We propose that P. ovale comprises 2 nonrecombining species that are sympatric in Africa and Asia. We speculate on possible scenarios that could have led to this speciation. Furthermore, the relatively high frequency of imported cases of symptomatic P. ovale infection in the United Kingdom suggests that the morbidity caused by ovale malaria has been underestimated.

Background Plasmodium vivax shows a small prevalence in West and Central Africa due to the high prevalence of Duffy negative people. However, Duffy negative individuals infected with P. vivax have been reported in areas of high prevalence of Duffy positive people who may serve as supply of P. vivax strains able to invade Duffy negative erythrocytes. We investigated the presence of P. vivax in two West African countries, using blood samples and mosquitoes collected during two on-going studies.Methodology/FindingsBlood samples from a total of 995 individuals were collected in seven villages in Angola and Equatorial Guinea, and 820 Anopheles mosquitoes were collected in Equatorial Guinea. Identification of the Plasmodium species was achieved by nested PCR amplification of the small-subunit rRNA genes; P. vivax was further characterized by csp gene analysis. Positive P. vivax-human isolates were genotyped for the Duffy blood group through the analysis of the DARC gene. Fifteen Duffy-negative individuals, 8 from Equatorial Guinea (out of 97) and 7 from Angola (out of 898), were infected with two different strains of P. vivax (VK210 and VK247).ConclusionsIn this study we demonstrated that P. vivax infections were found both in humans and mosquitoes, which means that active transmission is occurring. Given the high prevalence of infection in mosquitoes, we may speculate that this hypnozoite-forming species at liver may not be detected by the peripheral blood samples analysis. Also, this is the first report of Duffy negative individuals infected with two different strains of P. vivax (VK247 and classic strains) in Angola and Equatorial Guinea. This finding reinforces the idea that this parasite is able to use receptors other than Duffy to invade erythrocytes, which may have an enormous impact in P. vivax current distribution.

SummaryAn entomological study on vectors of malaria and their relative contribution to Plasmodium falciparum transmission in the semi-urban area of Ifakara, south-eastern Tanzania, was conducted. A total of 32 houses were randomly sampled from the area and light trap catches (LTC) performed in one room in each house every 2 weeks for 1 year. A total of 147 448 mosquitoes were caught from 789 LTC; 26 134 Anopheles gambiae s.l., 615 A. funestus, 718 other anophelines and 119 981 culicines. More than 60% of the total A. gambiae s.l. were found in five (0.6%) LTCs, with a maximum of 5889 caught in a single trap. Of 505 A. gambiae s.l. speciated by polymerase chain reaction, 91.5% were found to be A. arabiensis. Plasmodium falciparum sporozoite enzyme-linked immunosorbent assay tests were performed on 10 108 anopheles mosquitoes and 39 (0.38%) were positive. Entomological inoculation rate (EIR) estimates were generated using a standard method and an alternative method that allows the calculation of confidence intervals based on a negative binomial distribution of sporozoite positive mosquitoes. Overall EIR estimates were similar; 31 vs. 29 [95% confidence interval (CI): 19, 44] infectious bites per annum, respectively. The EIR ranged from 4 (95% CI: 1, 17) in the cool season to 108 (95% CI: 69, 170) in the wet season and from 54 (95% CI: 30, 97) in the east of the town to 15 (95% CI: 8, 30) in the town centre. These estimates show large variations over short distances in time and space. They are all markedly lower than those reported from nearby rural areas and for other parts of Tanzania.

Genetic diversity of malaria parasites represents a major issue in understanding several aspects of malaria infection and disease. Genotyping of Plasmodium falciparum infections with polymerase chain reaction @'CR)-based methods has therefore been introduced in epidemiological studies. Polymorphic regions of the mspl, msp2 and gluT genes are the most frequently used markers for genotyping, but methods may differ. A multicentre study was therefore conducted to evaluate the comparability of results from different laboratories when the same samples were analysed. Analyses of laboratory-cloned lines revealed high specificity but varying sensitivity. Detection of low-density clones was hampered in multiclonal infections. Analyses of isolates from Tanzania and Papua New Guinea revealed similar positivity rates with the same allelic types identified. The number of alleles detected per isolate, however, varied systematically between the laboratories especially at high parasite densities. When the analyses were repeated within the laboratories, high agreement was found in getting positive or negative results but with a random variation in the number of alleles detected. The msp2 locus appeared to be the most informative single marker for analyses of multiplicity of infection. Genotyping by PCR is a powerful tool for studies on genetic diversity of I? falciparum but this study has revealed limitations in comparing results on multiplicity of infection derived from different laboratories and emphasizes the need for highly standardized laboratory protocols.

In the malaria vector Anopheles gambiae Giles sensu stricto, two molecular forms denoted M and S are considered units of incipient speciation within this species. Very low hybrid frequencies and significant genetic differentiation have been found in sympatric M- and S-form populations. We studied the molecular form composition and the degree of genetic differentiation at 15 microsatellites in two samples of An. gambiae collected in two consecutive years from Bissau, Guinea Bissau. High frequencies of M/S hybrids (19-24%) were found in this area. Coincidently, very low levels of genetic differentiation were detected between forms when analysis involved microsatellites mapped at chromosome-3 (mean Fst, 0.000-0.002). The single exception was the X-linked AGXH678, for which high differentiation was measured (Fst, 0.158-0.301). This locus maps near the centromere of chromosome X, a low recombination region in which selection is likely to promote divergence between M and S forms. These results strongly suggest that the degree of isolation between M and S forms, considered the units of incipient speciation within An. gambiae, is not homogenous throughout the species distribution range.

Plasmodium vivax, the second most prevalent of the human malaria parasites, is estimated to affect 75 million people annually. It is very rare, however, in west and central Africa, due to the high prevalence of the Duffy negative phenotype in the human population. Due to its rarity in Africa, previous studies on the phylogeny of world-wide P. vivax have suffered from insufficient samples of African parasites. Here we compare the mitochondrial sequence diversity of parasites from Africa with those from other areas of the world, in order to investigate the origin of present-day African P. vivax. Mitochondrial genome sequencing revealed relatively little polymorphism within the African population compared to parasites from the rest of the world. This, combined with sequence similarity with parasites from India, suggests that the present day African P. vivax population in humans may have been introduced relatively recently from the Indian subcontinent. Haplotype network analysis also raises the possibility that parasites currently found in Africa and South America may be the closest extant relatives of the ancestors of the current world population. Lines of evidence are adduced that this ancestral population may be from an ancient stock of P. vivax in Africa.

Polymerase chain reaction (PCR) is now widely used in malaria research for analysis of field samples. However, little has been reported regarding loss of sensitivity due to field methodology. Therefore, studies were carried out in relation to blood sampling (anticoagulants, culture medium, filter paper), storage (temperature, time and immediate lysis) and handling (repeated thawing and freezing). The PCR was unaffected by citrate and EDTA but partly inhibited by heparin (inhibition was reversed by heparinase at optimal concentrations). Samples collected on filter paper showed a significant 100-fold lower sensitivity (compared to control samples frozen immediately after collection) when stored at 30 degrees C and 60% humidity; and the paper quality appeared to be critical. Storage of unprocessed whole blood at 4 degrees C, 20 degrees C or 30 degrees C rarely resulted in any loss of sensitivity. Repeated thawing generally resulted in 10-fold loss of sensitivity compared to blood kept frozen until DNA extraction. The presence of antimalarial drug did not apparently affect sensitivity. We conclude that the mode of collection and storage of blood samples may influence the sensitivity of detection of malaria parasites by PCR. This may be critical in studies including individuals with low parasitaemia, mixed infections and comparison of data from different settings.

We studied malaria transmission by comparing parasite populations in humans and mosquito vectors at the household level. Blood samples were collected from all inhabitants for microscopic detection of gametocytes and polymerase chain reaction analysis. The next morning, blood-fed resting mosquitoes were collected inside the bed nets used by the individuals surveyed the previous afternoon. After 8 days of maintenance, mosquitoes were dissected, and midguts and salivary glands were recovered for polymerase chain reaction analysis. Results showed that parasite distribution was the same in the 2 hosts when compared at each household but was different when whole populations were analyzed. Different associations of Plasmodium species seem to occur in humans (Plasmodium falciparum/ Plasmodium malariae) and mosquitoes (P. falciparum/Plasmodium ovale). Regarding P. falciparum infections, a higher proportion of single-genotype infections and less allele diversity are observed in mosquitoes than in humans.