Drug-resistant genotypes and multi-clonality in Plasmodium falciparum analysed by direct genome sequencing from peripheral blood of malaria patients.

Naturally acquired blood-stage infections of the malaria parasite Plasmodium falciparum typically harbour multiple haploid clones. The apparent number of clones observed in any single infection depends on the diversity of the polymorphic markers used for the analysis, and the relative abundance of rare clones, which frequently fail to be detected among PCR products derived from numerically dominant clones. However, minority clones are of clinical interest as they may harbour genes conferring drug resistance, leading to enhanced survival after treatment and the possibility of subsequent therapeutic failure. We deployed new generation sequencing to derive genome data for five non-propagated parasite isolates taken directly from 4 different patients treated for clinical malaria in a UK hospital. Analysis of depth of coverage and length of sequence intervals between paired reads identified both previously described and novel gene deletions and amplifications. Full-length sequence data was extracted for 6 loci considered to be under selection by antimalarial drugs, and both known and previously unknown amino acid substitutions were identified. Full mitochondrial genomes were extracted from the sequencing data for each isolate, and these are compared against a panel of polymorphic sites derived from published or unpublished but publicly available data. Finally, genome-wide analysis of clone multiplicity was performed, and the number of infecting parasite clones estimated for each isolate. Each patient harboured at least 3 clones of P. falciparum by this analysis, consistent with results obtained with conventional PCR analysis of polymorphic merozoite antigen loci. We conclude that genome sequencing of peripheral blood P. falciparum taken directly from malaria patients provides high quality data useful for drug resistance studies, genomic structural analyses and population genetics, and also robustly represents clonal multiplicity

Evaluation of allelic forms of the erythrocyte binding antigen 175 (EBA-175) in Plasmodium falciparum field isolates from Brazilian endemic area

<p>Abstract</p> <p>Background</p> <p>The <it>Plasmodium falciparum </it>Erythrocyte Binding Antigen-175 (EBA-175) is an antigen considered to be one of the leading malaria vaccine candidates. EBA-175 mediates sialic acid-dependent binding to glycophorin A on the erythrocytes playing a crucial role during invasion of the <it>P. falciparum </it>in the host cell. Dimorphic allele segments, termed C-fragment and F-fragment, have been found in high endemicity malaria areas and associations between the dimorphism and severe malaria have been described. In this study, the genetic dimorphism of EBA-175 was evaluated in <it>P. falciparum </it>field isolates from Brazilian malaria endemic area.</p> <p>Methods</p> <p>The study was carried out in rural villages situated near Porto Velho, Rondonia State in the Brazilian Amazon in three time points between 1993 and 2008. The allelic dimorphism of the EBA-175 was analysed by Nested PCR.</p> <p>Results</p> <p>The classical allelic dimorphism of the EBA-175 was identified in the studied area. Overall, C-fragment was amplified in a higher frequency than F-fragment. The same was observed in the three time points where C-fragment was observed in a higher frequency than F-fragment. Single infections (one fragment amplified) were more frequent than mixed infection (two fragments amplified).</p> <p>Conclusions</p> <p>These findings confirm the dimorphism of EBA175, since only the two types of fragments were amplified, C-fragment and F-fragment. Also, the results show the remarkable predominance of CAMP allele in the studied area. The comparative analysis in three time points indicates that the allelic dimorphism of the EBA-175 is stable over time.</p

Characterization of Within-Host Plasmodium falciparum Diversity Using Next-Generation Sequence Data

Our understanding of the composition of multi-clonal malarial infections and the epidemiological factors which shape their diversity remain poorly understood. Traditionally within-host diversity has been defined in terms of the multiplicity of infection (MOI) derived by PCR-based genotyping. Massively parallel, single molecule sequencing technologies now enable individual read counts to be derived on genome-wide datasets facilitating the development of new statistical approaches to describe within-host diversity. In this class of measures the FWS metric characterizes within-host diversity and its relationship to population level diversity. Utilizing P. falciparum field isolates from patients in West Africa we here explore the relationship between the traditional MOI and FWS approaches. FWS statistics were derived from read count data at 86,158 SNPs in 64 samples sequenced on the Illumina GA platform. MOI estimates were derived by PCR at the msp-1 and -2 loci. Significant correlations were observed between the two measures, particularly with the msp-1 locus (P = 5.92×10−5). The FWS metric should be more robust than the PCR-based approach owing to reduced sensitivity to potential locus-specific artifacts. Furthermore the FWS metric captures information on a range of parameters which influence out-crossing risk including the number of clones (MOI), their relative proportions and genetic divergence. This approach should provide novel insights into the factors which correlate with, and shape within-host diversity

Hookworm-Related Anaemia among Pregnant Women: A Systematic Review

Anaemia affects large numbers of pregnant women in developing countries and increases their risk of dying during pregnancy and delivering low birth weight babies, who in turn are at increased risk of dying. Human hookworm infection has long been recognized among the major causes of anaemia in poor communities, but understanding of the benefits of the management of hookworm infection in pregnancy has lagged behind the other major causes of maternal anaemia. Low coverage of anthelmintic treatment in maternal health programmes in many countries has been the result. After systematically reviewing the available literature we observed that increasing hookworm infection intensity is associated with lower haemoglobin levels in pregnant women. We also estimate that between a quarter and a third of pregnant women in sub-Saharan Africa are infected with hookworm and at risk of preventable hookworm-related anaemia. However, all identified intervention studies showed a benefit of deworming for maternal or child health and we argue that increased efforts should be made to increase the coverage of anthelmintic treatment among pregnant women

Factors determining the occurrence of submicroscopic malaria infections and their relevance for control.

Malaria parasite prevalence in endemic populations is an essential indicator for monitoring the progress of malaria control, and has traditionally been assessed by microscopy. However, surveys increasingly use sensitive molecular methods that detect higher numbers of infected individuals, questioning our understanding of the true infection burden and resources required to reduce it. Here we analyse a series of data sets to characterize the distribution and epidemiological factors associated with low-density, submicroscopic infections. We show that submicroscopic parasite carriage is common in adults, in low-endemic settings and in chronic infections. We find a strong, non-linear relationship between microscopy and PCR prevalence in population surveys (n=106), and provide a tool to relate these measures. When transmission reaches very low levels, submicroscopic carriers are estimated to be the source of 20-50% of all human-to-mosquito transmissions. Our findings challenge the idea that individuals with little previous malaria exposure have insufficient immunity to control parasitaemia and suggest a role for molecular screening