A Case of Plasmodium ovale wallikeri Infection in a Chinese Worker Returning from West Africa

In contrast to the gradual reduction in the number of locally transmitted malaria cases in China, the number of imported malaria cases has been increasing since 2008. Here, we report a case of a 39-year-old Chinese man who acquired Plasmodium ovale wallikeri infection while staying in Ghana, West Africa for 6 months in 2012. Microscopic examinations of Giemsa-stained thin and thick blood smears indicated Plasmodium vivax infection. However, the results of rapid diagnostic tests, which were conducted 3 times, were not in agreement with P. vivax. To further check the diagnosis, standard PCR analysis of the small-subunit rRNA gene was conducted, based on which a phylogeny tree was constructed. The results of gene sequencing indicated that this malaria is a variant of P. ovale (P. ovale wallikeri). The infection in this patient was not a new infection, but a relapse of the infection from the one that he had contracted in West Africa.     

Plasmodium ovale curtisi and Plasmodium ovale wallikeri circulate simultaneously in African communities

It has been proposed that ovale malaria in humans is caused by two closely related but distinct species of malaria parasite, Plasmodium ovale curtisi and Plasmodium ovale wallikeri. It was recently shown that these two parasite types are sympatric at the country level. However, it remains possible that localised geographic, temporal or ecological barriers exist within endemic countries which prevent recombination between the genomes of the two species. Here, using conventional and real-time quantitative PCR (qPCR) methods specifically designed to discriminate P. o. curtisi and P. o. wallikeri, it is shown that both species are present among clinic attendees in Congo-Brazzaville, and occur simultaneously both in lake-side and inland districts in Uganda and on Bioko Island, Equatorial Guinea. Thus P. o. curtisi and P. o. wallikeri in these localities are exactly sympatric in both time and space. These findings are consistent with the existence of a biological barrier, rather than geographical or ecological factors, preventing recombination between P. o. curtisi and P. o. wallikeri. In cross-sectional surveys carried out in Uganda and Bioko, our results show that infections with P. ovale spp. are more common than previously thought, occurring at a frequency of 1-6% in population samples, with both proposed species contributing to ovale malaria in six sites. Malaria elimination programmes in Africa need to include strategies for control of P. o. curtisi and P. o. wallikeri.     

How prevalent are Plasmodium ovale and P. malariae in East Asia?

Plasmodium ovale and Plasmodium malariae, two of the four human malaria parasites, are usually found at very low prevalence in East Asia, even in areas with intense malaria transmission. In this article, Fumihiko Kawamoto, Qing Liu, Marcelo Ferreira and Indah Tantular review data obtained in recent field surveys, using alternative diagnostic methods such as acridine orange staining and PCR-based methods, to evaluate the prevalence of these two malaria species in East Asia. They argue that these species might be much more prevalent in East Asia than reported previously. In addition, they discuss the implications of sequence variations found in the small subunit ribosomal RNA genes of the two species targeted by diagnostic PCR and compare morphological criteria for speciation of malaria parasites stained with Giemsa and acridine orange.     

Plasmodium ovale in Bangladesh: genetic diversity and the first known evidence of the sympatric distribution of Plasmodium ovale curtisi and Plasmodium ovale wallikeri in southern Asia

In spite of the high prevalence of malaria in Bangladesh and other southern Asian countries, there remains a substantial shortage of knowledge about the less common human malaria parasites. Recent studies indicate that Plasmodium ovale is made up of two species, namely Plasmodium ovale wallikeri and Plasmodium ovale curtisi. Genus- and species-specific nested PCR analyses of the ssrRNA gene was used to detect P. ovale infections among 2,246 diagnostic samples. Plasmodium ovale infections were further differentiated by nested PCR of the potra gene and multilocus sequence analysis of the cox1, porbp2 and the ssrRNA genes. Both P. ovale curtisi and P. ovale wallikeri occur sympatrically in the Chittagong Hill Tracts, Bangladesh and all patients presented with a mild or asymptomatic symptom complex at the time of diagnosis. The pathogens can be differentiated by nested PCRs targeting the ssrRNA and potra genes, and display dimorphism in multilocus sequence analyses. We believe that we report the first evidence of sympatric P. ovale curtisi and P. ovale wallikeri in southern Asia within a relatively confined study area of less than 5,000 km(2). High rates of mixed infections, the emergence of "new" human malaria parasite species and the evidence of zoonotic capability call for optimised diagnostic strategies for a new era of eradication.     

A New Real-Time PCR for the Detection of Plasmodium ovale wallikeri

It has been proposed that ovale malaria in humans is caused by two closely related but distinct species of malaria parasites: P. ovale curtisi and P. ovale wallikeri. We have extended and optimized a Real-time PCR assay targeting the parasite’s small subunit ribosomal RNA (ssrRNA) gene to detect both these species. When the assay was applied to 31 archival blood samples from patients diagnosed with P. ovale, it was found that the infection in 20 was due to P. ovale curtisi and in the remaining 11 to P. ovale wallikeri. Thus, this assay provides a useful tool that can be applied to epidemiological investigations of the two newly recognized distinct P. ovale species, that might reveal if these species also differ in their clinical manifestation, drugs susceptibility and relapse periodicity. The results presented confirm that P. ovale wallikeri is not confined to Southeast Asia, since the majority of the patients analyzed in this study had acquired their P. ovale infection in African countries, mostly situated in West Africa.     

Epidemiological evidence for an association between chloroquine resistance of Plasmodium falciparum and its immunological properties

The appearance of chloroquine-resistant genotypes o f Plasmodium falciparum has thwarted the goal of global eradication of malaria. Although much effort has been put into understanding the molecular mechanisms of chloroquine resistance, many questions about its distribution remain open: Why, some 30 years after the emergence o f chloroquine resistance, have resistant genotypes not taken over the population? Why have many parasites remained sensitive? Why, after its first appearance in Africa, has chloroquine resistance spread so rapidly through sub-Saharan Africa? In this paper Jacob Koella reviews epidemiological data that suggest that an answer to these questions may involve an association between chloroquine resistance and immunological properties o f malaria parasites.     

Risk of malaria in British residents returning from malarious areas.

OBJECTIVES--To identify which British residents travelling abroad are at greatest risk of malaria infection, and to determine the efficacy of malaria chemoprophylaxis for preventing P falciparum infections in tropical Africa. DESIGN--Prospective cohort study (case-base linkage) with routine national surveillance systems. Denominators (base population) were obtained from monitoring a random sample of returning British travellers with the international passenger survey. Numerators (cases) were obtained from reports of malaria infections in British residents, through the Malaria Reference Laboratory network. SETTING--International passenger survey conducted at passport control of international airports in Britain. Malaria reports received nationally were collated centrally in London. SUBJECTS--2948 British residents (0.2%) returning to Britain in 1987 randomly selected and questioned and 1052 British residents with microscopically confirmed malaria infections in 1987, whose case reports were reviewed and on whom additional data were collected by postal survey. MAIN OUTCOME MEASURES--Annual incidence subdivided by categories of risk. Chemoprophylactic efficacy for east and west Africa by principal regimens and compliance. RESULTS--Annual rates of reported infection per 100,000 travellers to Oceania were 4100; to west and east Africa were 375 and 172 respectively; to Latin America, the Far East, and the Middle East were 12, 2, and 1 respectively. Immigrants visiting friends and relatives in Ghana and Nigeria were at greatest risk (1303 and 952 per 100,000 respectively) in west Africa. Business travellers to Kenya experienced the highest attack rates in east Africa (465 per 100,000). Age-sex specific attack rates varied by region. No prophylaxis was reported to have been used by 23% of British visitors to west Africa, 17% to east Africa, 46% to central or southern Africa, and 58% visiting south Asia. The efficacy of chloroquine plus proguanil against P falciparum infection was 73% and 54% in west and east Africa respectively. Lower values were obtained for chloroquine alone and proguanil alone. The efficacy of Maloprim (pyrimethamine-dapsone) was 61% in west Africa, but only 9% in east Africa. Visitors to west Africa who did not comply with their chemoprophylactic regimen were at a 2.5-fold higher risk of infection than fully compliant users. Non-compliant visitors to east Africa had similar rates of infection as non-drug users. CONCLUSIONS--In 1987 chloroquine plus proguanil was the preferred chemoprophylactic regimen for P falciparum infection in Africa; antimalarial drugs must be taken regularly to be effective.     

Plasmodium malariae and Plasmodium ovale--the "bashful" malaria parasites

Although Plasmodium malariae was first described as an infectious disease of humans by Golgi in 1886 and Plasmodium ovale identified by Stevens in 1922, there are still large gaps in our knowledge of the importance of these infections as causes of malaria in different parts of the world. They have traditionally been thought of as mild illnesses that are caused by rare and, in case of P. ovale, short-lived parasites. However, recent advances in sensitive PCR diagnosis are causing a re-evaluation of this assumption. Low-level infection seems to be common across malaria-endemic areas, often as complex mixed infections. The potential interactions of P. malariae and P. ovale with Plasmodium falciparum and Plasmodium vivax might explain some basic questions of malaria epidemiology, and understanding these interactions could have an important influence on the deployment of interventions such as malaria vaccines.     

Usefulness of genus-specific PCR and Southern blot species-specific hybridization for the detection of imported malaria cases in Italy

A PCR method involving a genus-specific oligonucleotides set and Southern blot hybridization with four species-specific probes to P. falciparum, P. vivax, P. malariae and P. ovale was evaluated for the detection of malaria parasites in blood samples from 101 patients with clinically suspect malaria infection imported to Italy. Plasmodium falciparum was the main species detected. As determined by microscopy, 53 (52.4%) patients had malaria and of these: 40 (75.5%) were infected with P. falciparum; 7 (13.2%) with P. vivax; 1 (1.9%) with P. ovale; 3 (5.7%) with P. malariae; 1 (1.9%) with P. vivax or P. ovale; and 1 (1.9%) with P. falciparum or P. vivax. Ninety-seven out 101 blood samples were submitted to ParaSight-F test which showed a sensitivity of 94.73%, and a specificity of 93.22%, as compared to microscopy. The PCR assay using the genus-specific oligonucleotide primer set (pg-PCR) was able to detect 53 (52.4%) infections and showed a sensitivity of 100% and a specificity of 100%, when compared to microscopy. The parasite species were identified by Southern blot hybridization using species-specific probes and 40 (75.5%) samples were P. falciparum positive, 5 (9.4%) P. vivax positive, 4 (7.5%) P. ovale positive, and 2 (3.8%) P. malariae positive. When the Southern blot results were compared to those of blood-film diagnosis, we observed some disagreement. In particular, compared to Southern blot, microscopy underestimated P. ovale infection; blood film analysis recognised only 1 P. ovale sample, whereas Southern blot recognised 4 P. ovale positive samples (by microscopy, 2 of these were detected as P. vivax, 1 as P. ovale or P. vivax, and the other as P. falciparum or P. vivax). Southern blot hybridization was unable to identify one P. falciparum and one P. vivax positive case detected by microscopy. We also plan to use a reference nested-PCR assay to clarify the disagreement observed between microscopy and Southern blot hybridization.     

Real-time PCR assay for discrimination of Plasmodium ovale curtisi and Plasmodium ovale wallikeri in the Ivory Coast and in the Comoros Islands

ABSTRACT: BACKGROUND: Plasmodium ovale is one of the five malaria species infecting humans. Recent data have shown that the name of this neglected species masks two distinct genotypes also called curtisi and wallikeri. Some authors show that these species could be sympatric. These two subspecies are not differentiated by microscopy techniques and malaria rapid diagnostic tests. This diagnostic defect is the result of low parasitaemia, antigenic polymorphism and absence of antibodies performance and requires the use of sequencing techniques. An accurate and easy discrimination detection method is necessary. METHODS: A new molecular assay was developed to easily identify the two genotypes of P. ovale. This tool allowed the study of 90 blood samples containing P. ovale, confirmed by molecular biology techniques, which were obtained from patients with imported malaria. RESULTS: The new marker was validated on well genotyped samples. The genotype of 90 P. ovale samples mainly imported from the Ivory Coast and the Comoros Islands was easily and quickly realized. The distribution of the two subspecies was described with a significant number of samples and showed that the two genotypes were present in the studied countries. CONCLUSION: This work confirms the presence of the two species in the same country for the first time, in the Ivory Coast and the Comoros Islands. A better genotyping of P. ovale types may improve a better characterization of the clinical pathophysiology for each.     

Variant Plasmodium ovale isolated from a patient infected in Ghana

Recent data have found that Plasmodium ovale can be separated in two distinct species: classic and variant P. ovale based on multilocus typing of different genes. This study presents a P. ovale isolate from a patient infected in Ghana together with an analysis of the small subunit RNA, cytochrome b, cytochrome c oxidase I, cysteine protease and lactate dehydrogenase genes, which show that the sample is a variant P. ovale and identical or highly similar to variant P. ovale isolated from humans in South-East Asia and Africa, and from a chimpanzee in Cameroon. The split between the variant and classic P. ovale is estimated to have occurred 1.7 million years ago.     

Antimalarial activity of potential inhibitors of Plasmodium falciparum lactate dehydrogenase enzyme selected by docking studies

The Plasmodium falciparum lactate dehydrogenase enzyme (PfLDH) has been considered as a potential molecular target for antimalarials due to this parasite's dependence on glycolysis for energy production. Because the LDH enzymes found in P. vivax, P. malariae and P. ovale (pLDH) all exhibit ～90% identity to PfLDH, it would be desirable to have new anti-pLDH drugs, particularly ones that are effective against P. falciparum, the most virulent species of human malaria. Our present work used docking studies to select potential inhibitors of pLDH, which were then tested for antimalarial activity against P. falciparum in vitro and P. berghei malaria in mice. A virtual screening in DrugBank for analogs of NADH (an essential cofactor to pLDH) and computational studies were undertaken, and the potential binding of the selected compounds to the PfLDH active site was analyzed using Molegro Virtual Docker software. Fifty compounds were selected based on their similarity to NADH. The compounds with the best binding energies (itraconazole, atorvastatin and posaconazole) were tested against P. falciparum chloroquine-resistant blood parasites. All three compounds proved to be active in two immunoenzymatic assays performed in parallel using monoclonals specific to PfLDH or a histidine rich protein (HRP2). The IC(50) values for each drug in both tests were similar, were lowest for posaconazole (<5 μM) and were 40- and 100-fold less active than chloroquine. The compounds reduced P. berghei parasitemia in treated mice, in comparison to untreated controls; itraconazole was the least active compound. The results of these activity trials confirmed that molecular docking studies are an important strategy for discovering new antimalarial drugs. This approach is more practical and less expensive than discovering novel compounds that require studies on human toxicology, since these compounds are already commercially available and thus approved for human use.     

Hot topic or hot air? Climate change and malaria resurgence in East African highlands

Climate has a significant impact on malaria incidence and we have predicted that forecast climate changes might cause some modifications to the present global distribution of malaria close to its present boundaries. However, it is quite another matter to attribute recent resurgences of malaria in the highlands of East Africa to climate change. Analyses of malaria time-series at such sites have shown that malaria incidence has increased in the absence of co-varying changes in climate. We find the widespread increase in resistance of the malaria parasite to drugs and the decrease in vector control activities to be more likely driving forces behind the malaria resurgence.     

Evaluation of a new plate hybridization assay for the laboratory diagnosis of imported malaria in Italy

A new molecular diagnostic method "Malaria-IBRIDOGEN" (Amplimedical S.p.A.--Bioline Division, Turin, Italy) based on a plate-hybridization assay for the simultaneous detection and identification of human malaria parasites was evaluated in this study. A target DNA sequence of the plasmodial 18S ribosomal RNA gene was amplified by polymerase chain reaction (PCR) and hybridized in microtiter wells with five biotinylated probes each specific for Plasmodium falciparum, P. vivax, P. malariae, P. ovale and the beta-globine human gene, respectively. Compared to the nested-PCR actually used in our laboratory for the molecular diagnosis of malaria, "Malaria-IBRIDOGEN" revealed an overall sensitivity of 100% (51/51) for the four human Plasmodium species testing 100 whole blood samples from people with malaria-like symptoms and fever. Specificity was 92% (45/49) considering four discordant samples as "false positive" by "Malaria-IBRIDOGEN". The assay showed a threshold of parasite density (detection limit) of 0.07 P. falciparum parasites/microliter, 0.15-1.5 P. vivax parasites/microliter, 0.3 P. malariae parasites/microliter and 0.4 P. ovale parasites/microliter of whole blood, respectively. This assay could be successfully applied to the laboratory diagnosis of malaria as a useful aid to microscopy.     

Compensatory evolution in the human malaria parasite Plasmodium ovale

The fixation of neutral compensatory mutations in a population depends on the effective population size of the species, which can fluctuate dramatically within a few generations, the mutation rate, and the selection intensity associated with the individual mutations. We observe compensatory mutations and intermediate states in populations of the malaria parasite Plasmodium ovale. The appearance of compensatory mutations and intermediate states in P. ovale raises interesting questions about population structure that could have considerable impact on the control of the associated disease.     

Advances in the study of Anopheles funestus, a major vector of malaria in Africa

The recent literature on cytogenetic and molecular studies of Anopheles funestus, a major vector of malaria in Africa, is reviewed. Molecular data from West and Central Africa suggest a new species in the group closely allied to Anopheles rivulorum. Cytogenetic and molecular studies of populations from West, Central, East and southern Africa indicate considerable genetic structuring within An. funestus itself, which may well restrict the spread of pyrethroid resistance that has been demonstrated in southern Africa.     

Rare quadruple malaria infection in Irian Jaya Indonesia.

We report an exceptional finding from a blood slide collected in a remote area in the western half of New Guinea Island (Irian Jaya Province, Indonesia). One adolescent patient was found patently coinfected with all 4 known human malaria species, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale. Diagnostic erythrocytic stages of all 4 species were clearly seen in the peripheral blood. A nested polymerase chain reaction, using species-specific primer pairs to detect DNA, helped substantiate this finding. Previous reports from Africa, Thailand, and New Guinea have detected all 4 species in a population but not simultaneously in an individual with a patent, microscopically detectable infection. We believe this quadruple infection represents the first reported natural case of all 4 human malaria parasites observed concurrently in the peripheral blood from a single Giemsa-stained slide.     

Epidemic malaria and warmer temperatures in recent decades in an East African highland

Climate change impacts on malaria are typically assessed with scenarios for the long-term future. Here we focus instead on the recent past (1970-2003) to address whether warmer temperatures have already increased the incidence of malaria in a highland region of East Africa. Our analyses rely on a new coupled mosquito-human model of malaria, which we use to compare projected disease levels with and without the observed temperature trend. Predicted malaria cases exhibit a highly nonlinear response to warming, with a significant increase from the 1970s to the 1990s, although typical epidemic sizes are below those observed. These findings suggest that climate change has already played an important role in the exacerbation of malaria in this region. As the observed changes in malaria are even larger than those predicted by our model, other factors previously suggested to explain all of the increase in malaria may be enhancing the impact of climate change.     

Quantitative trait loci controlling refractoriness to Plasmodium falciparum in natural Anopheles gambiae mosquitoes from a malaria-endemic region in western Kenya

Natural anopheline populations exhibit much variation in ability to support malaria parasite development, but the genetic mechanisms underlying this variation are not clear. Previous studies in Mali, West Africa, identified two quantitative trait loci (QTL) in Anopheles gambiae mosquitoes that confer refractoriness (failure of oocyst development in mosquito midguts) to natural Plasmodium falciparum parasites. We hypothesize that new QTL may be involved in mosquito refractoriness to malaria parasites and that the frequency of natural refractoriness genotypes may be higher in the basin region of Lake Victoria, East Africa, where malaria transmission intensity and parasite genetic diversity are among the highest in the world. Using field-derived F2 isofemale families and microsatellite marker genotyping, two loci significantly affecting oocyst density were identified: one on chromosome 2 between markers AG2H135 and AG2H603 and the second on chromosome 3 near marker AG3H93. The first locus was detected in three of the five isofemale families studied and colocalized to the same region as Pen3 and pfin1 described in other studies. The second locus was detected in two of the five isofemale families, and it appears to be a new QTL. QTL on chromosome 2 showed significant additive effects while those on chromosome 3 exhibited significant dominant effects. Identification of P. falciparum-refractoriness QTL in natural An. gambiae mosquitoes is critical to the identification of the genes involved in malaria parasite transmission in nature and for understanding the coevolution between malaria parasites and mosquito vectors.