A high-throughput method to detect Plasmodium falciparum clones in limiting dilution microplates

<p>Abstract</p> <p>Background</p> <p>Molecular and cellular studies of <it>Plasmodium falciparum </it>require cloning of parasites by limiting dilution cultivation, typically performed in microplates. The parasite's slow replication rate combined with laborious methods for identification of positive wells has limited these studies. A new high-throughput method for detecting growth without compromising parasite viability is reported.</p> <p>Methods</p> <p><it>In vitro </it>parasite cultivation is associated with extracellular acidification. A survey of fluorescent pH indicators identified 5-(and-6)-carboxy SNARF-1 as a membrane-impermeant dye with a suitable <it>pK_a</it>value. Conditions for facile detection of viable parasites in 96-well microplates were optimized and used for limiting dilution cloning of genetic cross progeny and transfected parasites.</p> <p>Results</p> <p>5-(and-6)-carboxy SNARF-1 is a two-emission wavelength dye that accurately reported extracellular pH in parasite cultures. It readily detected parasite growth in microplate wells and yielded results comparable to labour-intensive examination of Giemsa-stained smears. The dye is non-toxic, allowing parasite detection without transfer of culture material to additional plates for separate assays. This dye was used with high-throughput limiting dilution culture to generate additional progeny clones from the HB3 × Dd2 genetic cross.</p> <p>Conclusions</p> <p>This fluorescence-based assay represents a low-cost, efficient method for detection of viable parasites in microplate wells; it can be easily expanded by automation.</p

Separate Ion Pathways in a Cl−/H+ Exchanger

CLC-ec1 is a prokaryotic CLC-type Cl−/H+ exchange transporter. Little is known about the mechanism of H+ coupling to Cl−. A critical glutamate residue, E148, was previously shown to be required for Cl−/H+ exchange by mediating proton transfer between the protein and the extracellular solution. To test whether an analogous H+ acceptor exists near the intracellular side of the protein, we performed a mutagenesis scan of inward-facing carboxyl-bearing residues and identified E203 as the unique residue whose neutralization abolishes H+ coupling to Cl− transport. Glutamate at this position is strictly conserved in all known CLCs of the transporter subclass, while valine is always found here in CLC channels. The x-ray crystal structure of the E203Q mutant is similar to that of the wild-type protein. Cl− transport rate in E203Q is inhibited at neutral pH, and the double mutant, E148A/E203Q, shows maximal Cl− transport, independent of pH, as does the single mutant E148A. The results argue that substrate exchange by CLC-ec1 involves two separate but partially overlapping permeation pathways, one for Cl− and one for H+. These pathways are congruent from the protein's extracellular surface to E148, and they diverge beyond this point toward the intracellular side. This picture demands a transport mechanism fundamentally different from familiar alternating-access schemes

Highly heterogeneous residual malaria risk in western Thailand

Over the past decades, the malaria burden in Thailand has substantially declined. Most infections now originate from the national border regions. In these areas, the prevalence of asymptomatic infections is still substantial and poses a challenge for the national malaria elimination program. To determine epidemiological parameters as well as risk factors for malaria infection in western Thailand, we carried out a cohort study in Kanchanaburi and Ratchaburi provinces on the Thailand-Myanmar border. Blood samples from 999 local participants were examined for malaria infection every 4 weeks between May 2013 and Jun 2014. Prevalence of Plasmodium falciparum and Plasmodium vivax was determined by quantitative PCR (qPCR) and showed a seasonal variation with values fluctuating from 1.7% to 4.2% for P. vivax and 0% to 1.3% for P. falciparum. Ninety percent of infections were asymptomatic. The annual molecular force of blood-stage infection (molFOB) was estimated by microsatellite genotyping to be 0.24 new infections per person-year for P. vivax and 0.02 new infections per person-year for P. falciparum. The distribution of infections was heterogenous, that is, the vast majority of infections (>80%) were found in a small number of individuals (<8% of the study population) who tested positive at multiple timepoints. Significant risk factors were detected for P. vivax infections, including previous clinical malaria, occupation in agriculture and travel to Myanmar. In contrast, indoor residual spraying was associated with a protection from infection. These findings provide a recent landscape of malaria epidemiology and emphasize the importance of novel strategies to target asymptomatic and imported infections

Intracellular Proton-Transfer Mutants in a CLC Cl−/H+ Exchanger

CLC-ec1, a bacterial homologue of the CLC family’s transporter subclass, catalyzes transmembrane exchange of Cl− and H+. Mutational analysis based on the known structure reveals several key residues required for coupling H+ to the stoichiometric countermovement of Cl−. E148 (Gluex) transfers protons between extracellular water and the protein interior, and E203 (Gluin) is thought to function analogously on the intracellular face of the protein. Mutation of either residue eliminates H+ transport while preserving Cl− transport. We tested the role of Gluin by examining structural and functional properties of mutants at this position. Certain dissociable side chains (E, D, H, K, R, but not C and Y) retain H+/Cl− exchanger activity to varying degrees, while other mutations (V, I, or C) abolish H+ coupling and severely inhibit Cl− flux. Transporters substituted with other nonprotonatable side chains (Q, S, and A) show highly impaired H+ transport with substantial Cl− transport. Influence on H+ transport of side chain length and acidity was assessed using a single-cysteine mutant to introduce non-natural side chains. Crystal structures of both coupled (E203H) and uncoupled (E203V) mutants are similar to wild type. The results support the idea that Gluin is the internal proton-transfer residue that delivers protons from intracellular solution to the protein interior, where they couple to Cl− movements to bring about Cl−/H+ exchange

Malaria Parasite clag3 Genes Determine Channel-Mediated Nutrient Uptake by Infected Red Blood Cells

SummaryDevelopment of malaria parasites within vertebrate erythrocytes requires nutrient uptake at the host cell membrane. The plasmodial surface anion channel (PSAC) mediates this transport and is an antimalarial target, but its molecular basis is unknown. We report a parasite gene family responsible for PSAC activity. We used high-throughput screening for nutrient uptake inhibitors to identify a compound highly specific for channels from the Dd2 line of the human pathogen P. falciparum. Inheritance of this compound's affinity in a Dd2 × HB3 genetic cross maps to a single parasite locus on chromosome 3. DNA transfection and in vitro selections indicate that PSAC-inhibitor interactions are encoded by two clag3 genes previously assumed to function in cytoadherence. These genes are conserved in plasmodia, exhibit expression switching, and encode an integral protein on the host membrane, as predicted by functional studies. This protein increases host cell permeability to diverse solutes.PaperFlic

Sensitive detection of Plasmodium vivax malaria by the rotating-crystal magneto-optical method in Thailand

The rotating-crystal magneto-optical detection (RMOD) method has been developed for the rapid and quantitative diagnosis of malaria and tested systematically on various malaria infection models. Very recently, an extended field trial in a high-transmission region of Papua New Guinea demonstrated its great potential for detecting malaria infections, in particular Plasmodium vivax. In the present small-scale field test, carried out in a low-transmission area of Thailand, RMOD confirmed malaria in all samples found to be infected with Plasmodium vivax by microscopy, our reference method. Moreover, the magneto-optical signal for this sample set was typically 1–3 orders of magnitude higher than the cut-off value of RMOD determined on uninfected samples. Based on the serial dilution of the original patient samples, we expect that the method can detect Plasmodium vivax malaria in blood samples with parasite densities as low as ∼5–10 parasites per microliter, a limit around the pyrogenic threshold of the infection. In addition, by investigating the correlation between the magnitude of the magneto-optical signal, the parasite density and the erythrocytic stage distribution, we estimate the relative hemozoin production rates of the ring and the trophozoite stages of in vivo Plasmodium vivax infections

Plasmodium vivax and Plasmodium falciparum infection dynamics: re-infections, recrudescences and relapses

Background: In malaria endemic populations, complex patterns of Plasmodium vivax and Plasmodium falciparum blood-stage infection dynamics may be observed. Genotyping samples from longitudinal cohort studies for merozoite surface protein (msp) variants increases the information available in the data, allowing multiple infecting parasite clones in a single individual to be identified. msp genotyped samples from two longitudinal cohorts in Papua New Guinea (PNG) and Thailand were analysed using a statistical model where the times of acquisition and clearance of each clone in every individual were estimated using a process of data augmentation. Results: For the populations analysed, the duration of blood-stage P. falciparum infection was estimated as 36 (95% Credible Interval (CrI): 29, 44) days in PNG, and 135 (95% CrI 94, 191) days in Thailand. Experiments on simulated data indicated that it was not possible to accurately estimate the duration of blood-stage P. vivax infections due to the lack of identifiability between a single blood-stage infection and multiple, sequential blood-stage infections caused by relapses. Despite this limitation, the method and data point towards short duration of blood-stage P. vivax infection with a lower bound of 24 days in PNG, and 29 days in Thailand. On an individual level, P. vivax recurrences cannot be definitively classified into re-infections, recrudescences or relapses, but a probabilistic relapse phenotype can be assigned to each P. vivax sample, allowing investigation of the association between epidemiological covariates and the incidence of relapses. Conclusion: The statistical model developed here provides a useful new tool for in-depth analysis of malaria data from longitudinal cohort studies, and future application to data sets with multi-locus genotyping will allow more detailed investigation of infection dynamics

Asymptomatic and sub-microscopic malaria infection in Kayah State, eastern Myanmar

Myanmar has the heaviest burden of malaria in the Greater Mekong Sub-region. Asymptomatic Plasmodium spp. infections are common in this region and may represent an important reservoir of transmission that must be targeted for malaria elimination.; A mass blood survey was conducted among 485 individuals from six villages in Kayah State, an area of endemic but low transmission malaria in eastern Myanmar. Malaria infection was screened by rapid diagnostic test (RDT), light microscopy and real-time polymerase chain reaction (PCR), and its association with demographic factors was explored.; The prevalence of asymptomatic Plasmodium spp. infection was 2.3% (11/485) by real-time PCR. Plasmodium vivax accounted for 72.7% (8/11) and Plasmodium falciparum for 27.3% (3/11) of infections. Men were at greater risk of infection by Plasmodium spp. than women. Individuals who worked as farmers or wood and bamboo cutters had an increased risk of infection.; A combination of RDT, light microscopy and PCR diagnostics were used to identify asymptomatic malaria infection, providing additional information on asymptomatic cases in addition to the routine statistics on symptomatic cases, so as to determine the true burden of disease in the area. Such information and risk factors can improve malaria risk stratification and guide decision-makers towards better design and delivery of targeted interventions in small villages, representative of Kayah State

The Plasmodium falciparum rhoptry protein RhopH3 plays essential roles in host cell invasion and nutrient uptake.

Merozoites of the protozoan parasite responsible for the most virulent form of malaria, Plasmodium falciparum, invade erythrocytes. Invasion involves discharge of rhoptries, specialized secretory organelles. Once intracellular, parasites induce increased nutrient uptake by generating new permeability pathways (NPP) including a Plasmodium surface anion channel (PSAC). RhopH1/Clag3, one member of the three-protein RhopH complex, is important for PSAC/NPP activity. However, the roles of the other members of the RhopH complex in PSAC/NPP establishment are unknown and it is unclear whether any of the RhopH proteins play a role in invasion. Here we demonstrate that RhopH3, the smallest component of the complex, is essential for parasite survival. Conditional truncation of RhopH3 substantially reduces invasive capacity. Those mutant parasites that do invade are defective in nutrient import and die. Our results identify a dual role for RhopH3 that links erythrocyte invasion to formation of the PSAC/NPP essential for parasite survival within host erythrocytes

Asymptomatic Plasmodium vivax infections induce robust IgG responses to multiple blood-stage proteins in a low-transmission region of western Thailand

BACKGROUND: Thailand is aiming to eliminate malaria by the year 2024. Plasmodium vivax has now become the dominant species causing malaria within the country, and a high proportion of infections are asymptomatic. A better understanding of antibody dynamics to P. vivax antigens in a low-transmission setting, where acquired immune responses are poorly characterized, will be pivotal for developing new strategies for elimination, such as improved surveillance methods and vaccines. The objective of this study was to characterize total IgG antibody levels to 11 key P. vivax proteins in a village of western Thailand. METHODS: Plasma samples from 546 volunteers enrolled in a cross-sectional survey conducted in 2012 in Kanchanaburi Province were utilized. Total IgG levels to 11 different proteins known or predicted to be involved in reticulocyte binding or invasion (ARP, GAMA, P41, P12, PVX_081550, and five members of the PvRBP family), as well as the leading pre-erythrocytic vaccine candidate (CSP) were measured using a multiplexed bead-based assay. Associations between IgG levels and infection status, age, and spatial location were explored. RESULTS: Individuals from a low-transmission region of western Thailand reacted to all 11 P. vivax recombinant proteins. Significantly greater IgG levels were observed in the presence of a current P. vivax infection, despite all infected individuals being asymptomatic. IgG levels were also higher in adults (18 years and older) than in children. For most of the proteins, higher IgG levels were observed in individuals living closer to the Myanmar border and further away from local health services. CONCLUSIONS: Robust IgG responses were observed to most proteins and IgG levels correlated with surrogates of exposure, suggesting these antigens may serve as potential biomarkers of exposure, immunity, or both