RESUMO
The malaria parasite, Plasmodium falciparum, was introduced into Hispaniola and other regions of the Americas through the slave trade spanning the 16th through the 19th centuries. During this period, more than 12 million Africans were brought across the Atlantic to the Caribbean and other regions of the Americas. Since malaria is holoendemic in West Africa, a substantial percentage of these individuals carried the parasite. St. Domingue on Hispaniola, now modern-day Haiti, was a major port of disembarkation, and malaria is still actively transmitted there. We undertook a detailed study of the phylogenetics of the Haitian parasites and those from Colombia and Peru utilizing whole-genome sequencing. Principal-component and phylogenetic analyses, based upon single nucleotide polymorphisms (SNPs) in protein coding regions, indicate that, despite the potential for millions of introductions from Africa, the Haitian parasites share an ancestral relationship within a well-supported monophyletic clade with parasites from South America, while belonging to a distinct lineage. This result, in stark contrast to the historical record of parasite introductions, is best explained by a severe population bottleneck experienced by the parasites introduced into the Americas. Here, evidence is presented for targeted selection of rare African alleles in genes which are expressed in the mosquito stages of the parasite's life cycle. These genetic markers support the hypothesis that the severe population bottleneck was caused by the required adaptation of the parasite to transmission by new definitive hosts among the Anopheles (Nyssorhynchus) spp. found in the Caribbean and South America.IMPORTANCE Historical data suggest that millions of P. falciparum parasite lineages were introduced into the Americas during the trans-Atlantic slave trade, which would suggest a paraphyletic origin of the extant isolates in the Western Hemisphere. Our analyses of whole-genome variants show that the American parasites belong to a well-supported monophyletic clade. We hypothesize that the required adaptation to American vectors created a severe bottleneck, reducing the effective introduction to a few lineages. In support of this hypothesis, we discovered genes expressed in the mosquito stages of the life cycle that have alleles with multiple, high-frequency or fixed, nonsynonymous mutations in the American populations which are rarely found in African isolates. These alleles appear to be in gene products critical for transmission through the anopheline vector. Thus, these results may inform efforts to develop novel transmission-blocking vaccines by identifying parasite proteins functionally interacting with the vector that are important for successful transmission. Further, to the best of our knowledge, these are the first whole-genome data available from Haitian P. falciparum isolates. Defining the genome of these parasites provides genetic markers useful for mapping parasite populations and monitoring parasite movements/introductions.
Assuntos
Adaptação Fisiológica/genética , Anopheles/parasitologia , Variação Genética , Filogenia , Plasmodium falciparum/genética , Animais , Marcadores Genéticos , Haiti , Malária Falciparum/parasitologia , Mosquitos Vetores/parasitologia , Mutação , Plasmodium falciparum/classificação , Plasmodium falciparum/fisiologia , América do Sul , Estados Unidos , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: Public health measures are poised for transition from malaria control to malaria elimination on the island of Hispaniola. Assessment of the reservoir of asymptomatic infections from which acute malaria cases may derive is critical to plan and evaluate elimination efforts. Current field technology is ill suited for detecting sub-microscopic infections, thus highly sensitive survey methods capable of detecting virtually all infections are needed. In this study the prevalence of infection with Plasmodium falciparum was determined in patients seeking medical care primarily for non-febrile conditions in six departments in Haiti using a newly designed qRT-PCR-based assay. METHODS: Three different methods of parasite detection were compared to assess their utility in approximating the prevalence of P. falciparum infections in the population: malaria rapid diagnostic test (RDT) designed to detect histidine-rich protein 2 (HRP2), thick smear microscopy, and a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay based upon the small sub-unit ribosomal RNA. The limit of detection of the qRT-PCR assay utilized was 0.0003 parasite/µL of blood. Venous blood was obtained from a total of 563 subjects from six departments in Haiti, all of whom were seeking medical attention without complaints consistent with malaria. Each subject was questioned for knowledge and behaviour using demographic and epidemiological survey to identify risk factors for disease transmission. RESULTS: Among the 563 samples tested, ten and 16 were found positive for malaria by RDT and microscopy, respectively. Using the qRT-PCR test to assess the infection status of these subjects, an additional 92 were identified for a total of 108. Based upon the qRT-PCR assay results, a wide variation in prevalence of infection in asymptomatic subjects was seen between geographic locations ranging from 4-41%. The prevalence of infection was highest in the Grand Anse, Nord and Sud-Est Departments, and demographic data from questionnaires provide evidence for focal disease transmission. CONCLUSIONS: The qRT-PCR assay is sufficiently sensitive to identify an unexpectedly large number of asymptomatic, submicroscopic infections. Identifying and clearing these infections presents a significant challenge to both control and elimination efforts, but the qRT-PCR assay offers a reliable method to identify them.
Assuntos
Infecções Assintomáticas/epidemiologia , Testes Diagnósticos de Rotina/métodos , Malária Falciparum/diagnóstico , Malária Falciparum/epidemiologia , Plasmodium falciparum/isolamento & purificação , Adulto , Estudos Transversais , Feminino , Haiti/epidemiologia , Humanos , Imunoensaio , Microscopia , Prevalência , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , População Rural , Adulto JovemRESUMO
BACKGROUND: In Haiti where chloroquine (CQ) is widely used for malaria treatment, reports of resistance are scarce. However, recent identification of CQ resistance genotypes in one site is suggestive of an emerging problem. Additional studies are needed to evaluate genetic mutations associated with CQ resistance, especially in the Plasmodium falciparum multi-drug resistance-1 gene (pfmdr1) while expanding the already available information on P. falciparum CQ transporter gene (pfcrt) in Haiti. METHODS: Blood samples were collected on Whatman filter cards (FTA) from eight clinics spread across Haiti. Following the confirmation of P. falciparum in the samples, PCR protocols were used to amplify regions of pfmdr1and pfcrt codons of interest, (86, 184, 1034, 1042, and 1246) and (72-76), respectively. Sequencing and site-specific restriction enzyme digestions were used to analyse these DNA fragments for the presence of single nucleotide polymorphisms (SNPs) known to confer resistance to anti-malarial drugs. RESULTS: P. falciparum infection was confirmed in160 samples by amplifying a segment of the P. falciparum 18S small subunit ribosomal RNA gene (pfssurrna). The sequence of pfmdr1 in 54 of these samples was determined between codons 86,184 codons 1034, 1042 and 1246. No sequence differences from that of the NF54 clone 3D7 were found among the 54 samples except at codon 184, where a non-silent mutation was found in all samples predicted to alter the amino acid sequence replacing tyrosine with phenylalanine (Y184F). This altered sequence was also confirmed by restriction enzyme digestion. The sequence of pfmdr1 at codons 86, 184, 1034 and 1042 encoded the NFSN haplotype. The sequence of pfcrt codons 72-76 from 79 samples was determined and found to encode CVMNK, consistent with a CQ sensitive genotype. CONCLUSION: The presence of the Y184F mutation in pfmdr1 of P. falciparum parasites in Haiti may have implications for resistance to antimalarial drugs. The absence of mutation in pfcrt at codon 76 among 79 isolates tested suggests that sensitivity to CQ in Haiti remains common. Wide-spread screening of the pfmdr1 and pfcrt especially among patients experiencing treatment failure may be a useful tool in early detection of the emergence of antimalarial drug resistance in Haiti.