% of the parasites still carried the pfcrt K76T mutation. All isolates were mutant at the K76T polymorphism. Based on combinations of MS markers, seven different Ethiopian CQR variants (E1-E7) were identified. Heterozygosity (He) for MS flanking the pfcrt chloroquine resistance allele ranged from 0.00 (mscrt -29, -29.268 kb) to 0.21 (mscrt -2, -2.814 kb). H-e ranged from 0.00 (msint 3, 0 kb) to 0.19 (msint 2, 0 kb) for MS within the pfcrt gene. Both intronic and MS flanking the pfcrt gene showed low levels of diversity. Conclusion: pfcrt CQR allele seems to be fixed in the study area. Of the different haplotypes associated with CQR, only the CVIET genotype was identified. No reversal to the wild-type has occurred in Ethiopia unlike in many Africa countries where CQR parasites declined after cessation of CQ use. Decreased diversity in CQR isolates surrounding pfcrt suggests CQ selection and homogenization among CQR parasite population. While mutation in msint 3 and mscrt -29 of the mutant pfcrt allele is being fixed, it seems that mutations in msint 2 and mscrt -2 are still evolving and may indicate the start of re-diversification of the population from a fixed 76 T population.

Place, publisher, year, edition, pages
2014. Vol. 13, 120- p.
Keyword [en]
pfcrt, Wild type, Drug resistance, Chloroquine, Plasmodium falciparum, Mutations, Heterozygosity, Microsatellite, Ethiopia
National Category
Infectious Medicine
URN: urn:nbn:se:uu:diva-225094DOI: 10.1186/1475-2875-13-120ISI: 000334807900004OAI: diva2:719977

Golassa and Enweji contributed equally to this work.

Available from: 2014-05-27 Created: 2014-05-27 Last updated: 2015-01-22Bibliographically approved
In thesis
1. Dynamics of Resistant Plasmodium falciparum Parasites
Open this publication in new window or tab >>Dynamics of Resistant Plasmodium falciparum Parasites
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Persistence of drug resistant Plasmodium falciparum is a major problem to management and control malaria in endemic areas. The focus of this thesis was to study the dynamics of resistant P. falciparum parasites. The study was performed in two African countries: 1) Sudan: Asar village in eastern Sudan, here we examined the persistence of drug sensitive and resistant P. falciparum genotypes among individuals with single-clone and multiple clones infection during the dry season. We genotyped microsatellite loci in the vicinity of the dihydrofolate reductase gene (dhfr) and the dihydropteroate synthase gene (dhps). Microsatellite investigation showed that asymptomatic parasitemia persisted in some patients for several months throughout the dry season and into the next transmission season. In some samples mixed infections were detected, and we noted several cases where the microsatellite haplotype varied from month to month, suggesting turnover of different parasite populations in the blood. This demonstrates that even during asymptomatic infections there can be dynamics within the parasite population in an individual. In addition, we calculated the parasite density throughout the dry season to the next transmission season by using allele-specific quantitative PCR. Parasite density during the dry season fluctuated, but was generally lower than in the first transmission season. A significant difference (P<0.05) between dry and first transmission season was found in regard to the parasite density, whereas no significant difference was observed when dry and second transmission season were compared (P>0.05). 2) Ethiopia: West Arsi zone, one of the malaria endemic zones of the Oromia region. In the first study we determined the prevalence of asymptomatic malaria carriages from November-December 2012. According to PCR the prevalence of sub-microscopic P. falciparum carriage was 19.2%, microscopy-based prevalence was 3.7% while the prevalence was 6.9% using RDT. Based on this, PCR was considered a better tool for measuring Plasmodium prevalence than microscopy and RDT. A second study addressed the genetic diversity of chloroquine resistance (CQR) in P. falciparum by analysing four microsatellite markers in and around the pfcrt gene. Although CQ was withdrawn for more than a decade, 100% of the parasites still carried the Pfcrt K76T mutation. Only the CVIET haplotype was identified. Based on combinations of MS markers, seven different Ethiopian CQR variants (E1-E7) were identified. Both intronic and MS flanking the pfcrt gene showed low levels of diversity.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 49 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1020
National Category
Other Basic Medicine
Research subject
Molecular Genetics
urn:nbn:se:uu:diva-230224 (URN)978-91-554-9007-2 (ISBN)
Public defence
2014-11-12, C8:301, BMC, Husargatan 3, Uppsala, 09:15 (English)
Available from: 2014-10-22 Created: 2014-08-20 Last updated: 2015-01-22

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