Genetics of chloroquine-resistant malaria: a haplotypic view

The development and rapid spread of chloroquine resistance (CQR) in Plasmodium falciparum have triggered the identification of several genetic target(s) in the P. falciparum genome. In particular, mutations in the Pfcrt gene, specifically, K76T and mutations in three other amino acids in the region adjoining K76 (residues 72, 74, 75 and 76), are considered to be highly related to CQR. These various mutations form several different haplotypes and Pfcrt gene polymorphisms and the global distribution of the different CQR- Pfcrt haplotypes in endemic and non-endemic regions of P. falciparum malaria have been the subject of extensive study. Despite the fact that the Pfcrt gene is considered to be the primary CQR gene in P. falciparum , several studies have suggested that this may not be the case. Furthermore, there is a poor correlation between the evolutionary implications of the Pfcrt haplotypes and the inferred migration of CQR P. falciparum based on CQR epidemiological surveillance data. The present paper aims to clarify the existing knowledge on the genetic basis of the different CQR- Pfcrt haplotypes that are prevalent in worldwide populations based on the published literature and to analyse the data to generate hypotheses on the genetics and evolution of CQR malaria.

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part I: Aspidosperma nitidum (Benth) used as a remedy to treat fever and malaria in the Amazon

Infusions of Aspidosperma nitidum (Apocynaceae) wood bark are used to treat fever and malaria in the Amazon Region. Several species of this family are known to possess indole alkaloids and other classes of secondary metabolites, whereas terpenoids, an inositol and the indole alkaloids harmane-3 acid and braznitidumine have been described in A. nitidum . In the present study, extracts from the wood bark, leaves and branches of this species were prepared for assays against malaria parasites and cytotoxicity testing using human hepatoma and normal monkey kidney cells. The wood bark extracts were active against Plasmodium falciparum and showed a low cytotoxicity in vitro, whereas the leaf and branch extracts and the pure alkaloid braznitidumine were inactive. A crude methanol extract was subjected to acid-base fractionation aimed at obtaining alkaloid-rich fractions, which were active at low concentrations against P. falciparum and in mice infected with and sensitive Plasmodium berghei parasites. Our data validate the antimalarial usefulness of A. nitidum wood bark, a remedy that can most likely help to control malaria. However, the molecules responsible for this antimalarial activity have not yet been identified. Considering their high selectivity index, the alkaloid-rich fractions from the plant bark might be useful in the development of new antimalarials.

First published record of urban malaria in Puerto Gaitan, Meta, Colombia

Patterns of malaria cases were compared between the department of Meta and the municipality of Puerto Gaitán, Colombia, to examine temporal change in malaria from 2005-2010. During this time frame in Meta the mean ratio was 2.53; in contrast, in Puerto Gaitán it was 1.41, meaning that a surprisingly high proportion of Plasmodium falciparum cases were reported from this municipality. A detailed analysis of data from Puerto Gaitán for 2009 and 2010 detected a significant difference (χ2, p < 0.001) in the distribution of plasmodia, with Plasmodium vivax more prevalent in 2009 and P. falciparum in 2010. Males had the highest number of cases but there was no difference in the distribution of cases between sexes and years. In both years, for both sexes, people 16-40 accounted for the majority of cases (58.9% in 2009; 60.4% in 2010). There were significant differences in the distribution of both P. vivax (χ2, p < 0.01) and P. falciparum cases (χ2, p < 0.05) by geographic setting (urban vs. non-urban) between years. Urban cases of both P. vivax and P. falciparum are recorded in this study for the first time in Puerto Gaitán, possibly the result of area wide displacement and migration due to armed conflict.

Anthropophilic biting behaviour of Anopheles (Kerteszia) neivai Howard, Dyar & Knab associated with Fishermen’s activities in a malaria-endemic area in the Colombian Pacific

On the southwest Pacific Coast of Colombia, a field study was initiated to determine the human-vector association between Anopheles (Kerteszia) neivai and fishermen, including their nearby houses. Mosquitoes were collected over 24-h periods from mangrove swamps, marshlands and fishing vessels in three locations, as well as in and around the houses of fishermen. A total of 6,382 mosquitoes were collected. An. neivai was most abundant in mangroves and fishing canoes (90.8%), while Anopheles albimanus was found indoors (82%) and outdoors (73%). One An. neivai and one An. albimanus collected during fishing activities in canoes were positive for Plasmodium vivax , whereas one female An. neivai collected in a mangrove was positive for P. vivax . In the mangroves and fishing canoes, An. neivai demonstrated biting activity throughout the day, peaking between 06:00 pm-07:00 pm and there were two minor peaks at dusk and dawn. These peaks coincided with fishing activities in the marshlands and mangroves, a situation that places the fishermen at risk of contracting malaria when they are performing their daily activities. It is recommended that protective measures be implemented to reduce the risk that fishermen will contract malaria.

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.

Immune response to the mumps component of the MMR vaccine in the routine of immunisation services in the Brazilian National Immunisation Program

A non-controlled longitudinal study was conducted to evaluate the combined vaccine against measles, mumps and rubella (MMR) immunogenicity in 150 children vaccinated in the routine of three health units in the city of Rio de Janeiro, Brazil, 2008-2009, without other vaccines administered during the period from 30 days before to 30 days after vaccination. A previous study conducted in Brazil in 2007, in 1,769 children ranging from 12-15 months of age vaccinated against yellow fever and MMR simultaneously or at intervals of 30 days or more between doses, had shown low seroconversion for mumps regardless of the interval between administration of the two vaccines. The current study showed 89.5% (95% confidence interval: 83.3; 94.0) seroconversion rate for mumps. All children seroconverted for measles and rubella. After revaccination, high antibody titres and seroconversion rates were achieved against mumps. The results of this study and others suggest that two MMR doses confer optimal immunoresponses for all three antigens and the possible need for additional doses should be studied taking into account not only serological, but also epidemiological data, as there is no serological correlate of protection for mumps.

Anthropophilic Anopheles species composition and malaria in Tierradentro, Córdoba, Colombia

Malaria is still a primary health problem in Colombia. The locality of Tierradentro is situated in the municipality of Montelíbano, Córdoba, in the northwest of Colombia, and has one of the highest annual parasite index of malaria nationwide. However, the vectors involved in malaria transmission in this locality have not yet been identified. In this study, the local anthropophilic Anopheles composition and natural infectivity with Plasmodium were investigated. In August 2009, 927 female Anopheles mosquitoes were collected in eight localities using the human landing catch method and identified based on their morphology. Cryptic species were determined by restriction fragment length polymorphism-internal transcribed spacer (ITS)2 molecular analysis. Eight species [Anopheles nuneztovari s.l. (92.8%), Anopheles darlingi (5.1%), Anopheles triannulatus s.l. (1.8%), Anopheles pseudopunctipennis s.l. (0.2%), Anopheles punctimacula s.l. (0.2%), Anopheles apicimacula (0.1%), Anopheles albimanus (0.1%) and Anopheles rangeli (0.1%)] were identified and species identity was confirmed by ITS2 sequencing. This is the first report of An. albimanus, An. rangeli and An. apicimacula in Tierradentro. Natural infectivity with Plasmodium was determined by ELISA. None of the mosquitoes was infectious for Plasmodium. An. nuneztovari s.l. was the predominant species and is considered the primary malaria vector; An. darlingi and An. triannulatus s.l. could serve as secondary vectors.

Getting ready for malaria elimination: a check list of critical issues to consider

In recent years, a renewed interest in malaria elimination and eradication has emerged and seems to be rooting in the minds of the scientific community, public health specialists, funding bodies, policy makers and politicians. Malaria eradication will certainly benefit from improved and innovative tools; notwithstanding novel knowledge in fields ranging from basic science to mathematical modelling and health systems research. However, the elimination of malaria also encompasses a broad range of essential aspects that countries and other actors need to consider when thinking of embarking on such an adventure, including the implementation of innovative strategies, the ability to incorporate the most up-to-date evidence into policy, the integration of malaria into the broader health agenda, the strengthening of surveillance and health systems, capacity building, funding, advocacy and, very importantly, research. While in some cases this enthusiasm is clearly justified, some countries are still a long way from realistically advancing towards elimination. This paper attempts to provide guidance on all the necessary issues that should be considered when initiating a malaria elimination program.

Plasmodium vivax malaria elimination: should innovative ideas from the past be revisited?

In the 1950s, the strategy of adding chloroquine to food salt as a prophylaxis against malaria was considered to be a successful tool. However, with the development of Plasmodium resistance in the Brazilian Amazon, this control strategy was abandoned. More than 50 years later, asexual stage resistance can be avoided by screening for antimalarial drugs that have a selective action against gametocytes, thus old prophylactic measures can be revisited. The efficacy of the old methods should be tested as complementary tools for the elimination of malaria.

Malaria on the Guiana Shield: a review of the situation in French Guiana

In a climate of growing concern that Plasmodium falciparum may be developing a drug resistance to artemisinin derivatives in the Guiana Shield, this review details our current knowledge of malaria and control strategy in one part of the Shield, French Guiana. Local epidemiology, test-treat-track strategy, the state of parasite drug resistance and vector control measures are summarised. Current issues in terms of mobile populations and legislative limitations are also discussed.

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.

Influence of age on the haemoglobin concentration of malaria-infected patients in a reference centre in the Brazilian Amazon

Anaemia is amongst the major complications of malaria, a major public health problem in the Amazon Region in Latin America. We examined the haemoglobin (Hb) concentrations of malaria-infected patients and compared it to that of malaria-negative febrile patients and afebrile controls. The haematological parameters of febrile patients who had a thick-blood-smear performed at an infectious diseases reference centre of the Brazilian Amazon between December 2009-January 2012 were retrieved together with clinical data. An afebrile community control group was composed from a survey performed in a malaria-endemic area. Hb concentrations and anaemia prevalence were analysed according to clinical-epidemiological status and demographic characteristics. In total, 7,831 observations were included. Patients with Plasmodium falciparum infection had lower mean Hb concentrations (10.5 g/dL) followed by P. vivax-infected individuals (12.4 g/dL), community controls (12.8 g/dL) and malaria-negative febrile patients (13.1 g/dL) (p < 0.001). Age, gender and clinical-epidemiological status were strong independent predictors for both outcomes. Amongst malaria-infected individuals, women in the reproductive age had considerably lower Hb concentrations. In this moderate transmission intensity setting, both vivax and falciparum malaria are associated with reduced Hb concentrations and risk of anaemia throughout a wide age range.

Vascular dysfunction as a target for adjuvant therapy in cerebral malaria

Cerebral malaria (CM) is a life-threatening complication of Plasmodium falciparum malaria that continues to be a major global health problem. Brain vascular dysfunction is a main factor underlying the pathogenesis of CM and can be a target for the development of adjuvant therapies for the disease. Vascular occlusion by parasitised red blood cells and vasoconstriction/vascular dysfunction results in impaired cerebral blood flow, ischaemia, hypoxia, acidosis and death. In this review, we discuss the mechanisms of vascular dysfunction in CM and the roles of low nitric oxide bioavailability, high levels of endothelin-1 and dysfunction of the angiopoietin-Tie2 axis. We also discuss the usefulness and relevance of the murine experimental model of CM by Plasmodium berghei ANKA to identify mechanisms of disease and to screen potential therapeutic interventions.

Metabolomics in the fight against malaria

Metabolomics uses high-resolution mass spectrometry to provide a chemical fingerprint of thousands of metabolites present in cells, tissues or body fluids. Such metabolic phenotyping has been successfully used to study various biologic processes and disease states. High-resolution metabolomics can shed new light on the intricacies of host-parasite interactions in each stage of the Plasmodium life cycle and the downstream ramifications on the host’s metabolism, pathogenesis and disease. Such data can become integrated with other large datasets generated using top-down systems biology approaches and be utilised by computational biologists to develop and enhance models of malaria pathogenesis relevant for identifying new drug targets or intervention strategies. Here, we focus on the promise of metabolomics to complement systems biology approaches in the quest for novel interventions in the fight against malaria. We introduce the Malaria Host-Pathogen Interaction Center (MaHPIC), a new systems biology research coalition. A primary goal of the MaHPIC is to generate systems biology datasets relating to human and non-human primate (NHP) malaria parasites and their hosts making these openly available from an online relational database. Metabolomic data from NHP infections and clinical malaria infections from around the world will comprise a unique global resource.