Michelle L. Gatton

A large proportion of asymptomatic Plasmodium infections with low and sub-microscopic parasite densities in the low transmission setting of Temotu Province, Solomon Islands: challenges for malaria diagnostics in an elimination setting

BackgroundMany countries are scaling up malaria interventions towards elimination. This transition changes demands on malaria diagnostics from diagnosing ill patients to detecting parasites in all carriers including asymptomatic infections and infections with low parasite densities. Detection methods suitable to local malaria epidemiology must be selected prior to transitioning a malaria control programme to elimination. A baseline malaria survey conducted in Temotu Province, Solomon Islands in late 2008, as the first step in a provincial malaria elimination programme, provided malaria epidemiology data and an opportunity to assess how well different diagnostic methods performed in this setting.MethodsDuring the survey, 9,491 blood samples were collected and examined by microscopy for Plasmodium species and density, with a subset also examined by polymerase chain reaction (PCR) and rapid diagnostic tests (RDTs). The performances of these diagnostic methods were compared.ResultsA total of 256 samples were positive by microscopy, giving a point prevalence of 2.7%. The species distribution was 17.5% Plasmodium falciparum and 82.4% Plasmodium vivax. In this low transmission setting, only 17.8% of the P. falciparum and 2.9% of P. vivax infected subjects were febrile (≥38°C) at the time of the survey. A significant proportion of infections detected by microscopy, 40% and 65.6% for P. falciparum and P. vivax respectively, had parasite density below 100/μL. There was an age correlation for the proportion of parasite density below 100/μL for P. vivax infections, but not for P. falciparum infections. PCR detected substantially more infections than microscopy (point prevalence of 8.71%), indicating a large number of subjects had sub-microscopic parasitemia. The concordance between PCR and microscopy in detecting single species was greater for P. vivax (135/162) compared to P. falciparum (36/118). The malaria RDT detected the 12 microscopy and PCR positive P. falciparum, but failed to detect 12/13 microscopy and PCR positive P. vivax infections.ConclusionAsymptomatic malaria infections and infections with low and sub-microscopic parasite densities are highly prevalent in Temotu province where malaria transmission is low. This presents a challenge for elimination since the large proportion of the parasite reservoir will not be detected by standard active and passive case detection. Therefore effective mass screening and treatment campaigns will most likely need more sensitive assays such as a field deployable molecular based assay.

Genetic Diversity of Plasmodium falciparum Histidine-Rich Protein 2 (PfHRP2) and Its Effect on the Performance of PfHRP2-Based Rapid Diagnostic Tests

Rising costs of antimalarial agents are increasing the demand for accurate diagnosis of malaria. Rapid diagnostic tests (RDTs) offer great potential to improve the diagnosis of malaria, particularly in remote areas. Many RDTs are based on the detection of Plasmodium falciparum histidine-rich protein (PfHRP) 2, but reports from field tests have questioned their sensitivity and reliability. We hypothesize that the variability in the results of PfHRP2-based RDTs is related to the variability in the target antigen. We tested this hypothesis by examining the genetic diversity of PfHRP2, which includes numerous amino acid repeats, in 75 P. falciparum lines and isolates originating from 19 countries and testing a subset of parasites by use of 2 PfHRP2-based RDTs. We observed extensive diversity in PfHRP2 sequences, both within and between countries. Logistic regression analysis indicated that 2 types of repeats were predictive of RDT detection sensitivity (87.5% accuracy), with predictions suggesting that only 84% of P. falciparum parasites in the Asia-Pacific region are likely to be detected at densities < or = 250 parasites/microL. Our data also indicated that PfHRP3 may play a role in the performance of PfHRP2-based RDTs. These findings provide an alternative explanation for the variable sensitivity in field tests of malaria RDTs that is not due to the quality of the RDTs.

A systematic review of mathematical models of mosquito-borne pathogen transmission: 1970-2010

Mathematical models of mosquito-borne pathogen transmission originated in the early twentieth century to provide insights into how to most effectively combat malaria. The foundations of the Ross–Macdonald theory were established by 1970. Since then, there has been a growing interest in reducing the public health burden of mosquito-borne pathogens and an expanding use of models to guide their control. To assess how theory has changed to confront evolving public health challenges, we compiled a bibliography of 325 publications from 1970 through 2010 that included at least one mathematical model of mosquito-borne pathogen transmission and then used a 79-part questionnaire to classify each of 388 associated models according to its biological assumptions. As a composite measure to interpret the multidimensional results of our survey, we assigned a numerical value to each model that measured its similarity to 15 core assumptions of the Ross–Macdonald model. Although the analysis illustrated a growing acknowledgement of geographical, ecological and epidemiological complexities in modelling transmission, most models during the past 40 years closely resemble the Ross–Macdonald model. Modern theory would benefit from an expansion around the concepts of heterogeneous mosquito biting, poorly mixed mosquito-host encounters, spatial heterogeneity and temporal variation in the transmission process.

THE IMPORTANCE OF MOSQUITO BEHAVIOURAL ADAPTATIONS TO MALARIA CONTROL IN AFRICA

Over the past decade the use of long‐lasting insecticidal nets (LLINs), in combination with improved drug therapies, indoor residual spraying (IRS), and better health infrastructure, has helped reduce malaria in many African countries for the first time in a generation. However, insecticide resistance in the vector is an evolving threat to these gains. We review emerging and historical data on behavioral resistance in response to LLINs and IRS. Overall the current literature suggests behavioral and species changes may be emerging, but the data are sparse and, at times unconvincing. However, preliminary modeling has demonstrated that behavioral resistance could have significant impacts on the effectiveness of malaria control. We propose seven recommendations to improve understanding of resistance in malaria vectors. Determining the public health impact of physiological and behavioral insecticide resistance is an urgent priority if we are to maintain the significant gains made in reducing malaria morbidity and mortality.

Relapses of Plasmodium vivax Infection Result from Clonal Hypnozoites Activated at Predetermined Intervals

Plasmodium vivax infections are characterized by varying numbers of relapses occurring at different intervals as a result of activation of liver-stage hypnozoites. Parasite or host factors that determine the number and timing of relapses are unclear. In the present article, we report the analysis of relapse patterns and molecular characterization of parasites collected from Australian soldiers experiencing relapses of vivax malaria after exposure in East Timor. Although high molecular diversity was observed, a single allelic type was identified in association with 99% of relapses. Importantly, in 71% of patients experiencing >1 relapse, the allelic types were clonal and different in the 2 different relapses. These results, combined with those from a computer simulation model, suggest that a single hypnozoite clone was activated, causing a relapse, and that multiple relapses most likely arose from coordinated activation of hypnozoites originating from different parasite strains. These findings suggest remarkable regulation of relapse intervals in vivax malaria.

Artemisinin-Induced Dormancy in Plasmodium falciparum: Duration, Recovery Rates, and Implications in Treatment Failure

BACKGROUND Despite the remarkable activity of artemisinin and its derivatives, monotherapy with these agents has been associated with high rates of recrudescence. The temporary arrest of the growth of ring-stage parasites (dormancy) after exposure to artemisinin drugs provides a plausible explanation for this phenomenon. METHODS Ring-stage parasites of several Plasmodium falciparum lines were exposed to different doses of dihydroartemisinin (DHA) alone or in combination with mefloquine. For each regime, the proportion of recovering parasites was determined daily for 20 days. RESULTS Parasite development was abruptly arrested after a single exposure to DHA, with some parasites being dormant for up to 20 days. Approximately 50% of dormant parasites recovered to resume growth within the first 9 days. The overall proportion of parasites recovering was dose dependent, with recovery rates ranging from 0.044% to 1.313%. Repeated treatment with DHA or with DHA in combination with mefloquine led to a delay in recovery and an approximately 10-fold reduction in total recovery. Strains with different genetic backgrounds appeared to vary in their capacity to recover. CONCLUSIONS These results imply that artemisinin-induced arrest of growth occurs readily in laboratory-treated parasites and may be a key factor in P. falciparum malaria treatment failure.

Effect of sequence variation in Plasmodium falciparum Histidine-Rich protein 2 on binding of specific monoclonal antibodies: Implications for rapid diagnostic tests for malaria

ABSTRACT The ability to accurately diagnose malaria infections, particularly in settings where laboratory facilities are not well developed, is of key importance in the control of this disease. Rapid diagnostic tests (RDTs) offer great potential to address this need. Reports of significant variation in the field performance of RDTs based on the detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) (PfHRP2) and of significant sequence polymorphism in PfHRP2 led us to evaluate the binding of four HRP2-specific monoclonal antibodies (MABs) to parasite proteins from geographically distinct P. falciparum isolates, define the epitopes recognized by these MABs, and relate the copy number of the epitopes to MAB reactivity. We observed a significant difference in the reactivity of the same MAB to different isolates and between different MABs tested with single isolates. When the target epitopes of three of the MABs were determined and mapped onto the peptide sequences of the field isolates, significant variability in the frequency of these epitopes was observed. These findings support the role of sequence variation as an explanation for variations in the performance of HRP2-based RDTs and point toward possible approaches to improve their diagnostic sensitivities.

High diversity and rapid changeover of expressed var genes during the acute phase of Plasmodium falciparum infections in human volunteers

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins expressed on the surface of P. falciparum-infected erythrocytes undergo antigenic variation by switching the gene expressed within a repertoire of approximately 50 var genes per haploid genome. The switching of PfEMP1 plays an important role in the survival and pathogenesis of the parasite. To understand how a parasite switches its var gene expression in human infections, we investigated the composition and change of var gene transcripts during the acute phase of well-defined laboratory-induced P. falciparum infections in naïve human hosts. Multiple var transcripts, with the same dominant transcript, were identified in samples collected after three to four asexual-parasite cycles in two volunteers infected with cloned 3D7 P. falciparum via mosquito bites. A major change in composition and frequency of var gene transcripts was observed between the culture used to infect the mosquitoes and the parasites recovered from the infected volunteers. A further change was seen when infected blood from a mosquito-infected volunteer was either passaged to other volunteers or cultured in vitro. The diversity of var transcripts did not increase with time. The results suggest that the switch of var gene expression is reinitiated after mosquito transmission and that var genes may rapidly switch from the first gene expressed after liver stage, but subsequent switching occurs at a much lower rate.

Suppression of mRNAs Encoding Tegument Tetraspanins from Schistosoma mansoni Results in Impaired Tegument Turnover

Schistosomes express a family of integral membrane proteins, called tetraspanins (TSPs), in the outer surface membranes of the tegument. Two of these tetraspanins, Sm-TSP-1 and Sm-TSP-2, confer protection as vaccines in mice, and individuals who are naturally resistant to S. mansoni infection mount a strong IgG response to Sm-TSP-2. To determine their functions in the tegument of S. mansoni we used RNA interference to silence expression of Sm-tsp-1 and Sm-tsp-2 mRNAs. Soaking of parasites in Sm-tsp dsRNAs resulted in 61% (p = 0.009) and 74% (p = 0.009) reductions in Sm-tsp-1 and Sm-tsp-2 transcription levels, respectively, in adult worms, and 67%–75% (p = 0.011) and 69%–89% (p = 0.004) reductions in Sm-tsp-1 and Sm-tsp-2 transcription levels, respectively, in schistosomula compared to worms treated with irrelevant control (luciferase) dsRNA. Ultrastructural morphology of adult worms treated in vitro with Sm-tsp-2 dsRNA displayed a distinctly vacuolated and thinner tegument compared with controls. Schistosomula exposed in vitro to Sm-tsp-2 dsRNA had a significantly thinner and more vacuolated tegument, and morphology consistent with a failure of tegumentary invaginations to close. Injection of mice with schistosomula that had been electroporated with Sm-tsp-1 and Sm-tsp-2 dsRNAs resulted in 61% (p = 0.005) and 83% (p = 0.002) reductions in the numbers of parasites recovered from the mesenteries four weeks later when compared to dsRNA-treated controls. These results imply that tetraspanins play important structural roles impacting tegument development, maturation or stability.

Recasting the theory of mosquito-borne pathogen transmission dynamics and control

Mosquito-borne diseases pose some of the greatest challenges in public health, especially in tropical and sub-tropical regions of the world. Efforts to control these diseases have been underpinned by a theoretical framework developed for malaria by Ross and Macdonald, including models, metrics for measuring transmission, and theory of control that identifies key vulnerabilities in the transmission cycle. That framework, especially Macdonalds formula for R0 and its entomological derivative, vectorial capacity, are now used to study dynamics and design interventions for many mosquito-borne diseases. A systematic review of 388 models published between 1970 and 2010 found that the vast majority adopted the Ross–Macdonald assumption of homogeneous transmission in a well-mixed population. Studies comparing models and data question these assumptions and point to the capacity to model heterogeneous, focal transmission as the most important but relatively unexplored component in current theory. Fine-scale heterogeneity causes transmission dynamics to be nonlinear, and poses problems for modeling, epidemiology and measurement. Novel mathematical approaches show how heterogeneity arises from the biology and the landscape on which the processes of mosquito biting and pathogen transmission unfold. Emerging theory focuses attention on the ecological and social context for mosquito blood feeding, the movement of both hosts and mosquitoes, and the relevant spatial scales for measuring transmission and for modeling dynamics and control.