Can Wolbachia be used to control malaria?

Malaria is a mosquito-borne infectious disease caused by Plasmodium parasites transmitted by the infectious bite of Anopheles mosquitoes. Vector control of malaria has predominantly focused on targeting the adult mosquito through insecticides and bed nets. However, current vector control methods are often not sustainable for long periods so alternative methods are needed. A novel biocontrol approach for mosquito-borne diseases has recently been proposed, it uses maternally inherited endosymbiotic Wolbachia bacteria transinfected into mosquitoes in order to interfere with pathogen transmission. Transinfected Wolbachia strains in Aedes aegypti mosquitoes, the primary vector of dengue fever, directly inhibit pathogen replication, including Plasmodium gallinaceum, and also affect mosquito reproduction to allow Wolbachia to spread through mosquito populations. In addition, transient Wolbachia infections in Anopheles gambiae significantly reduce Plasmodium levels. Here we review the prospects of using a Wolbachia-based approach to reduce human malaria transmission through transinfection of Anopheles mosquitoes.

Enhanced T cell activation in Plasmodium falciparum malaria-infected human immunodeficiency virus-1 patients from Mozambique

Human immunodeficiency virus (HIV)-1 infection has an important impact on malaria. Plasmodium falciparum and HIV-1 co-infected patients (Pf/HIV) present with a high degree of anaemia, enhanced parasitaemia and decreased CD4+ T cell counts, which increase the risk of developing severe malaria. In addition, infection with either Pf or HIV-1 alone causes extensive immune activation. Our hypothesis was that lymphocyte activation is potentiated in Pf/HIV co-infected patients, consequently worsening their immunosuppressed state. To test this hypothesis, 22 Pf/HIV patients, 34 malaria patients, 29 HIV/AIDS patients and 10 healthy controls without malaria or HIV/acquired immune deficiency syndrome (AIDS) from Maputo/Mozambique were recruited for this study. As expected, anaemia was most prevalent in the Pf/HIV group. A significant variation in parasite density was observed in the Pf/HIV co-infected group (110-75,000 parasites/µL), although the median values were similar to those of the malaria only patients. The CD4+ T cell counts were significantly lower in the Pf/HIV group than in the HIV/AIDS only or malaria only patients. Lymphocyte activation was evaluated by the percentage of activation-associated molecules [CD38 expression on CD8+ and human leukocyte antigen-DR expression on CD3+ T cells]. The highest CD38 expression was detected in the Pf/HIV co-infected patients (median = 78.2%). The malaria only (median = 50%) and HIV/AIDS only (median = 52%) patients also exhibited elevated levels of these molecules, although the values were lower than those of the Pf/HIV co-infected cases. Our findings suggest that enhanced T-cell activation in co-infected patients can worsen the immune response to both diseases.

Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.

Blood shizonticidal activities of phenazines and naphthoquinoidal compounds against Plasmodium falciparum in vitro and in mice malaria studies

Due to the recent advances of atovaquone, a naphthoquinone, through clinical trials as treatment for malarial infection, 19 quinone derivatives with previously reported structures were also evaluated for blood schizonticide activity against the malaria parasite Plasmodium falciparum. These compounds include 2-hydroxy-3-methylamino naphthoquinones (2-9), lapachol (10), nor-lapachol (11), iso-lapachol (12), phthiocol (13) and phenazines (12-20). Their cytotoxicities were also evaluated against human hepatoma and normal monkey kidney cell lines. Compounds 2 and 5 showed the highest activity against P. falciparum chloroquine-resistant blood-stage parasites (clone W2), indicated by their low inhibitory concentration for 50% (IC50) of parasite growth. The therapeutic potential of the active compounds was evaluated according to the selectivity index, which is a ratio of the cytotoxicity minimum lethal dose which eliminates 50% of cells and the in vitro IC50. Naphthoquinones 2 and 5, with activities similar to the reference antimalarial chloroquine, were also active against malaria in mice and suppressed parasitaemia by more than 60% in contrast to compound 11 which was inactive. Based on their in vitro and in vivo activities, compounds 2 and 5 are considered promising molecules for antimalarial treatment and warrant further study.

Malaria in Brazil: what happens outside the Amazonian endemic region

Brazil, a country of continental proportions, presents three profiles of malaria transmission. The first and most important numerically, occurs inside the Amazon. The Amazon accounts for approximately 60% of the nation’s territory and approximately 13% of the Brazilian population. This region hosts 99.5% of the nation’s malaria cases, which are predominantly caused by Plasmodium vivax (i.e., 82% of cases in 2013). The second involves imported malaria, which corresponds to malaria cases acquired outside the region where the individuals live or the diagnosis was made. These cases are imported from endemic regions of Brazil (i.e., the Amazon) or from other countries in South and Central America, Africa and Asia. Imported malaria comprised 89% of the cases found outside the area of active transmission in Brazil in 2013. These cases highlight an important question with respect to both therapeutic and epidemiological issues because patients, especially those with falciparum malaria, arriving in a region where the health professionals may not have experience with the clinical manifestations of malaria and its diagnosis could suffer dramatic consequences associated with a potential delay in treatment. Additionally, because the Anopheles vectors exist in most of the country, even a single case of malaria, if not diagnosed and treated immediately, may result in introduced cases, causing outbreaks and even introducing or reintroducing the disease to a non-endemic, receptive region. Cases introduced outside the Amazon usually occur in areas in which malaria was formerly endemic and are transmitted by competent vectors belonging to the subgenus Nyssorhynchus (i.e., Anopheles darlingi, Anopheles aquasalis and species of the Albitarsis complex). The third type of transmission accounts for only 0.05% of all cases and is caused by autochthonous malaria in the Atlantic Forest, located primarily along the southeastern Atlantic Coast. They are caused by parasites that seem to be (or to be very close to) P. vivax and, in a less extent, by Plasmodium malariae and it is transmitted by the bromeliad mosquito Anopheles (Kerteszia) cruzii. This paper deals mainly with the two profiles of malaria found outside the Amazon: the imported and ensuing introduced cases and the autochthonous cases. We also provide an update regarding the situation in Brazil and the Brazilian endemic Amazon.

The remarkable journey of adaptation of the Plasmodium falciparum malaria parasite to New World anopheline mosquitoes

Plasmodium falciparum originated in Africa, dispersed around the world as a result of human migration and had to adapt to several different indigenous anopheline mosquitoes. Anophelines from the New World are evolutionary distant form African ones and this probably resulted in a more stringent selection of Plasmodium as it adapted to these vectors. It is thought that Plasmodium has been genetically selected by some anopheline species through unknown mechanisms. The mosquito immune system can greatly limit infection and P. falciparum evolved a strategy to evade these responses, at least in part mediated by Pfs47, a highly polymorphic gene. We propose that adaptation of P. falciparum to new vectors may require evasion of their immune system. Parasites with a Pfs47 haplotype compatible with the indigenous mosquito vector would be able to survive and be transmitted. The mosquito antiplasmodial response could be an important determinant of P. falciparum population structure and could affect malaria transmission in the Americas.

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.

Disease in the dark: molecular characterization of Polychromophilus murinus in temperate zone bats revealed a worldwide distribution of this malaria-like disease.

For a better understanding of the complex coevolutionary processes between hosts and parasites, accurate identification of the actors involved in the interaction is of fundamental importance. Blood parasites of the Order Haemosporidia, responsible for malaria, have become the focus of a broad range of studies in evolutionary biology. Interestingly, molecular-based studies on avian malaria have revealed much higher species diversity than previously inferred with morphology. Meanwhile, studies on bat haemosporidian have been largely neglected. In Europe, only one genus (Polychromophilus) and two species have been morphologically described. To evaluate the presence of potential cryptic species and parasite prevalence, we undertook a molecular characterization of Polychromophilus in temperate zone bats. We used a nested-PCR approach on the cytochrome b mitochondrial gene to detect the presence of parasites in 237 bats belonging to four different species and in the dipteran bat fly Nycteribia kolenatii, previously described as being the vector of Polychromophilus. Polychromophilus murinus was found in the four bat species and in the insect vector with prevalence ranging from 4% for Myotis myotis to 51% for M. daubentoni. By sequencing 682 bp, we then investigated the phylogenetic relationships of Polychromophilus to other published malarial lineages. Seven haplotypes were found, all very closely related, suggesting the presence of a single species in our samples. These haplotypes formed a well-defined clade together with Haemosporidia of tropical bats, revealing a worldwide distribution of this parasite mostly neglected by malarial studies since the 1980s.

Residual antimalarials in malaria patients from Tanzania--implications on drug efficacy assessment and spread of parasite resistance.

Repeated antimalarial treatment for febrile episodes and self-treatment are common in malaria-endemic areas. The intake of antimalarials prior to participating in an in vivo study may alter treatment outcome and affect the interpretation of both efficacy and safety outcomes. We report the findings from baseline plasma sampling of malaria patients prior to inclusion into an in vivo study in Tanzania and discuss the implications of residual concentrations of antimalarials in this setting. In an in vivo study conducted in a rural area of Tanzania in 2008, baseline plasma samples from patients reporting no antimalarial intake within the last 28 days were screened for the presence of 14 antimalarials (parent drugs or metabolites) using liquid chromatography-tandem mass spectrometry. Among the 148 patients enrolled, 110 (74.3%) had at least one antimalarial in their plasma: 80 (54.1%) had lumefantrine above the lower limit of calibration (LLC = 4 ng/mL), 7 (4.7%) desbutyl-lumefantrine (4 ng/mL), 77 (52.0%) sulfadoxine (0.5 ng/mL), 15 (10.1%) pyrimethamine (0.5 ng/mL), 16 (10.8%) quinine (2.5 ng/mL) and none chloroquine (2.5 ng/mL). The proportion of patients with detectable antimalarial drug levels prior to enrollment into the study is worrying. Indeed artemether-lumefantrine was supposed to be available only at government health facilities. Although sulfadoxine-pyrimethamine is only recommended for intermittent preventive treatment in pregnancy (IPTp), it was still widely used in public and private health facilities and sold in drug shops. Self-reporting of previous drug intake is unreliable and thus screening for the presence of antimalarial drug levels should be considered in future in vivo studies to allow for accurate assessment of treatment outcome. Furthermore, persisting sub-therapeutic drug levels of antimalarials in a population could promote the spread of drug resistance. The knowledge on drug pressure in a given population is important to monitor standard treatment policy implementation.

A clonal Plasmodium falciparum population in an isolated outbreak of malaria in the Republic of Cabo Verde

We present the first parasitological, molecular and longitudinal analysis of an isolated outbreak of malaria. This outbreak occurred on Santiago Island (Republic of Cabo Verde), a region where malaria is hypoendemic and controlled, and thus the population is considered non-immune. Blood samples were collected from the inhabitants over 1 month and during cross-sectional surveys in the following year. The presence and nature of the parasites was determined by PCR. Plasmodium falciparum was the only species detected. Genetic analysis revealed that the circulating parasites were genetically homogeneous, and probably clonal. Gametocytes were found throughout this period. Our data suggest that this represented a focal outbreak, resulting in the infection of at least 40% of the villagers with a clonal parasite line. Thus, P. falciparum infections can persist for at least 1 year in a substantial proportion (10%) of the hosts. Implications for malaria control and the interpretation of epidemiological data are discussed.

Assessement of Malaria Transmission in an Area with Very Low Mosquito Density

The increase in world travel in recent years, especially to and from areas where vector-borne diseases are endemic, has resulted in a substantial rise in imported cases of those diseases. In particular, malaria is a cause of concern. In those countries at the edge of its distribution, it can be difficult to distinguish between autochthonous and imported cases. However, distinguishing between the two is important because of the different allocation of resources to combat the disease that each requires. In general, observation of the various stages of parasite development in wild-caught female mosquitoes is considered evidence of autochthonous transmission. Observation of oocysts in the mosquito mid-gut testifies that mosquitoes are susceptible to infection but conclusions cannot be reached about their ability to complete the transmission cycle. Perhaps the best indication of autochthonous transmission is microscopic observation of sporozoites in mosquito salivary glands, since this detects parasites ready to be inoculated (BELER et al., 1990). Detection of circumsporozoite protein (CSP)(BURKOT, WILLIAMS & SCHNEIDER, 1984) in dry mosquito thoraxes, by Enzyme Linked Immunosorbent Assay (ELISA) is also widely used to determine transmission, especially when large numbers of mosquitoes need to be processed. Such assays provide information about the parasite species infecting the mosquito (BURKOT & WIRTZ, 1986; WIRTZ et al., 1987; BELER et al., 1990).

Malaria importée: des médicaments toujours plus efficaces et plus sûrs [Imported malaria: safer and more efficacious drugs?].

In response to the spread of parasite resistance to old antimalarial drugs, the large-scale implementation of artemisinine-based combinations has allowed to improving patient survival and reducing parasite transmission. Even though decreased susceptibility of parasites to artemisinine has been observed in South-East Asia, this phenomenon has no practical implications for travelers with uncomplicated malaria. The combination of artemether-lumefantrine is still very effective and safe, be it for P. falciparum or vivax. Intravenous administration of artesunate has allowed to significantly reducing case fatality rate of severe malaria patients when compared to quinine treatment in endemic areas. Artesunate is also recommended in travelers, but with close monitoring, especially for hematological parameters, in order to confirm its superiority.

Malaria haplotype frequency estimation.

We present a Bayesian approach for estimating the relative frequencies of multi-single nucleotide polymorphism (SNP) haplotypes in populations of the malaria parasite Plasmodium falciparum by using microarray SNP data from human blood samples. Each sample comes from a malaria patient and contains one or several parasite clones that may genetically differ. Samples containing multiple parasite clones with different genetic markers pose a special challenge. The situation is comparable with a polyploid organism. The data from each blood sample indicates whether the parasites in the blood carry a mutant or a wildtype allele at various selected genomic positions. If both mutant and wildtype alleles are detected at a given position in a multiply infected sample, the data indicates the presence of both alleles, but the ratio is unknown. Thus, the data only partially reveals which specific combinations of genetic markers (i.e. haplotypes across the examined SNPs) occur in distinct parasite clones. In addition, SNP data may contain errors at non-negligible rates. We use a multinomial mixture model with partially missing observations to represent this data and a Markov chain Monte Carlo method to estimate the haplotype frequencies in a population. Our approach addresses both challenges, multiple infections and data errors.

High prevalence and lineage diversity of avian malaria in wild populations of great tits (Parus major) and mosquitoes (Culex pipiens).

Avian malaria studies have taken a prominent place in different aspects of evolutionary ecology. Despite a recent interest in the role of vectors within the complex interaction system of the malaria parasite, they have largely been ignored in most epidemiological studies. Epidemiology of the disease is however strongly related to the vector's ecology and behaviour, and there is a need for basic investigations to obtain a better picture of the natural associations between Plasmodium lineages, vector species and bird hosts. The aim of the present study was to identify the mosquito species involved in the transmission of the haemosporidian parasites Plasmodium spp. in two wild populations of breeding great tits (Parus major) in western Switzerland. Additionally, we compared Plasmodium lineages, based on mitochondrial DNA cytochrome b sequences, between the vertebrate and dipteran hosts, and evaluated the prevalence of the parasite in the mosquito populations. Plasmodium spp. were detected in Culex pipiens only, with an overall 6.6% prevalence. Among the six cytochrome b lineages of Plasmodium identified in the mosquitoes, three were also present in great tits. The results provide evidence for the first time that C. pipiens can act as a natural vector of avian malaria in Europe and yield baseline data for future research on the epidemiology of avian malaria in European countries.

Temporal changes in mosquito abundance (Culex pipiens), avian malaria prevalence and lineage composition

Background: Knowledge on the temporal dynamics of host/vector/parasite interactions is a pre-requisite to further address relevant questions in the fields of epidemiology and evolutionary ecology of infectious diseases. In studies of avian malaria, the natural history of Plasmodium parasites with their natural mosquito vectors, however, is mostly unknown. Methods: Using artificial water containers placed in the field, we monitored the relative abundance of parous females of Culex pipiens mosquitoes during two years (2010-2011), in a population in western Switzerland. Additionally, we used molecular tools to examine changes in avian malaria prevalence and Plasmodium lineage composition in female C. pipiens caught throughout one field season (April-August) in 2011. Results: C. pipiens relative abundance varied both between years and months, and was associated with temperature fluctuations. Total Plasmodium prevalence was high and increased from spring to summer months (13.1-20.3%). The Plasmodium community was composed of seven different lineages including P. relictum (SGS1, GRW11 and PADOM02 lineages), P. vaughani (lineage SYAT05) and other Plasmodium spp. (AFTRU5, PADOM1, COLL1). The most prevalent lineages, P. vaughani (lineage SYAT05) and P. relictum (lineage SGS1), were consistently found between years, although they had antagonistic dominance patterns during the season survey. Conclusions: Our results suggest that the time window of analysis is critical in evaluating changes in the community of avian malaria lineages infecting mosquitoes. The potential determinants of the observed changes as well as their implications for future prospects on avian malaria are discussed.