Using CF11 cellulose columns to inexpensively and effectively remove human DNA from Plasmodium falciparum-infected whole blood samples

ABSTRACT

Artemisinin-Resistant Malaria: Research Challenges, Opportunities, and Public Health Implications

Artemisinin-based combination therapies are the most effective drugs to treat Plasmodium falciparum malaria. Reduced sensitivity to artemisinin monotherapy, coupled with the emergence of parasite resistance to all partner drugs, threaten to place millions of patients at risk of inadequate treatment of malaria. Recognizing the significance and immediacy of this possibility, the Fogarty International Center and the National Institute of Allergy and Infectious Diseases of the U.S. National Institutes of Health convened a conference in November 2010 to bring together the diverse array of stakeholders responding to the growing threat of artemisinin resistance, including scientists from malarious countries in peril. This conference encouraged and enabled experts to share their recent unpublished data from studies that may improve our understanding of artemisinin resistance. Conference sessions addressed research priorities to forestall artemisinin resistance and fostered collaborations between field- and laboratory-based researchers and international programs, with the aim of translating new scientific evidence into public health solutions. Inspired by this conference, this review summarizes novel findings and perspectives on artemisinin resistance, approaches for translating research data into relevant public health information, and opportunities for interdisciplinary collaboration to combat artemisinin resistance

An ultrasensitive reverse transcription polymerase chain reaction assay to detect asymptomatic low-density Plasmodium falciparum and Plasmodium vivax infections in small volume blood samples.

BackgroundHighly sensitive, scalable diagnostic methods are needed to guide malaria elimination interventions. While traditional microscopy and rapid diagnostic tests (RDTs) are suitable for the diagnosis of symptomatic malaria infection, more sensitive tests are needed to screen for low-density, asymptomatic infections that are targeted by interventions aiming to eliminate the entire reservoir of malaria infection in humans.MethodsA reverse transcription polymerase chain reaction (RT- PCR) was developed for multiplexed detection of the 18S ribosomal RNA gene and ribosomal RNA of Plasmodium falciparum and Plasmodium vivax. Simulated field samples stored for 14 days with sample preservation buffer were used to assess the analytical sensitivity and specificity. Additionally, 1750 field samples from Southeastern Myanmar were tested both by RDT and ultrasensitive RT-PCR.ResultsLimits of detection (LoD) were determined under simulated field conditions. When 0.3 mL blood samples were stored for 14 days at 28 °C and 80% humidity, the LoD was less than 16 parasites/mL for P. falciparum and 19.7 copies/µL for P. vivax (using a plasmid surrogate), about 10,000-fold lower than RDTs. Of the 1739 samples successfully evaluated by both ultrasensitive RT-PCR and RDT, only two were RDT positive while 24 were positive for P. falciparum, 108 were positive for P. vivax, and 127 were positive for either P. vivax and/or P. falciparum using ultrasensitive RT-PCR.ConclusionsThis ultrasensitive RT-PCR method is a robust, field-tested screening method that is vastly more sensitive than RDTs. Further optimization may result in a truly scalable tool suitable for widespread surveillance of low-level asymptomatic P. falciparum and P. vivax parasitaemia

Lack of allele-specific efficacy of a bivalent AMA1 malaria vaccine

<p>Abstract</p> <p>Background</p> <p>Extensive genetic diversity in vaccine antigens may contribute to the lack of efficacy of blood stage malaria vaccines. Apical membrane antigen-1 (AMA1) is a leading blood stage malaria vaccine candidate with extreme diversity, potentially limiting its efficacy against infection and disease caused by <it>Plasmodium falciparum </it>parasites with diverse forms of AMA1.</p> <p>Methods</p> <p>Three hundred Malian children participated in a Phase 2 clinical trial of a bivalent malaria vaccine that found no protective efficacy. The vaccine consists of recombinant AMA1 based on the 3D7 and FVO strains of <it>P. falciparum </it>adjuvanted with aluminum hydroxide (AMA1-C1). The gene encoding AMA1 was sequenced from <it>P. falciparum </it>infections experienced before and after immunization with the study vaccine or a control vaccine. Sequences of <it>ama1 </it>from infections in the malaria vaccine and control groups were compared with regard to similarity to the vaccine antigens using several measures of genetic diversity. Time to infection with parasites carrying AMA1 haplotypes similar to the vaccine strains with respect to immunologically important polymorphisms and the risk of infection with vaccine strain haplotypes were compared.</p> <p>Results</p> <p>Based on 62 polymorphic AMA1 residues, 186 unique <it>ama1 </it>haplotypes were identified among 315 <it>ama1 </it>sequences that were included in the analysis. Eight infections had <it>ama1 </it>sequences identical to 3D7 while none were identical to FVO. Several measures of genetic diversity showed that <it>ama1 </it>sequences in the malaria vaccine and control groups were comparable both at baseline and during follow up period. Pre- and post-immunization <it>ama1 </it>sequences in both groups all had a similar degree of genetic distance from FVO and 3D7 <it>ama1</it>. No differences were found in the time of first clinical episode or risk of infection with an AMA1 haplotype similar to 3D7 or FVO with respect to a limited set of immunologically important polymorphisms found in the cluster 1 loop of domain I of AMA1.</p> <p>Conclusion</p> <p>This Phase 2 trial of a bivalent AMA1 malaria vaccine found no evidence of vaccine selection or strain-specific efficacy, suggesting that the extreme genetic diversity of AMA1 did not account for failure of the vaccine to provide protection.</p

High levels of Plasmodium falciparum rosetting in all clinical forms of severe malaria in African children

Plasmodium falciparum rosetting (the spontaneous binding of infected erythrocytes to uninfected erythrocytes) is a well-recognized parasite virulence factor. However, it is currently unclear whether rosetting is associated with all clinical forms of severe malaria, or only with specific syndromes such as cerebral malaria. We investigated the relationship between rosetting and clinical malaria in 209 Malian children enrolled in a case-control study of severe malaria. Rosetting was significantly higher in parasite isolates from severe malaria cases compared with non-severe hyperparasitemia and uncomplicated malaria controls (F(2,117) = 8.15, P < 0.001). Analysis of sub-categories of severe malaria (unrousable coma, severe anemia, non-comatose neurological impairment, repeated seizures or a small heterogeneous group with signs of renal failure or jaundice) showed high levels of rosetting in all sub-categories, and no statistically significant differences in rosetting between sub-categories (F(4,67) = 1.28, P = 0.28). Thus rosetting may contribute to the pathogenesis of all severe malaria syndromes in African children, and interventions to disrupt rosetting could be potential adjunctive therapies for all forms of severe malaria in Africa

World Antimalarial Resistance Network (WARN) III: Molecular Markers for Drug Resistant Malaria

Molecular markers for drug resistant malaria represent public health tools of great but mostly unrealized potential value. A key reason for the failure of molecular resistance markers to live up to their potential is that data on the their prevalence is scattered in disparate databases with no linkage to the clinical, in vitro and pharmacokinetic data that are needed to relate the genetic data to relevant phenotypes. The ongoing replacement of older monotherapies for malaria by new, more effective combination therapies presents an opportunity to create an open access database that brings together standardized data on molecular markers of drug resistant malaria from around the world. This paper presents a rationale for creating a global database of molecular markers for drug resistant malaria and for linking it to similar databases containing results from clinical trials of drug efficacy, in vitro studies of drug susceptibility, and pharmacokinetic studies of antimalarial drugs, in a World Antimalarial Resistance Network (WARN). This database will be a global resource, guiding the selection of first line drugs for treating uncomplicated malaria, for preventing malaria in travelers and for intermittent preventive treatment of malaria in pregnant women, infants and other vulnerable groups. Perhaps most important, a global database for molecular markers of drug resistant malaria will accelerate the identification and validation of markers for resistance to artemisinin-based combination therapies and, thereby, potentially prolong the useful therapeutic lives of these important new drugs

Platelet-mediated clumping of Plasmodium falciparum infected erythrocytes is associated with high parasitemia but not severe clinical manifestations of malaria in African children

Platelet-mediated clumping of Plasmodium falciparum infected erythrocytes is an adhesive phenotype commonly found in field isolates that has previously been associated with severe malaria. Here, clumping was assessed in 131 isolates from Malian children. The clumping phenotype was seen in 6% (n=51) of uncomplicated malaria, 24% (n=51) of severe malaria, and 45% (n=29) of high parasitemia non-severe malaria isolates. Multivariate analysis indicated that clumping was strongly positively associated with parasitemia (F(1,122)=24.1, p<0.001) but not with disease category (F(2,122)=1.8, p=0.17). Therefore platelet-mediated clumping in Malian P. falciparum isolates is primarily associated with high parasitemia and not with severe clinical manifestations of malaria

Prevalence and distribution of human Plasmodium infection in Pakistan

Background: Both Plasmodium vivax and Plasmodium falciparum are prevalent in Pakistan, yet up-to-date data on the epidemiology of malaria in Pakistan are not available. This study was undertaken to determine the current prevalence and distribution of Plasmodium species across the country. Methods: A malariometric population survey was conducted in 2011 using blood samples collected from 801 febrile patients of all ages in four provinces and the capital city of Islamabad. Microscopically confirmed Plasmodium-positive blood samples were reconfirmed by polymerase chain reaction (PCR). Confirmed parasite-positive samples were subjected to species-specific PCR capable of detecting four species of human malaria. Results: Of the 707 PCR-positive samples, 128 (18%) were P. falciparum, 536 (76%) were P. vivax, and 43 (6%) were mixed P. falciparum and P. vivax. Ninety-four microscopy-positive samples were PCR-negative, and Plasmodium malariae and Plasmodium ovale were not detected. Prevalence of P. vivax ranged from 2.4 % in Punjab Province to 10.8 % in Sindh Province and prevalence of P. falciparum ranged from 0.1 % in Islamabad to 3.8 % in Balochistan. Conclusions: Plasmodium infections in Pakistan are largely attributed to P. vivax but P. falciparum and mixed species infections are also prevalent. In addition, regional variation in the prevalence and species composition of malaria is high

Extent and Dynamics of Polymorphism in the Malaria Vaccine Candidate Plasmodium falciparum Reticulocyte-Binding Protein Homologue-5 in Kalifabougou, Mali

Reticulocyte-binding homologues (RH) are a ligand family that mediates merozoite invasion of erythrocytes in Plasmodium falciparum. Among the five members of this family identified so far, only P. falciparum reticulocyte-binding homologue-5 (PfRH5) has been found to be essential for parasite survival across strains that differ in virulence and route of host-cell invasion. Based on its essential role in invasion and early evidence of sequence conservation, PfRH5 has been prioritized for development as a vaccine candidate. However, little is known about the extent of genetic variability of RH5 in the field and the potential impact of such diversity on clinical outcomes or on vaccine evasion. Samples collected during a prospective cohort study of malaria incidence conducted in Kalifabougou, in southwestern Mali, were used to estimate genetic diversity, measure haplotype prevalence, and assess the within-host dynamics of PfRH5 variants over time and in relation to clinical malaria. A total of 10 nonsynonymous polymorphic sites were identified in the Pfrh5 gene, resulting in 13 haplotypes encoding unique protein variants. Four of these variants have not been previously observed. Plasmodium falciparum reticulocyte-binding homologue-5 had low amino acid haplotype (h = 0.58) and nucleotide (π = 0.00061) diversity. By contrast to other leading blood-stage malaria vaccine candidate antigens, amino acid differences were not associated with changes in the risk of febrile malaria in consecutive infections. Conserved B- and T-cell epitopes were identified. These results support the prioritization of PfRH5 for possible inclusion in a broadly cross-protective vaccine