David T. McNamara

R5 HIV productively infects Langerhans cells, and infection levels are regulated by compound CCR5 polymorphisms

Langerhans cells (LCs) are suspected to be initial targets for HIV after sexual exposure (by becoming infected or by capturing virus). Here, productive R5 HIV infection of LC ex vivo and LC-mediated transmission of virus to CD4+ T cells were both found to depend on CCR5. By contrast, infection of monocyte-derived dendritic cells and transfer of infection from monocyte-derived dendritic cells to CD4+ T cells were mediated by CCR5-dependent as well as DC-specific ICAM-3-grabbing nonintegrin-dependent pathways. Furthermore, in 62 healthy individuals, R5 HIV infection levels in LCs ex vivo were associated with CCR5 genotype. Specifically, genotyping for ORFΔ32 revealed that LCs isolated from ORFΔ32/wt individuals were significantly less susceptible to HIV when compared with LCs isolated from ORFwt/wt individuals (P = 0.016). Strikingly, further genetic analyses of the A-2459G CCR5 promoter polymorphism in ORFΔ32/wt heterozygous individuals revealed that LCs isolated from -2459A/G + ORFΔ32/wt individuals were markedly less susceptible to HIV than were LCs from -2459A/A + ORFΔ32/wt individuals (P = 0.012). Interestingly, these genetic susceptibility data in LCs parallel those of genetic susceptibility studies performed in cohorts of HIV-infected individuals. Thus, we suggest that CCR5-mediated infection of LCs, and not capture of virus by LCs, provides a biologic basis for understanding certain aspects of host genetic susceptibility to initial HIV infection.

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.

High sensitivity detection of Plasmodium species reveals positive correlations between infections of different species, shifts in age distribution and reduced local variation in Papua New Guinea.

BackgroundWhen diagnosed by standard light microscopy (LM), malaria prevalence can vary significantly between sites, even at local scale, and mixed species infections are consistently less common than expect in areas co-endemic for Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae. The development of a high-throughput molecular species diagnostic assay now enables routine PCR-based surveillance of malaria infections in large field and intervention studies, and improves resolution of species distribution within and between communities.MethodsThis study reports differences in the prevalence of infections with all four human malarial species and of mixed infections as diagnosed by LM and post-PCR ligase detection reaction – fluorescent microsphere (LDR-FMA) assay in 15 villages in the central Sepik area of Papua New Guinea.ResultsSignificantly higher rates of infection by P. falciparum, P. vivax, P. malariae and Plasmodium ovale were observed in LDR-FMA compared to LM diagnosis (p < 0.001). Increases were particularly pronounced for P. malariae (3.9% vs 13.4%) and P. ovale (0.0% vs 4.8%). In contrast to LM diagnosis, which suggested a significant deficit of mixed species infections, a significant excess of mixed infections over expectation was detected by LDR-FMA (p < 0.001). Age of peak prevalence shifted to older age groups in LDR-FMA diagnosed infections for P. falciparum (LM: 7–9 yrs 47.5%, LDR-FMA: 10–19 yrs 74.2%) and P. vivax (LM: 4–6 yrs 24.2%, LDR-FMA: 7–9 yrs 50.9%) but not P. malariae infections (10–19 yrs, LM: 7.7% LDR-FMA: 21.6%). Significant geographical variation in prevalence was found for all species (except for LM-diagnosed P. falciparum), with the extent of this variation greater in LDR-FMA than LM diagnosed infections (overall, 84.4% vs. 37.6%). Insecticide-treated bednet (ITN) coverage was also the dominant factor linked to geographical differences in Plasmodium species infection prevalence explaining between 60.6% – 74.5% of this variation for LDR-FMA and 81.8% – 90.0% for LM (except P. falciparum), respectively.ConclusionThe present study demonstrates that application of molecular diagnosis reveals patterns of malaria risk that are significantly different from those obtained by standard LM. Results provide insight relevant to design of malaria control and eradication strategies.

Development of a Multiplex PCR-Ligase Detection Reaction Assay for Diagnosis of Infection by the Four Parasite Species Causing Malaria in Humans

ABSTRACT The diagnosis of infections caused by Plasmodium species is critical for understanding the nature of malarial disease, treatment efficacy, malaria control, and public health. The demands of field-based epidemiological studies of malaria will require faster and more sensitive diagnostic methods as new antimalarial drugs and vaccines are explored. We have developed a multiplex PCR-ligase detection reaction (LDR) assay that allows the simultaneous diagnosis of infection by all four parasite species causing malaria in humans. This assay exhibits sensitivity and specificity equal to those of other PCR-based assays, identifying all four human malaria parasite species at levels of parasitemias equal to 1 parasitized erythrocyte/μl of blood. The multiplex PCR-LDR assay goes beyond other PCR-based assays by reducing technical procedures and by detecting intraindividual differences in species-specific levels of parasitemia. Application of the multiplex PCR-LDR assay will provide the sensitivity and specificity expected of PCR-based diagnostic assays and will contribute new insight regarding relationships between the human malaria parasite species and the human host in future epidemiological studies.

Reduced Plasmodium vivax Erythrocyte Infection in PNG Duffy-Negative Heterozygotes

Background Erythrocyte Duffy blood group negativity reaches fixation in African populations where Plasmodium vivax (Pv) is uncommon. While it is known that Duffy-negative individuals are highly resistant to Pv erythrocyte infection, little is known regarding Pv susceptibility among heterozygous carriers of a Duffy-negative allele (+/−). Our limited knowledge of the selective advantages or disadvantages associated with this genotype constrains our understanding of the effect that interventions against Pv may have on the health of people living in malaria-endemic regions. Methods and Findings We conducted cross-sectional malaria prevalence surveys in Papua New Guinea (PNG), where we have previously identified a new Duffy-negative allele among individuals living in a region endemic for all four human malaria parasite species. We evaluated infection status by conventional blood smear light microscopy and semi-quantitative PCR-based strategies. Analysis of a longitudinal cohort constructed from our surveys showed that Duffy heterozygous (+/−) individuals were protected from Pv erythrocyte infection compared to those homozygous for wild-type alleles (+/+) (log-rank tests: LM, p = 0.049; PCR, p = 0.065). Evaluation of Pv parasitemia, determined by semi-quantitative PCR-based methods, was significantly lower in Duffy +/− vs. +/+ individuals (Mann-Whitney U: p = 0.023). Overall, we observed no association between susceptibility to P. falciparum erythrocyte infection and Duffy genotype. Conclusions Our findings provide the first evidence that Duffy-negative heterozygosity reduces erythrocyte susceptibility to Pv infection. As this reduction was not associated with greater susceptibility to Pf malaria, our in vivo observations provide evidence that Pv-targeted control measures can be developed safely.

CR1 Knops blood group alleles are not associated with severe malaria in the Gambia

The Knops blood group antigen erythrocyte polymorphisms have been associated with reduced falciparum malaria-based in vitro rosette formation (putative malaria virulence factor). Having previously identified single-nucleotide polymorphisms (SNPs) in the human complement receptor 1 (CR1/CD35) gene underlying the Knops antithetical antigens Sl1/Sl2 and McCa/McCb, we have now performed genotype comparisons to test associations between these two molecular variants and severe malaria in West African children living in the Gambia. While SNPs associated with Sl:2 and McC(b+) were equally distributed among malaria-infected children with severe malaria and control children not infected with malaria parasites, high allele frequencies for Sl 2 (0.800, 1365/1706) and McCb (0.385, 658/1706) were observed. Further, when compared to the Sl 1/McCa allele observed in all populations, the African Sl 2/McCb allele appears to have evolved as a result of positive selection (modified Nei–Gojobori test Ka−Ks/s.e.=1.77, P-value <0.05). Given the role of CR1 in host defense, our findings suggest that Sl 2 and McCb have arisen to confer a selective advantage against infectious disease that, in view of these case–control study data, was not solely Plasmodium falciparum malaria. Factors underlying the lack of association between Sl 2 and McCb with severe malaria may involve variation in CR1 expression levels.

A Multiplex Ligase Detection Reaction-Fluorescent Microsphere Assay for Simultaneous Detection of Single Nucleotide Polymorphisms Associated with Plasmodium falciparum Drug Resistance

ABSTRACT Incomplete malaria control efforts have resulted in a worldwide increase in resistance to drugs used to treat the disease. A complex array of mutations underlying antimalarial drug resistance complicates efficient monitoring of parasite populations and limits the success of malaria control efforts in regions of endemicity. To improve the surveillance of Plasmodium falciparum drug resistance, we developed a multiplex ligase detection reaction-fluorescent-microsphere-based assay (LDR-FMA) that identifies single nucleotide polymorphisms (SNPs) in the P. falciparum dhfr (9 alleles), dhps (10 alleles), and pfcrt (3 alleles) genes associated with resistance to Fansidar and chloroquine. We evaluated 1,121 blood samples from study participants in the Wosera region of Papua New Guinea, where malaria is endemic. Results showed that 468 samples were P. falciparum negative and 453 samples were P. falciparum positive by a Plasmodium species assay and all three gene assays (concordance, 82.2%). For P. falciparum infections where the assay for each gene was positive, 2 samples carried resistance alleles for all three genes, 299 carried resistance alleles for dhfr and pfcrt, 131 carried resistance alleles for only one gene (dhfr [n = 40], dhps [n = 1], or pfcrt [n = 90]), and 21 carried only sensitive alleles at all three genes. Mixed-strain infections characterized 100 samples. Overall, 95.4% (432/453) of P. falciparum-infected samples carried at least one allele associated with resistance to Fansidar or chloroquine. In view of the fact that 86.3% (391/453) of P. falciparum-infected samples carried pfcrt mutations, chloroquine is largely ineffective against P. falciparum in Papua New Guinea. Surveillance of additional dhfr and dhps polymorphisms in order to monitor the continued effectiveness of Fansidar is recommended.

High-throughput molecular diagnosis of circumsporozoite variants VK210 and VK247 detects complex Plasmodium vivax infections in malaria endemic populations in Papua New Guinea

Malaria is endemic in lowland and coastal regions of Papua New Guinea (PNG), and is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. Infection by P. vivax is attributed to distinct strains, VK210 and VK247, which differ in the sequence of the circumsporozoite protein (pvcsp). Here, based upon sequence polymorphisms in pvcsp, we developed a post-PCR ligation detection reaction-fluorescent microsphere assay (LDR-FMA) to distinguish these P. vivax strains. This diagnostic assay was designed to detect the presence of both VK210 and VK247 P. vivax strains simultaneously in a high-throughput 96-well format. Using this assay, we analyzed human blood samples from the Wosera (n=703) and Mugil (n=986) regions to evaluate the prevalence of these P. vivax strains. VK210 and VK247 strains were found in both study sites. In the Wosera, single infections with VK210 strain were observed to be most common (41.7%), followed by mixed-strain (36.8%) and VK247 single-strain infections (21.5%). Similarly, in Mugil, VK210 single-strain infections were most common (51.6%), followed by mixed-strain (34.4%) and VK247 single-strain infections (14%). These results suggest that the distribution of P. vivax infections was similar between the two study sites. Interestingly, we observed a non-random distribution of these two P. vivax strains, as mixed-strain infections were significantly more prevalent than expected in both study sites (Wosera and Mugil χ(2)p-value<0.001). Additionally, DNA sequence analysis of a subset of P. vivax infections showed that no individual pvcsp alleles were shared between the two study sites. Overall, our results illustrate that PNG malaria-endemic regions harbor a complex mixture of P. vivax strains, and emphasize the importance of malaria control strategies that would be effective against a highly diverse parasite population.