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Factors Influencing Evolution to Antimalarial Drug Resistance in Plasmodium falciparum in Sudan and The Gambia
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Göte Swedberg)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Drug resistance is a major obstacle to management and control of malaria and currently progressing at a rapid rate across Africa. This thesis has examined factors influencing evolution of resistant P. falciparum at two sites in Africa, including parasite migration, cross mating and fitness cost of resistance. In Asar village, eastern Sudan, the frequencies of drug sensitive and resistant parasites were monitored throughout the dry season in the absence of anti-malarial drug usage to examine whether persistence of resistant parasites is reduced in the absence of drug pressure. Two cohorts of P. falciparum infected patients were treated with chloroquine in the transmission season (Oct-Dec), and followed monthly in the dry season into the next transmission season. A large proportion of the cohort maintained sub-patent asymptomatic P. falciparum infections throughout the entire study period. Alleles of the chloroquine resistance transporter (Pfcrt) and multi-drug resistance protein (Pfmdr1) were examined. Mutant alleles of Pfcrt reached fixation following CQ treatment and remained high in the transmission season. However, at the start of the dry season, wild type alleles of both genes started to emerge and increased significantly in frequency as the season progressed. The mutant Pfcrt haplotype was invariably CVIET, indicating migration of CQ resistant parasites into an area; otherwise the CVMNK haplotype is normal. In addition, microsatellite haplotypes of dihydrofolate reductase (dhfr) gene and dihydropteroate synthase (dhps) genes, which control the parasite response to pyrimethamine and sulfadoxine respectively, were characterized. One major dhfr haplotype with double dhfr mutations and two major mutant dhps haplotypes were seen in eastern Sudan. These haplotypes are distinct from those prevailing in other African countries, suggesting the likely local origin of dhfr and dhps haplotypes conferring drug resistance.

Transmission capacities of different P. falciparum clones within a single infection in The Gambia have a high ability to produce gametocytes and infect Anopheles mosquitoes even when they exist at levels not detectable by microscopy and PCR. These findings emphasize the crucial role of gametocyte complexity and infectivity in generating the remarkable diversity of P. falciparum genotypes seen in infected people. Parasites with different resistant dihydrofolate reductase (dhfr) haplotypes have the ability to infect Anopheles mosquitoes following drug treatment, and cross-mating between parasites with different dhfr haplotypes was detected. Our results showed that the major dhfr haplotype in the Gambia is similar to the common one seen in other African countries, suggesting that parasite migration plays a major role in spread of resistance. Indeed, the dominant resistant haplotype seen in infected patients was readily transmitted to infect mosquitoes.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2011. , 54 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 661
Keyword [en]
cross-mating, fitness, microsatellite haplotypes, mosquito infectivity
National Category
Infectious Medicine
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:uu:diva-150254ISBN: 978-91-554-8044-8OAI: oai:DiVA.org:uu-150254DiVA: diva2:407123
Public defence
2011-05-12, C10:301, Biomedical Center (BMC), Husaratan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2011-04-20 Created: 2011-03-28 Last updated: 2011-05-05Bibliographically approved
List of papers
1. Fitness cost of drug resistance in malaria parasites
Open this publication in new window or tab >>Fitness cost of drug resistance in malaria parasites
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

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Many field surveys have demonstrated a decline in the prevalence of drug resistant parasites following withdrawal of drugs, implying fitness cost of mutations causing resistance. However, for clear ethical reason, within-host dynamics of drug-resistant mutants and sensitive forms in the absence of the drug has not been investigated in nature.   Here we monitored longevity of drug resistant Plasmodium falciparum parasites in a small village in eastern Sudan with a long dry period followed by a brief annual rains and appearance of Anopheles mosquitoes. This allows tracing the fate of drug resistant P. falciparum clones, in the absence of selective drug pressure, over a period of 7 to 8 months, in the dry season. Two cohorts were examined, (a) 83 patients enrolled in October 1993 and monitored to December 1994, and (b) 121 patients recruited in October 2001 and followed-up to December 2002. Patients in both cohorts were treated, on diagnosis, with chloroquine (25mg/kg) and then followed monthly, in each visit a blood sample was collected and  a patient was treated only if fever and visible parasitaemia were detected. We used PCR and RT-PCR to detect parasites and gametocytes that persisted at sub-patent levels in the dry season, respectively. In addition, we examined alleles of genes controlling the response of P. falciparum to chloroquine; the chloroquine resistance transporter (Pfcrt76T) and the multi-drug resistance protein (Pfmdr186Y).  A large proportion of both cohorts maintained gametocytes producing sub-patent asymptomatic P. falciparum infections throughout the dry season. Mutant alleles of Pfcrt76T reached fixation following CQ treatment and remained high in the transmission season, a reflection of selection. However, at the start of the dry season, wild type alleles of both genes (Pfcrt76K and Pfmdr186N) started to emerge and increased significantly in frequency as the season progressed. Some members of both cohorts, encountered malaria episode in the ensuing transmission season, all of them were found to harbour parasite with wild type alleles for both genes.  The above data has clearly shown selective disadvantage of CQ resistant genotypes (Pfcrt76T, Pfmdr186Y) in the absence of the drug. The data were discussed in the context of fitness of drug resistant parasite and its epidemiological impact.

Keyword
Plasmodium falciparum, fitness cost, chloroquine resistance, pfcrt, pfmdr1
National Category
Infectious Medicine
Research subject
Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-150253 (URN)
Available from: 2011-03-29 Created: 2011-03-28 Last updated: 2011-06-28Bibliographically approved
2. Distinct haplotypes of dhfr and dhps among Plasmodium falciparum isolates in an area of high level of sulfadoxine-pyrimethamine (SP) resistance in eastern Sudan
Open this publication in new window or tab >>Distinct haplotypes of dhfr and dhps among Plasmodium falciparum isolates in an area of high level of sulfadoxine-pyrimethamine (SP) resistance in eastern Sudan
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2009 (English)In: Infection, Genetics and Evolution, ISSN 1567-1348, E-ISSN 1567-7257, Vol. 9, no 5, 778-783 p.Article in journal (Refereed) Published
Abstract [en]

Typing of polymorphic microsatellites that are linked to drug resistance genes has shed light on the origin and pattern of spread of some anti-malarial drugs. Recent surveys revealed spread of a high-level pyrimethemine resistant lineage of Plasmodium falciparum, of Asian origin, across Africa. Here, we examined mutations in dihydrofolate reductase, dhfr [chromsosome 4], the dihydropteroate synthase, dhps [chromosome 8] associated with resistance to sulfadoxine-pyrimethamine (SP), and neighboring microsatellites among P. falciparum isolates in Asar village, eastern Sudan. This area lies at the fringes of malaria endemicity, where the remote P. falciparum parasites have some distinct genetic characteristics. Overall, 89% (84/94) of the examined isolates carried double mutations at dhfr (N51I and S108N), but the 59R and I164L mutations were not seen. Similarly, the majority, 43% (35/81) of the isolates carried double mutations at dhps (437G, 540E). Analysis of neighboring microsatellites revealed one major dhfr haplotype with mutations (51I, 108N) and one dhps haplotype with mutations (436S, 437G, 540E). These haplotypes differ from the major ones thought to drive resistance to SP across Africa. The resistant haplotypes of dhfr and dhps, in Asar, share some microsatellites with the wild genotypes suggesting that they were generated locally. Among isolates successfully examined, 40% shared identical haplotypes of the 2 loci, comprising a dominant resistant lineage. Undoubtedly, this lineage plays an important role in clinical failure to SP in this area.

Keyword
dhfr, dhps haplotypes, Pyrimethamine, Sulfadoxine resistance, Plasmodium falciparum, Evolution, Sudan
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-119510 (URN)10.1016/j.meegid.2009.04.010 (DOI)000269271400006 ()19379843 (PubMedID)
Available from: 2010-02-26 Created: 2010-02-26 Last updated: 2011-06-28Bibliographically approved
3. High gametocyte complexity and mosquito infectivity of Plasmodium falciparum in The Gambia
Open this publication in new window or tab >>High gametocyte complexity and mosquito infectivity of Plasmodium falciparum in The Gambia
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2008 (English)In: International journal for parasitology, ISSN 0020-7519, Vol. 38, no 2, 219-227 p.Article in journal (Refereed) Published
Abstract [en]

The purpose of this work was to determine the infectivity to mosquitoes of genetically diverse Plasmodium falciparum clones seen in natural infections in the Gambia. Two principal questions were addressed: (i) how infectious are gametocytes of sub-patent infections, particularly at the end of the dry season; and (ii) are all clones in multiclonal infections equally capable of infecting mosquitoes? The work was carried out with two cohorts of infected individuals. Firstly, a group of 31 P. falciparum-infected people were recruited in the middle of the dry season (May, 2003), then examined for P. falciparum at the beginning (August 2003) and middle (October, 2003) of the transmission season. On each occasion, we examined the genotypes of asexual forms and gametocytes by PCR and RT-PCR, as well as their infectivity to Anopheles gambiae using membrane feeds. One individual gave rise to infected mosquitoes in May, and two in August. Different gametocyte genotypes co-existed in the same infection and fluctuated over time. The mean multiplicity of infection was 1.4, 1.7 and 1.5 clones in May, August and October, respectively. Second, a group of patients undergoing drug-treatment during August 2003 was tested for asexual and gametocyte genotypes and their infectivity to mosquitoes. Forty-three out of 100 feeds produced infections. The genetic complexity of the parasites in mosquitoes was sometimes greater than that detectable in the blood on which the mosquitoes had fed. This suggested that gametocytes of clones existing in the blood below PCR detection limits at the time of the feed were at least as infectious to the mosquitoes as the more abundant clones. These findings emphasise the crucial role of gametocyte complexity and infectivity in generating the remarkable diversity of P. falciparum genotypes seen in infected people, even in an area of seasonal transmission.

Keyword
Plasmodium falciparum, Gametocytes, RT-PCR, Anopheles mosquito infectivity
National Category
Infectious Medicine
Research subject
Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-150252 (URN)10.1016/j.ipara.2007.07.003 (DOI)000253033900010 ()
Available from: 2011-05-04 Created: 2011-03-28 Last updated: 2011-05-04Bibliographically approved
4. Transmission and Cross-Mating of High-Level Resistance Plasmodium falciparum Dihydrofolate Reductase Haplotypes in The Gambia
Open this publication in new window or tab >>Transmission and Cross-Mating of High-Level Resistance Plasmodium falciparum Dihydrofolate Reductase Haplotypes in The Gambia
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2010 (English)In: American Journal of Tropical Medicine and Hygiene, ISSN 0002-9637, ISSN 20348495, Vol. 82, no 4, 535-541 p.Article in journal (Refereed) Published
Abstract [en]

A high-level pyrimethamine resistance Plasmodium falciparum lineage with triple dihydrofolate reductase (dhfr) mutations prevails across Africa. However, additional minority lineages were seen. We examined transmission success of mutant dhfr haplotypes among 22 children in The Gambia and 60 infected Anopheles gambiae mosquitoes fed on their blood. Additional polymorphic genes of the gametocyte-specific protein (pfg377) and merozoite surface protein-1 (MSP-1) were examined. Similarities were seen between pfg377 and MSP-1 alleles in children and mosquitoes and evidence of cross-mating between different parasite genotypes was seen in some infected mosquitoes, reflecting high transmission success of existing clones. With regard to dhfr, 16 haplotypes were seen among the children: 2 carried double mutations and 14 carried triple mutations. However, only nine haplotypes, all with triple mutations, were detected among mosquitoes. A single triple-mutant dhfr haplotype, similar to that in other countries in Africa, predominated among children (42%) and mosquitoes (60%), supporting the hypothesis of migration of this haplotype across Africa. However, evidence of cross-mating between the above haplotypes signifies the role of local evolution.

National Category
Medical and Health Sciences Biological Sciences
Identifiers
urn:nbn:se:uu:diva-137624 (URN)10.4269/ajtmh.2010.09-0378 (DOI)000276219700006 ()
Available from: 2010-12-15 Created: 2010-12-15 Last updated: 2011-06-28Bibliographically approved

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