Molecular analysis of Plasmodium falciparum recrudescent malaria infections in children treated with chloroquine in Nigeria

Parasite genotyping by a polymerase chain reaction was used to distinguish recrudescent from newly acquired Plasmodium falciparum infections in 50 of 160 Nigerian children taking part in a chloroquine efficacy study in Ibadan, Nigeria. A finger prick blood sample was taken from each child before and after treatment to identify recrudescent parasites. By investigating allelic variation in three polymorphic antigen loci, merozoite surface protein-1 (MSP-1), MSP-2, and glutamate-rich protein (GLURP), we determined parasite diversity in the population and in the infected host. DNA from pretreatment and post-treatment samples from 47 of the 50 patients who failed therapy was successfully amplified by the PCR. The MSP-1, MSP-2, and GLURP genotypes in all samples showed extensive diversity, indicating polyclonal infections. The average number of clones per infection in pre-treatment sample was 2.5 with MSP-1, 4.9 with MSP-2, and 2 with GLURP. The extent of multiplicity decreased significantly (P = 0.016) in posttreatment samples. Multiplicity of infection and initial parasite density were not age dependent. Comparison of the variant alleles in pretreatment and post-treatment samples of each patient indicates that 26 of the 47 children had genuinely recrudescent disease. Conversely, post-treatment samples from five children showed completely new genotypes, indicating either a previously sequestered population of parasites or a newly acquired infection. Overall, this study has shown the diversity and complexity of P. falciparum population in Ibadan, Nigeria. The study has also shown the dynamics of P. falciparum infections in this population before and after chloroquine treatment in an area of high malaria transmission.     

Genetic diversity in the merozoite surface protein 1 gene of Plasmodium falciparum in different malaria-endemic localities

A number of stage-specific antigens have been characterized for vaccine development in Plasmodium falciparum malaria. The polymorphic merozoite surface protein 1 (MSP-1) of Plasmodium falciparum is a major asexual blood stage malaria vaccine candidate antigen. In the present study, we analyzed the impact of hyperendemic malaria transmission, mesoendemic malaria transmission, and multiple infection on allelic diversity. We have used a simple strategy of polymerase chain reaction amplification and slot-blot hybridization to analyze variable regions of block-2, block-4 and blocks 6-10 of the MSP-1 gene. The allelic types of isolates collected from regions of hyperendemic malaria transmission (RHEMT) and mesoendemic malaria transmission (RMEMT) were compared. In RHEMT, 20 of 24 possible gene types were found among 163 isolates and more than one allelic type was found in 82 (50.3%) of the isolates. Thirteen of 24 possible gene types were found among 125 isolates in RMEMT and 27 (21.6%) of them contained more than one allele type. Our results suggest for the first time that the allelic distribution or allelic diversity and chances of finding multi-strain parasites in isolates in an area vary with the rate of transmission. Analyses of isolates containing more than one strain of parasite suggest that allelic types are randomly distributed, no specific type of alleles predominately show multi-strain infection, and neither strain of the parasite affect the process of infection and development of another.     

Genetic diversity of Plasmodium falciparum isolates from naturally infected children in north-central Nigeria using the merozoite surface protein-2 as molecular marker

ObjectiveTo characterize the genetic diversity of Plasmodium falciparum (P. falciparum) field isolates in children from Lafia, North-central Nigeria, using the highly polymorphic P. falciparum merozoite surface protein 2 (MSP-2) gene as molecular marker.MethodsThree hundred and twenty children were enrolled into the study between 2005 and 2006. These included 140 children who presented with uncomplicated malaria at the Dalhatu Araf Specialist Hospital, Lafia and another 180 children from the study area with asymptomatic infection. DNA was extracted from blood spot on filter paper and MSP-2 genes were genotyped using allele-specific nested PCR in order to analyze the genetic diversity of parasite isolates.ResultsA total of 31 and 34 distinct MSP-2 alleles were identified in the asymptomatic and uncomplicated malaria groups respectively. No difference was found between the multiplicity of infection in the asymptomatic group and that of the uncomplicated malaria group (P>0.05). However, isolates of the FC27 allele type were dominant in the asymptomatic group whereas isolates of the 3D7 allele type were dominant in the uncomplicated malaria group.ConclusionsThis study showed a high genetic diversity of P. falciparum isolates in North-central Nigeria and is comparable to reports from similar areas with high malaria transmission intensity.     

Geographical patterns of allelic diversity in the Plasmodium falciparum malaria-vaccine candidate, merozoite surface protein-2

The polymorphic merozoite surface protein-2 (MSP-2) of Plasmodium falciparum is a major malaria-vaccine candidate. In the present study, PCR and hybridization with allelic-specific probes were used to type the Msp-2 gene from isolates from hypo-endemic Brazil (N = 113), meso-endemic Vietnam (N = 208) and holo-endemic Tanzania (N = 67). The typing methods were designed to group isolates into the dimorphic allelic families FC27 and IC1 and to detect possible between-family recombination events. The analysis was complemented by a comparison of 156 Msp-2 sequences from the GenBank database with 12 additional sequences obtained during the present study. Statistically significant differences were detected in pair-wise comparisons of the distribution of Msp-2 allelic types in Brazil and Vietnam, and in Brazil and Tanzania, but not in Vietnam and Tanzania. The extent of allelic diversity in the Msp-2 gene, as estimated by the total number of different alleles found in a given parasite population and the mean multiplicity of infections, clearly paralleled the levels of malaria endemicity in the study areas. However, no correlation between age and multiplicity of infections was found in the subjects. The patterns of Msp-2 diversity in Brazil appeared to be temporally stable, since no significant difference was observed in the distribution of Msp-2 allelic types among isolates collected, 10--13 years apart, in the same area of Rond?nia. Despite the extensive sequence diversity found in Msp-2 alleles, especially in the central repetitive region of the molecule, several instances of identical or nearly identical alleles were found among isolates from different countries and regions, possibly as a result of extensive homoplasy. No recombinant allele was detected by molecular typing in any of the study sites, and the GenBank database included only 12 recombinant sequences (representing 7% of all reported Msp-2 sequences), all of them with an IC1-type 5' end and an FC27-type 3' end. A single, putative, crossover site was characterised for all recombinant alleles. Most of the allelic diversity observed was therefore attributable to variation in the repetitive region of the gene, instead of recombination between alleles of dimorphic families (as commonly found, for example, in the Msp-1 gene). The implications of these findings for studies on the genetic and antigenic diversity of malarial parasites are discussed.     

Plasmodium falciparum parasites in French Guiana: limited genetic diversity and high selfing rate

The genetic characteristics of Plasmodium falciparum isolates collected in French Guiana, where malaria transmission is low and occurs in isolated foci, were studied. Blood samples were collected from 142 patients with symptomatic malaria and typed using a polymerase chain reaction-based strategy for merozoite surface protein-(MSP-1) block 2, the MSP-2 central domain, and glutamate-rich protein (GLURP) repeat domain polymorphism. This showed that the parasite population circulating in French Guiana presented a limited number of allelic forms (4, 2, and 3 for MSP-1 block 2, MSP-1, and GLURP, respectively) and a small number of mixed infections, contrasting with the large genetic diversity of parasite populations and infection complexity reported for Africa, Asia, and other parts of South America. Two groups of isolates displaying identical 3 loci allele combinations were further studied for the Pf332 antigen, histidine-rich protein-1, thrombospondin-related anonymous protein, and Pf60 multigene family polymorphism. Within each group, most isolates were identical for all markers tested. This suggests a high rate of self-fertilization of P. falciparum parasites in French Guiana, resulting in homogenization of the population. The implications of these findings for malaria control in areas of low endemicity are discussed.     

New emerging Plasmodium falciparum genotypes in children during the transition phase from asymptomatic parasitemia to malaria

Semi-immunity against Plasmodium falciparum occurs after many infections. In areas of high malaria transmission, the prevalence of asymptomatic parasite carriers increases with age. We investigated P. falciparum genotypes in a cohort of asymptomatic carriers who were followed until they became symptomatic. Blood spots on filter paper and blood smears were collected daily from 10 children in Lambaréné, Gabon. The parasite genotypes present on successive days were determined by a polymerase chain reaction using the polymorphic region of the merozoite surface antigen-2 for typing. The same parasite genotypes persisted in eight out of ten children and parasite densities were low throughout the asymptomatic phase indicating inhibition of parasite growth. Appearance of symptoms was associated with an increase in parasitemia and appearance of novel parasite genotypes. The results suggest that the parasites causing a clinical episode are those against which a child has not yet mounted an efficient protective immune response.     

Distinguishing Plasmodium falciparum treatment failures from re-infections by using polymerase chain reaction genotyping in a holoendemic area in northeastern Tanzania.

An in vivo drug sensitivity study was conducted in Magoda village in northeastern Tanzania to evaluate the usefulness of polymerase chain reaction (PCR)-based genotyping of Plasmodium falciparum parasites to distinguish between re-infection and treatment failure. The study tested P. falciparum susceptibility to a combination of sulfadoxine/pyrimethamine (Fansidar; F. Hoffmann La Roche, Basel, Switzerland). Blood samples were collected before treatment and on days 7, 14, or 28 post-treatment in 51 asymptomatic children, of which 26 could not clear parasitemia within seven days post-treatment. Among the remaining 25 children who had no detectable parasites on day 7, only five remained parasite negative up to day 28. Primary and recrudescent P. falciparum parasites were analyzed by PCR using family specific primers for merozoite surface protein-1 (MSP-1), MSP-2, and glutamate-rich protein (GLURP). All samples contained multiple P. falciparum infections. For all children with recrudescent P. falciparum, common alleles were detected in both the primary and recrudescent samples. However, in no child were the exact same alleles detected in both samples, indicating that probably at least some of the recrudescing parasites originated from new infections. The study demonstrates the general usefulness of PCR genotyping technique in distinguishing re-infections from true recrudescences following therapeutic drug treatment.     

Markers for population genetic analysis of human plasmodia species, P. falciparum and P. vivax

Present report deals with the genetic diversity existing among the field isolates of Plasmodium falciparum and P. vivax in India. Isoenzymes and molecular markers were used to analyse field isolates of P. falciparum and P. vivax. High level of length polymorphism was observed in repeat nucleotide sequences of MSP-1, MSP-2 and GLURP in P. falciparum isolates and CSP, GAM-1 and MSP-3 alpha in P. vivax isolates. In study populations a high proportion of isolates (up to 60%) were comprised of more than one genetically distinct parasite type--multiclonal. Presence of identical allelic forms of enzyme and DNA variations in different geographical areas and in different years suggest that isolates belong to a single random mating population of P. vivax and P. falciparum. Observed random combination of alleles in the field isolates suggest the unlinked nature of loci studied. Study supports the feasibility of using molecular markers for the identification of recrudescence in P. falciparum from fresh infection.     

间日疟原虫MSP-1 和 CSP 基因遗传多样性的分析

目的比较间日疟原虫两种主要分子标志(MSP-1 和 CSP)的遗传多样性. 方法分别用MSP-1和CSP基因分型方法鉴定间日疟原虫现场分离株,并进行基因多态性比较和分析. 结果共检测32份海南省现场确诊的间日疟病人血样,MSP-1等位基因型混合感染率为18.75%,平均克隆数1.16;CSP基因型混合感染率为35.29%,平均克隆数为1.47.如果同时考虑两种基因型,混合感染率则为50.00%.空间对应分析发现,热带族与Sal-1型关系密切,PvⅡ型与重组Ⅲ型分布靠近,其他基因型则较分散. 结论同时用MSP-1和CSP两种分子标志检测间日疟原虫,其基因型混合感染率高于用单一标志检测,两种标志检测结果存在一定对应关系.     

High frequency of recombination-driven allelic diversity and temporal variation of Plasmodium falciparum msp1 in Tanzania

A major mechanism for the generation allelic diversity in the Plasmodium falciparum msp1 gene is meiotic recombination in the Anopheles mosquito. The frequency of recombination events is dependent on the intensity of transmission. Herein we investigate the frequency of recombination-driven allelic diversity and temporal variation of msp1 in Rufiji, eastern coastal Tanzania, where malaria transmission is intense. We identified 5' recombinant types, 3' sequence types, and msp1 haplotypes (unique associations of 5' recombinant types and 3' sequence types) to measure the extent and temporal variation of msp1 allelic diversity. The results show that msp1 haplotype diversity is higher in Tanzania as compared with areas with lower transmission rates. The frequencies of individual polymorphic regions/sites remained stable during the study period. However, the frequency distribution of msp1 haplotypes varied between 1993 and 1998. These results suggest that frequent recombination events between msp1 alleles intermittently generate novel alleles in high transmission areas.     

Transmission and Cross-Mating of High-Level Resistance Plasmodium falciparum Dihydrofolate Reductase Haplotypes in The Gambia

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.     

Are Plasmodium falciparum parasites present in peripheral blood genetically the same as those sequestered in the tissues?

Since quinine does not inhibit the growth of Plasmodium falciparum ring stages or mature schizonts, parasites may continue to emerge from sequestration sites after starting treatment. We used polymerase chain reaction amplification of P. falciparum merozoite surface protein 1 (MSP-1) and MSP-2 alleles to distinguish genotypes infecting 58 children with severe malaria. To examine changes in parasite populations in peripheral blood over time, we compared changes in number and spectrum of genotypes in samples on admission to a hospital to those obtained up to 24 hours later. Thirty-four children lost genotypes, 21 retained genotypes, and 3 gained an extra P. falciparum genotype at one locus but not the other. The lack of novel genotypes emerging suggests that among children with severe malaria the dominant clones sequestered in deep organs are usually the same as those in peripheral circulation.     

Diversity of Plasmodium falciparum clones infecting children living in a holoendemic area in north-eastern Tanzania

The diversity of Plasmodium falciparum clones and their role in progression from asymptomatic to symptomatic condition in children have been investigated. Attempts to identify whether particular parasite genotypes were associated with the development of clinical symptoms have been made. A cohort of 34 initially asymptomatic parasitaemic children aged 1-5 years were followed daily for 31 days. Clinical examinations were made each day for signs and symptoms of clinical malaria, followed by parasitological investigation. Nineteen children developed symptoms suggestive of clinical malaria during this period. Daily blood parasite samples from 13 children who developed clinical malaria symptoms and 7 who remained asymptomatic were genotyped by PCR-amplification of the polymorphic regions of the merozoite surface proteins 1 and 2 (MSP1 and MSP2) and the glutamate rich protein (GLURP) genes. Infections were found to be highly complex in both groups of children. Every isolate examined from both groups had a mixture of parasite clones. Daily changes were observed in both parasite density and genotypic pattern. The mean number of genotypes per individual was estimated at 4.9 and 2.7 for asymptomatic and symptomatic groups of children, respectively. Analysis of allele frequency distributions showed that these differed significantly for the MSP1 locus only.     

Frequent and persistent, asymptomatic Plasmodium falciparum infections in African infants, characterized by multilocus genotyping

To determine the duration and complexity of naturally acquired Plasmodium falciparum infections in small children, a longitudinal cohort study of 143 newborns was conducted in coastal Ghana. On average, children experienced 2 episodes of infection in their first 2 years of life, the median duration of an asymptomatic infection was <4 weeks, and estimates of the mean number of parasite genotypes per infection were 1.15-2.28. Nevertheless, 40% of the children experienced infections lasting 5 months old. The ability of very young children to clear or control malaria infections indicates the presence of effective innate or immune antiparasite mechanisms.     

Genetic diversity of Plasmodium falciparum and its relationship to parasite density in an area with different malaria endemicities in West Uganda

Field populations of Plasmodium falciparum can be effectively genotyped by PCR-amplification of selected fragments of the Merozoite Surface Proteins 1 and 2 (MSP1 and MSP2). Genetic diversity of P. falciparum populations in areas with different transmission levels (holo- vs. mesoendemic) was investigated in Kabarole District, West Uganda. 225 samples positive for P. falciparum were analysed by amplification of polymorphic regions and classified according to prevalence of allelic families. A large number of alleles was detected for each locus: 22 for MSP1 block 2 and 24 for MSP2 and, 175 (78%) of MSP1 alleles and 143 (64%) of MSP2 showed multiple infections within a range of 2-8 clones. Significant differences between holoendemic and mesoendemic areas in regards of population structure and number of multiclonal infections of P. falciparum were not apparent. However, a significant correlation between parasite density, selected MSP2 loci and differences between parasite density in monoclonal vs. multiclonal infections occurred. Multiplicity of infection was age-dependent.     

Long-term asymptomatic carriage of Plasmodium falciparum protects from malaria attacks: a prospective study among Senegalese children.

BACKGROUND: In areas of seasonal malaria transmission, long-term asymptomatic carriage of Plasmodium falciparum throughout the dry season has been primarily studied in terms of the parasites, and the clinical consequences of persistent parasite carriage are unknown. METHODS: A prospective study was conducted in Senegal, from 2001 through 2003 among 1356 children living in areas where malaria is endemic, with seasonal transmission occurring from August through December. Cross-sectional parasitological measurements and detection of active malaria attacks were performed. A malaria attack was defined as an axillary temperature > or =37.5 degrees C, associated with a parasite density >2500 trophozoites/microL. Children harboring P. falciparum in June who did not have clinical signs were defined as asymptomatic carriers. The association of asymptomatic carriage with parasite densities and with the occurrence of malaria attacks during the rainy season were analyzed separately for the years 2002 and 2003, taking into account potential confounding covariates and use of antimalarial drugs. RESULTS: The prevalence of asymptomatic carriage was 32% (332 of 1025 persons) in June 2002 and 23% (208 of 912 persons) in June 2003. Asymptomatic P. falciparum carriers had a significantly higher mean parasite density and a significantly lower probability of developing a malaria attack during the subsequent rainy season than did noncarriers (adjusted odds ratio in 2002, 0.56; P = .01; adjusted odds ratio in 2003, 0.50; P = .01). CONCLUSIONS: These results suggest that in areas of seasonal transmission, asymptomatic carriage of P. falciparum may protect against clinical malaria. Further studies are needed to understand the immune effectors and host susceptibility that could be involved in this phenomenon.     

Plasmodium falciparum populations from northeastern Myanmar display high levels of genetic diversity at multiple antigenic loci

Levels of genetic diversity of the malaria parasites and multiclonal infections are correlated with transmission intensity. In order to monitor the effect of strengthened malaria control efforts in recent years at the China–Myanmar border area, we followed the temporal dynamics of genetic diversity of three polymorphic antigenic markers msp1, msp2, and glurp in the Plasmodium falciparum populations. Despite reduced malaria prevalence in the region, parasite populations exhibited high levels of genetic diversity. Genotyping 258 clinical samples collected in four years detected a total of 22 PCR size alleles. Multiclonal infections were detected in 45.7% of the patient samples, giving a minimum multiplicity of infection of 1.41. The majority of alleles experienced significant temporal fluctuations through the years. Haplotype diversity based on the three-locus genotypes ranged from the lowest in 2009 at 0.33 to the highest in 2010 at 0.80. Sequencing of msp1 fragments from 36 random samples of five allele size groups detected 13 different sequences, revealing an additional layer of genetic complexity. This study suggests that despite reduced prevalence of malaria infections in this region, the parasite population size and transmission intensity remained high enough to allow effective genetic recombination of the parasites and continued maintenance of genetic diversity.     

Genetic diversity in the merozoite surface protein 1 and 2 genes of Plasmodium falciparum from the Artibonite Valley of Haiti

Describing genetic diversity of the Plasmodium falciparum parasite provides important information about the local epidemiology of malaria. In this study, we examined the genetic diversity of P. falciparum isolates from the Artibonite Valley in Haiti using the allelic families of merozoite surface protein 1 and 2 genes (msp-1 and msp-2). The majority of study subjects infected with P. falciparum had a single parasite genotype (56% for msp-1 and 69% for msp-2: n=79); 9 distinct msp-1 genotypes were identified by size differences on agarose gels. K1 was the most polymorphic allelic family with 5 genotypes (amplicons from 100 to 300 base pairs [bp]); RO33 was the least polymorphic, with a single genotype (120-bp). Although both msp-2 alleles (3D7/IC1, FC27) had similar number of genotypes (n=4), 3D7/IC1 was more frequent (85% vs. 26%). All samples were screened for the presence of the K76T mutation on the P. falciparum chloroquine resistance transporter (pfcrt) gene with 10 of 79 samples positive. Of the 2 (out of 10) samples from individuals follow-up for 21 days, P. falciparum parasites were present through day 7 after treatment with chloroquine. No parasites were found on day 21. Our results suggest that the level of genetic diversity is low in this area of Haiti, which is consistent with an area of low transmission.     

Alterations in Plasmodium falciparum genotypes during sequential infections suggest the presence of strain specific immunity

Many of the asexual stage Plasmodium falciparum proteins that are the targets of host protective responses are markedly polymorphic. The full repertoire of diversity is not defined for any antigen. Most studies have focused on the genes encoding merozoite surface proteins 1 and 2 (MSP1, MSP2). We explored the extent of diversity of some of the less studied merozoite surface antigens and analyzed the degree of complexity of malaria field isolates by deriving nucleotide sequences of several antigens. We have determined the genotype of apical membrane antigen 1 (AMA1) in a group of 30 field samples, collected over 29 months, from individuals living in an area of intense malaria transmission in Irian Jaya, identifying 14 different alleles. AMA1 genotyping was combined with previously determined MSP2 typing. AMA1 had the greatest power in distinguishing between isolates but methodological problems, especially when mixed infections are present, suggest it is not an ideal typing target. MSP1, MSP3, and glutamate-rich protein genotypes were also determined from a smaller group of samples, and all results were combined to derive an extended antigenic haplotype. Within this subset of 10 patients, nine different genotypes could be discerned; however, five patients were all infected with the same strain. This strain was present in individuals from two separate villages and was still present 12 months later. This strain was predominant at the first time point but had disappeared at the fourth time point. This significant change in malaria genotypes could be due to strain-specific immunity developing in this population.     

Short report: Molecular evaluation of the efficacy of chloroquine treatment of uncomplicated Plasmodium falciparum malaria in East Timor

The efficacy of chloroquine treatment of uncomplicated Plasmodium falciparum malaria in East Timor was investigated via molecular tools. Genotyping of the polymorphic markers msp1 and msp2 was performed to investigate the number and type of parasite alleles in pre- and posttreatment blood samples collected from 48 patients. Patients were infected with a minimum of 8 msp1 and 14 msp2 allelic types of parasite, and 43% of the patients had more than one allelic type before treatment. The genotyping also revealed that 66.7% of the patients were infected with at least one identical allelic type of parasite before and after treatment and therefore were likely to have experienced recrudescence. All parasites in pre- and posttreatment blood samples carried the K76T mutation in pfcrt, regardless of the clinical response to chloroquine. The sequence polymorphism patterns in pfcrt in the majority of parasites examined were identical to those observed in Bougainville, Papua New Guinea.