Genetic diversity and signatures of selection of drug resistance in Plasmodium populations from both human and mosquito hosts in continental Equatorial Guinea

BACKGROUND: In Plasmodium, the high level of genetic diversity and the interactions established by co-infecting parasite populations within the same host may be a source of selection on pathogen virulence and drug resistance. As different patterns have already been described in humans and mosquitoes, parasite diversity and population structure should be studied in both hosts to properly assess their effects on infection and transmission dynamics. This study aimed to characterize the circulating populations of Plasmodium spp and Plasmodium falciparum from a combined set of human blood and mosquito samples gathered in mainland Equatorial Guinea. Further, the origin and evolution of anti-malarial resistance in this area, where malaria remains a major public health problem were traced. METHODS: Plasmodium species infecting humans and mosquitoes were identified by nested-PCR of chelex-extracted DNA from dried blood spot samples and mosquitoes. Analysis of Pfmsp2 gene, anti-malarial-resistance associated genes, Pfdhps, Pfdhfr, Pfcrt and Pfmdr1, neutral microsatellites (STR) loci and Pfdhfr and Pfdhps flanking STR was undertaken to evaluate P. falciparum diversity. RESULTS: Prevalence of infection remains high in mainland Equatorial Guinea. No differences in parasite formula or significant genetic differentiation were seen in the parasite populations in both human and mosquito samples. Point mutations in all genes associated with anti-malarial resistance were highly prevalent. A high prevalence was observed for the Pfdhfr triple mutant in particular, associated with pyrimethamine resistance.Analysis of Pfdhps and Pfdhfr flanking STR revealed a decrease in the genetic diversity. This finding along with multiple independent introductions of Pfdhps mutant haplotypes suggest a soft selective sweep and an increased differentiation at Pfdhfr flanking microsatellites hints a model of positive directional selection for this gene. CONCLUSIONS: Chloroquine is no longer recommended for malaria treatment in Equatorial Guinea but sulphadoxine-pyrimethamine (SP) remains in use in combination with artesunate and is the only drug recommended in preventive chemotherapy in pregnancy. The high prevalence of point mutations in Pfdhfr and Pfdhps points to the danger of an eventual reduction in the efficacy of SP combined therapy in P. falciparum populations in Equatorial Guinea and to the essential continuous monitoring of these two genes.This study was supported by PEst-OE/SAU/LA0018/2011 - Proj. Estratégico LA0018 2011/2012 (http://cmdt.ihmt.unl.pt/index.php/pt/) and PTDC/SAU-EPI/113326/2009, “Fundacão para a Ciência e Tecnologia/Ministério da Educação e Ciência”, FCT/MEC (http://alfa.fct.mctes.pt/index.phtml.pt), Portugal and by “Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación”, Madrid, Spain. C. Mendes and P. Salgueiro hold FCT grants (SRFH/BD/41473/2007 and SFRH/BPD/72532/2010, respectively).S

A Molecular Epidemiological Study of var Gene Diversity to Characterize the Reservoir of Plasmodium falciparum in Humans in Africa

BACKGROUND: The reservoir of Plasmodium infection in humans has traditionally been defined by blood slide positivity. This study was designed to characterize the local reservoir of infection in relation to the diverse var genes that encode the major surface antigen of Plasmodium falciparum blood stages and underlie the parasite's ability to establish chronic infection and transmit from human to mosquito. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the molecular epidemiology of the var multigene family at local sites in Gabon, Senegal and Kenya which differ in parasite prevalence and transmission intensity. 1839 distinct var gene types were defined by sequencing DBLα domains in the three sites. Only 76 (4.1%) var types were found in more than one population indicating spatial heterogeneity in var types across the African continent. The majority of var types appeared only once in the population sample. Non-parametric statistical estimators predict in each population at minimum five to seven thousand distinct var types. Similar diversity of var types was seen in sites with different parasite prevalences. CONCLUSIONS/SIGNIFICANCE: Var population genomics provides new insights into the epidemiology of P. falciparum in Africa where malaria has never been conquered. In particular, we have described the extensive reservoir of infection in local African sites and discovered a unique var population structure that can facilitate superinfection through minimal overlap in var repertoires among parasite genomes. Our findings show that var typing as a molecular surveillance system defines the extent of genetic complexity in the reservoir of infection to complement measures of malaria prevalence. The observed small scale spatial diversity of var genes suggests that var genetics could greatly inform current malaria mapping approaches and predict complex malaria population dynamics due to the import of var types to areas where no widespread pre-existing immunity in the population exists

Rapid Dissemination of Plasmodium falciparum Drug Resistance Despite Strictly Controlled Antimalarial Use

BACKGROUND: Inadequate treatment practices with antimalarials are considered major contributors to Plasmodium falciparum resistance to chloroquine, pyrimethamine and sulfadoxine. The longitudinal survey conducted in Dielmo, a rural Senegalese community, offers a unique frame to explore the impact of strictly controlled and quantified antimalarial use for diagnosed malaria on drug resistance. METHODOLOGY/PRINCIPAL FINDINGS: We conducted on a yearly basis a retrospective survey over a ten-year period that included two successive treatment policies, namely quinine during 1990–1994, and chloroquine (CQ) and sulfadoxine/pyrimethamine (SP) as first and second line treatments, respectively, during 1995–1999. Molecular beacon-based genotyping, gene sequencing and microsatellite analysis showed a low prevalence of Pfcrt and Pfdhfr-ts resistance alleles of Southeast Asian origin by the end of 1994 and their effective dissemination within one year of CQ and SP implementation. The Pfcrt resistant allele rose from 9% to 46% prevalence during the first year of CQ reintroduction, i.e., after a mean of 1.66 CQ treatment courses/person/year. The Pfdhfr-ts triple mutant rose from 0% to 20% by end 1996, after a mean of 0.35 SP treatment courses/person in a 16-month period. Both resistance alleles were observed at a younger age than all other alleles. Their spreading was associated with enhanced in vitro resistance and rapidly translated in an increased incidence of clinical malaria episodes during the early post-treatment period. CONCLUSION/SIGNIFICANCE: In such a highly endemic setting, selection of drug-resistant parasites took a single year after drug implementation, resulting in a rapid progression of the incidence of clinical malaria during the early post-treatment period. Controlled antimalarial use at the community level did not prevent dissemination of resistance haplotypes. This data pleads against reintroduction of CQ in places where resistant allele frequency has dropped to a very low level after CQ use has been discontinued, unless drastic measures are put in place to prevent selection and spreading of mutants during the post-treatment period

Defining the origin of Plasmodium falciparum resistant dhfr isolates in Senegal.

We previously reported a high baseline prevalence of mutations in the dhfr and dhps genes of Plasmodium falciparum throughout Senegal. The highest prevalence of the triple dhfr pyrimethamine associated mutations were found in isolates obtained in the western part of the country near the capital city of Dakar. In this study, we sought out to determine the relatedness of dhfr wild type and mutated strains by analyzing three microsatellite regions upstream of the dhfr locus. Twenty-six of the 31 wild type strains had a unique microsatellite pattern. In contrast, of the 17 isolates containing the triple mutation in dhfr, 11 had an identical microsatellite pattern. Diverse geographical isolates in Senegal containing the triple dhfr mutation have arisen from a limited number of ancestral strains. In addition, we demonstrate that these isolates have shared ancestry with the previously reported triple mutation haplotype found in Tanzania, South Africa, and southeast Asia. This common ancestry may have implications for the malaria control strategy for reducing the spread of sulfadoxine-pyrimethamine resistance in Senegal and elsewhere in Africa

Contribuição para o estudo de resistência aos antimaláricos e análise de marcadores moleculares de Plasmodium falciparum e do hospedeiro humano, em Maputo, Moçambique

A malária é de longe a doença parasitária mais importante em Moçambique, constituindo um grave problema de saúde pública no País. Apesar de se considerar que um diagnóstico atempado e um tratamento correcto são os elementos básicos para um programa de controlo da malária bem sucedido, nas últimas décadas, o controlo e tratamento têm sido bastante dificultados pelo aparecimento e disseminação da resistência parasitária aos antimaláricos mais utilizados. Os mecanismos que conferem ao parasita a capacidade de resistir à maioria dos antimaláricos disponíveis não se encontram completamente elucidados. Deste modo, este trabalho teve como objectivo principal avaliar o envolvimento dos genes pfdhfr, pfdhps, pfcrt, pfmdr1 e pfATPase6 na resistência aos antimaláricos em Moçambique, recorrendo a populações naturais de parasitas em isolados colhidos de doentes com malária em Maputo, Moçambique. Neste contexto, foi efectuada a caracterização clínica dos pacientes com diferentes formas clínicas de malária e os perfis genotípicos relacionados com a resistência em P. falciparum para quatro antimaláricos: cloroquina, sulfadoxina/pirimetamina, amodiaquina e artemisinina, através da técnica de PCR-RFLP, para os polimorfismos pfcrt K76T e N75E, pfdhfr N51I, C59R, S108N e I164L, pfdhps A437G e K540E, pfmdr1 N86Y e N1246Y e pfATPase6, G1916 A, G110A, A2694T e G2306A. Os dados genotípicos foram subsequentemente analisados estatísticamente, no intuito de detectar associações significativas entre a presença de um determinado marcador antes e depois do tratamento com os diferentes antimaláricos utilizados. Foram também avaliados os polimorfismos em dois marcadores moleculares (ICAM-1 e CD36) do hospedeiro relacionados com susceptibilidade/resistência à malária, em isolados de pacientes com e sem malária. Os polimorfismos da variante CYP-450 (CYP2C8), relacionada com o metabolismo dos fármacos antimaláricos em pacientes com malária, foram também aqui analisados. Os resultados demonstraram a gravidade do problema de resistência a antimaláricos, evidenciado pelas elevadas prevalências dos alelos mutantes antes e depois dos tratamentos efectuados. Foi aqui observada uma elevação significativa de amostras contendo o alelo mutado pfdhps 437G após do tratamento com Fansidar® e com Fansidar®+Amodiaquina. Verificou-se existir uma correlação positiva entre o quíntuplo mutante e o número de isolados, depois do tratamento com Fansidar®. Estes resultados indicam reservas na utilização do Fansidar ® como uma componente da primeira linha de tratamento antimalárico no País e apontam o polimorfismo pfdhps 437G como um possível marcador para a monitorização da resistência a este fármaco.Apesar de não significativos, os resultados da análise do ICAM-1 mostraram a existência de uma possível associação entre a presença da mutação ICAM-1kilifi e a infecção malárica nas suas formas grave e não grave, enquanto para o CD36 foi notória a ausência da mutação T1264G no grupo controle (sem malária). Os resultados da análise dos polimorfismos no gene CYP2C8 demonstraram alguma inconclusividade, tendo no entanto permitido a obtenção de conhecimentos para estudos que possam ser realizados no futuro.Malaria is the most important parasitic disease and a public health problem in Mozambique. Though a correct therapy and diagnosis are basic for any control program, in the last decades, these have been hampered by the appearance and dissemination of drug resistance to most antimalarials. Mechanisms of drug resistance have not been clarified. This work had the objective to study the relation between a number of molecular markers pfdhfr, pfdhps, pfcrt, pfmdr1 and pfATPase6 in drug resistance in Mozambique, in parasite populations collected in Maputo. Clinical characterization of patients and their profiles associated to resistance in P. falciparum was carried out for 4 drugs, chloroquine, combined sulfadoxine/pyrimethamine, amodiaquine and artemisinin, through PCR-RFLP, for polymorphisms in pfcrt K76T and N75E, pfdhfr N51I, C59R, S108N and I164L, pfdhps A437G and K540E, pfmdr1 N86Y and N1246Y and pfATPase6, G1916 A, G110A, A2694T and G2306A. Statistical analysis was also carried out for a correct evaluation of presence of certain markers and response to treatment. Two other polymorphisms were also studied (ICAM-1 and CD36) in association to susceptibility to infection in isolates from people with or without malaria. Finally, polymorphisms of the gene CYP2C8, associated to drug metabolization in malaria patients was also investigated Results confirmed the severity of the problem of drug resistance in malaria, with very high presence and prevalence of mutant alleles in parasite populations before and after treatment. Pfdhps 437G after Fansidar® or Fansidar®+Amodiaquine treatment was significantly visible. There was also a positive correlation between a quintuple mutation association and number of isolates Fansidar® treatment. These results recommend reduction of the use of this drug as a component of first-line antimalarial treatment in Mozambique and indicate that pfdhps 437G can be used as a marker for resistance in clinical work. Though not significant, data from the studies with ICAM-1 showed a possible association between the presence of mutation of ICAM-1kilifi and malaria infection in their severe and non severe forms, while for CD36 it there was a negative association due to non presence of T1264G in the non malaria control group. The results regarding polymorphisms of the gene CYP2C8 were limiting in their interpretation, but nevertheless, contributed to the gathering of information that may be used in future studies