Moses J. Bockarie

Twelve microsatellite markers for characterization of Plasmodium falciparum from finger-prick blood samples

Multiple, selectively neutral genetic markers are the most appropriate tools for analysis of parasite population structure and epidemiology, but yet existing methods for characterization of malaria field samples utilize a limited number of antigen encoding genes, which appear to be under strong selection. We describe protocols for characterization of 12 microsatellite markers from finger-prick blood samples infected with Plasmodium falciparum. A two-step, heminested strategy was used to amplify all loci, and products were visualized by fluorescent end-labelling of internal primers. This procedure allows amplification from low levels of template, while eliminating the problem of spurious products due to primer carry over from the primary round of PCR. The loci can be conveniently multiplexed, while accurate sizing and quantification of PCR products can be automated using the GENOTYPER software. The primers do not amplify co-infecting malaria species such as P. vivax and P. malariae. To demonstrate the utility of these markers, we characterized 57 infected finger-prick blood samples from the village of Mebat in Papua New Guinea for all 12 loci, and all samples were genotyped a second time to measure reproducibility. Numbers of alleles per locus range from 4 to 10 in this population, while heterozygosities range from 0.21 to 0.87. Reproducibility (measured as concordance between predominant alleles detected in replicate samples) ranged from 92 to 98% for the 12 loci. The composition of PCR products from infections containing multiple malaria clones could also be defined using strict criteria and scored in a highly repeatable manner.

A human complement receptor 1 polymorphism that reduces Plasmodium falciparum rosetting confers protection against severe malaria

Parasitized red blood cells (RBCs) from children suffering from severe malaria often adhere to complement receptor 1 (CR1) on uninfected RBCs to form clumps of cells known as “rosettes.” Despite a well documented association between rosetting and severe malaria, it is controversial whether rosetting is a cause or a correlate of parasite virulence. CR1-deficient RBC show greatly reduced rosetting; therefore, we hypothesized that, if rosetting is a direct cause of malaria pathology, CR1-deficient individuals should be protected against severe disease. In this study, we show that RBC CR1 deficiency occurs in up to 80% of healthy individuals from the malaria-endemic regions of Papua New Guinea. This RBC CR1 deficiency is associated with polymorphisms in the CR1 gene and, unexpectedly, with α-thalassemia, a common genetic disorder in Melanesian populations. Analysis of a case-control study demonstrated that the CR1 polymorphisms and α-thalassemia independently confer protection against severe malaria. We have therefore identified CR1 as a new malaria resistance gene and provided compelling evidence that rosetting is an important parasite virulence phenotype that should be a target for drug and vaccine development.

Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype in association with pfcrt polymorphism in Papua New Guinea and South America

The mechanistic basis for chloroquine resistance (CQR) in Plasmodium falciparum recently has been linked to the polymorphic gene pfcrt. Alleles associated with CQR in natural parasite isolates harbor threonine (T), as opposed to lysine (K) at amino acid 76. P. falciparum CQR strains of African and Southeast Asian origin carry pfcrt alleles encoding an amino acid haplotype of CVIET (residues 72–76), whereas most South American CQR strains studied carry an allele encoding an SVMNT haplotype; chloroquine-sensitive strains from malarious regions around the world carry a CVMNK haplotype. Upon investigating the origin of pfcrt alleles in Papua New Guinean (PNG) P. falciparum we found either the chloroquine-sensitive-associated CVMNK or CQR-associated SVMNT haplotypes previously seen in Brazilian isolates. Remarkably we did not find the CVIET haplotype observed in CQR strains from Southeast Asian regions more proximal to PNG. Further we found a previously undescribed CQR phenotype to be associated with the SVMNT haplotype from PNG and South America. This CQR phenotype is significantly less responsive to verapamil chemosensitization compared with the effect associated with the CVIET haplotype. Consistent with this, we observed that verapamil treatment of P. falciparum isolates carrying pfcrt SVMNT is associated with an attenuated increase in digestive vacuole pH relative to CVIET pfcrt-carrying isolates. These data suggest a key role for pH-dependent changes in hematin receptor concentration in the P. falciparum CQR mechanism. Our findings also suggest that P. falciparum CQR has arisen through multiple evolutionary pathways associated with pfcrt K76T.

The epidemiology of malaria in Papua New Guinea

Papua New Guinea (PNG) is a patchwork of different ecological zones, inhabited by human populations of exceptional cultural and linguistic diversity. This results in complex variations in vector ecology and malaria epidemiology. Malaria is the main cause of morbidity in many health facilities in lowland areas, but it is absent in much of the highlands. All four human malaria species occur, but endemicity varies widely, with Plasmodium falciparum locally reaching holo-endemic levels that are rarely found outside sub-Saharan Africa. The high frequency of Plasmodium vivax is an important difference to most African situations. PNG is therefore a prime location for studies of interactions between different parasite species, and of the biology of local human genetic adaptation and its implications for malaria morbidity and mortality.

Randomised community-based trial of annual single-dose diethylcarbamazine with or without ivermectin against Wuchereria bancrofti infection in human beings and mosquitoes

BACKGROUNDnWHO has targeted lymphatic filariasis for elimination. Studies of vector-parasite relations of Wuchereria bancrofti suggest that a reduction in the microfilarial reservoir by mass chemotherapy may interrupt transmission and thereby eliminate infection. However, no field data exist on the impact of chemotherapy alone on vector efficiency and transmission intensity of W bancrofti. We compared the impact of an annual community-wide single-dose treatment with diethylcarbamazine alone or with ivermectin on rate and intensity of microfilaraemia, and transmission intensity in an area of Papua New Guinea endemic for intense W bancrofti transmission.nnnMETHODSnWe carried out clinical and parasitological surveys in 14 communities in matched pairs. People aged 5 years or older in seven communities received randomly assigned diethylcarbamazine 6 mg/kg and people in the other seven communities received diethylcarbamazine 6 mg/kg plus ivermectin 400 micrograms/kg. We made physical examinations for hydroceles and leg oedema and investigated microfilarial densities by membrane filtration before and after treatment. We selected five communities for monthly entomological surveys between September, 1993, and September, 1995. Mosquitoes were collected in these communities by the all-night landing catch method and were individually dissected to identify rates of infection and infectiveness.nnnFINDINGSn2219 (87.6%) of 2534 eligible people received treatment. Microfilarial rate and density had decreased 1 year after treatment in all 14 communities; this decrease was significantly higher in communities given combined therapy than in those given diethylcarbamazine alone (mean decreases 57.5% and 30.6%, respectively; p = 0.0013). Greater decreases were also seen in community-specific microfilarial intensity with combined therapy (mean reductions 91.1% and 69.8%, respectively; p = 0.0047). The rate of leg oedema was not altered, but the frequency of advanced hydroceles decreased by 47% with combined therapy and 56% with diethylcarbamazine alone. 26,641 Anopheles punctulatus mosquitoes were caught during 499 person-nights of landing catches. Exposure to infective third-stage larvae decreased in all monitored five communities. Annual transmission potential decreased by between 75.7% and 98.8% in combined-therapy communities and between 75.6% and 79.4% in communities given diethylcarbamazine alone. Transmission was almost interrupted in two communities treated with combined therapy.nnnINTERPRETATIONnAnnual single-dose community-wide treatment with diethylcarbamazine alone or with ivermectin is effective for the control of lymphatic filariasis in highly endemic areas, but combination therapy brings about greater decreases in rates and intensity of microfilaraemia.

Prevalence of CYP2B6 alleles in malaria-endemic populations of West Africa and Papua New Guinea

ObjectiveCytochrome P450 2B6 (CYP2B6) is involved in the metabolism of artemisinin drugs, a novel series of antimalarials. Our aim was to analyze the prevalence of the most commonly observed CYP2B6 alleles in malaria-endemic populations of West Africa (WA) and Papua New Guinea (PNG). MethodsUsing a post-PCR ligation detection reaction-fluorescent microsphere assay, frequencies of CYP2B6*1A, *2, *3, *4, *5, *6, *7, and *9 were determined in WA (n=166) and PNG (n=174). To compare with the results of previous studies, we also determined the allele frequencies in 291 North Americans of various ethnic groups.ResultsSignificant differences were observed between WA and PNG for the frequencies of alleles CYP2B6*1A (45% vs 33%, P = 0.003), *2 (4% vs. 0%, P<0.001), *6 (42% vs 62%, P<0.001), and *9 (8% vs 1%, P<0.001), and genotypes *1A/*9 (9% vs 0%, P<0.001) and *6/*6 (17% vs 43%, P<0.001). The frequencies of CYP2B6 genotypes in the populations were in Hardy-Weinberg equilibrium, except for PNG where an overall significant deficit of heterozygosity was observed (HO=0.431, HE=0.505, P=0.004). The allele frequencies in Asian-Americans and Caucasians-Americans were comparable to those documented for Japanese and Caucasian populations.ConclusionsCYP2B6 variants, previously shown to affect metabolism of a variety of drugs, occur in WA and PNG, and there are significant genetic differences at the CYP2B6 locus in these populations. It may be important to determine if these differences alter the efficacy of artemisinin drugs.

Antigenic Drift in the Ligand Domain of Plasmodium vivax Duffy Binding Protein Confers Resistance to Inhibitory Antibodies

Interaction of the Duffy binding protein (DBP) with its erythrocyte receptor is critical for maintaining Plasmodium vivax blood-stage infections, making DBP an appealing vaccine candidate. The cysteine-rich region II is the ligand domain of DBP and a target of vaccine development. Interestingly, most of the allelic diversity observed in DBP is due to the high rate of nonsynonymous polymorphisms in this critical domain for receptor recognition. Similar to the hypervariability in influenza hemagglutinin, this pattern of polymorphisms in the DBP ligand domain suggests that this variation is a mechanism to evade antibody neutralization. To evaluate the role that dbp allelic diversity plays in strain-specific immunity, we examined the ability of an anti-Sal1 DBP serum to inhibit the erythrocyte-binding function of variant dbp alleles expressed on COS cells. We observed that the PNG-7.18 allele was significantly less sensitive to immune inhibition of its erythrocyte-binding activity than were the Sal1 and PNG-27.16 alleles. This result suggested that the unique polymorphisms of resistant PNG-7.18 were part of a protective epitope on the DBP ligand. To confirm this, Sal1 was converted to the refractory phenotype by introduction of 3 polymorphisms unique to PNG-7.18, via site-directed mutagenesis. The results of the present study indicate that linked polymorphisms have an additive, synergistic effect on DBP antigenic character.

The impact of repeated rounds of mass drug-administration with diethylcarbamazine plus albendazole on bancroftian filariasis in Papua New Guinea

Background This study employed various monitoring methods to assess the impact of repeated rounds of mass drug administration (MDA) on bancroftian filariasis in Papua New Guinea, which has the largest filariasis problem in the Pacific region. Methodology/Principal Findings Residents of rural villages near Madang were studied prior to and one year after each of three rounds of MDA with diethylcarbamazine plus albendazole administered per World Health Organization (WHO) guidelines. The mean MDA compliance rate was 72.9%. Three rounds of MDA decreased microfilaremia rates (Mf, 1 ml night blood by filter) from 18.6% pre-MDA to 1.3% after the third MDA (a 94% decrease). Mf clearance rates in infected persons were 71%, 90.7%, and 98.1% after 1, 2, and 3 rounds of MDA. Rates of filarial antigenemia assessed by card test (a marker for adult worm infection) decreased from 47.5% to 17.1% (a 64% decrease) after 3 rounds of MDA. The filarial antibody rate (IgG4 antibodies to Bm14, an indicator of filarial infection status and/or exposure to mosquito-borne infective larvae) decreased from 59.3% to 25.1% (a 54.6% decrease). Mf, antigen, and antibody rates decreased more rapidly in children <11 years of age (by 100%, 84.2%, and 76.8%, respectively) relative to older individuals, perhaps reflecting their lighter infections and shorter durations of exposure/infection prior to MDA. Incidence rates for microfilaremia, filarial antigenemia, and antifilarial antibodies also decreased significantly after MDA. Filarial DNA rates in Anopheles punctulatus mosquitoes that had recently taken a blood meal decreased from 15.1% to 1.0% (a 92.3% decrease). Conclusions/Significance MDA had dramatic effects on all filariasis parameters in the study area and also reduced incidence rates. Follow-up studies will be needed to determine whether residual infection rates in residents of these villages are sufficient to support sustained transmission by the An. punctulatus vector. Lymphatic filariasis elimination should be feasible in Papua New Guinea if MDA can be effectively delivered to endemic populations.

Discordant Patterns of Genetic Variation at Two Chloroquine Resistance Loci in Worldwide Populations of the Malaria Parasite Plasmodium falciparum

ABSTRACT Mutations in the chloroquine resistance (CQR) transporter gene of Plasmodium falciparum (Pfcrt; chromosome 7) play a key role in CQR, while mutations in the multidrug resistance gene (Pfmdr1; chromosome 5) play a significant role in the parasites resistance to a variety of antimalarials and also modulate CQR. To compare patterns of genetic variation at Pfcrt and Pfmdr1 loci, we investigated 460 blood samples from P. falciparum-infected patients from four Asian, three African, and three South American countries, analyzing microsatellite (MS) loci flanking Pfcrt (five loci [∼40 kb]) and Pfmdr1 (either two loci [∼5 kb] or four loci [∼10 kb]). CQR Pfmdr1 allele-associated MS haplotypes showed considerably higher genetic diversity and higher levels of subdivision than CQR Pfcrt allele-associated MS haplotypes in both Asian and African parasite populations. However, both Pfcrt and Pfmdr1 MS haplotypes showed similar levels of low diversity in South American parasite populations. Median-joining network analyses showed that the Pfcrt MS haplotypes correlated well with geography and CQR Pfcrt alleles, whereas there was no distinct Pfmdr1 MS haplotype that correlated with geography and/or CQR Pfmdr1 alleles. Furthermore, multiple independent origins of CQR Pfmdr1 alleles in Asia and Africa were inferred. These results suggest that variation at Pfcrt and Pfmdr1 loci in both Asian and African parasite populations is generated and/or maintained via substantially different mechanisms. Since Pfmdr1 mutations may be associated with resistance to artemisinin combination therapies that are replacing CQ, particularly in Africa, it is important to determine if, and how, the genetic characteristics of this locus change over time.

Age-Acquired Immunity to a Plasmodium vivax Invasion Ligand, the Duffy Binding Protein

The interaction between the Plasmodium vivax merozoite Duffy binding protein region II (DBPII) and the human erythrocyte Duffy antigen leads to infection. Highly polymorphic regions of this protein may have arisen as a mechanism to avoid host immunity. To examine whether immunity to P. vivax is directed against these polymorphic regions of DBPII, age-associated changes in the frequency of specific DBPII alleles among 358 P. vivax-positive Papua New Guineans were examined. Although the overall number and diversity of DBPII haplotypes simultaneously infecting an individual decreased with increasing age, only certain alleles at particular loci declined in frequency, indicating preferential immune selection against these alleles. One such polymorphic locus formed part of a B cell epitope, and antibodies from exposed individuals differentially recognized alleles at this locus. Therefore, acquisition of strain-specific age-acquired immunity is partially directed against polymorphic motifs within P. vivax DBPII, suggesting that these polymorphisms are maintained and likely arose under immune pressure in the host.