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      Ultra-Sensitive Detection of Plasmodium falciparum by Amplification of Multi-Copy Subtelomeric Targets

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          Abstract

          Background

          Planning and evaluating malaria control strategies relies on accurate definition of parasite prevalence in the population. A large proportion of asymptomatic parasite infections can only be identified by surveillance with molecular methods, yet these infections also contribute to onward transmission to mosquitoes. The sensitivity of molecular detection by PCR is limited by the abundance of the target sequence in a DNA sample; thus, detection becomes imperfect at low densities. We aimed to increase PCR diagnostic sensitivity by targeting multi-copy genomic sequences for reliable detection of low-density infections, and investigated the impact of these PCR assays on community prevalence data.

          Methods and Findings

          Two quantitative PCR (qPCR) assays were developed for ultra-sensitive detection of Plasmodium falciparum, targeting the high-copy telomere-associated repetitive element 2 (TARE-2, ∼250 copies/genome) and the var gene acidic terminal sequence ( varATS, 59 copies/genome). Our assays reached a limit of detection of 0.03 to 0.15 parasites/μl blood and were 10× more sensitive than standard 18S rRNA qPCR. In a population cross-sectional study in Tanzania, 295/498 samples tested positive using ultra-sensitive assays. Light microscopy missed 169 infections (57%). 18S rRNA qPCR failed to identify 48 infections (16%), of which 40% carried gametocytes detected by pfs25 quantitative reverse-transcription PCR. To judge the suitability of the TARE-2 and varATS assays for high-throughput screens, their performance was tested on sample pools. Both ultra-sensitive assays correctly detected all pools containing one low-density P. falciparum–positive sample, which went undetected by 18S rRNA qPCR, among nine negatives. TARE-2 and varATS qPCRs improve estimates of prevalence rates, yet other infections might still remain undetected when absent in the limited blood volume sampled.

          Conclusions

          Measured malaria prevalence in communities is largely determined by the sensitivity of the diagnostic tool used. Even when applying standard molecular diagnostics, prevalence in our study population was underestimated by 8% compared to the new assays. Our findings highlight the need for highly sensitive tools such as TARE-2 and varATS qPCR in community surveillance and for monitoring interventions to better describe malaria epidemiology and inform malaria elimination efforts.

          Abstract

          Ingrid Felger and colleagues developed an assay that targets multi-copy genomic sequences and can detect low-density infections with falciparum malaria parasites.

          Editors' Summary

          Background

          Nearly half the world's population is at risk of malaria, and more than 600,000 people die from this mosquito-borne parasitic infection every year. Most of these deaths are caused by Plasmodium falciparum, which is transmitted to people by night-flying Anopheles mosquitoes. These insects inject “sporozoites” into people, a parasitic form that replicates inside human liver cells. After a few days, the liver cells release “merozoites,” which invade red blood cells, where they replicate rapidly before bursting out and infecting more red blood cells. This increase in parasitic burden causes malaria's characteristic fever, which needs to be treated promptly to prevent anemia and organ damage. Infected red blood cells also release “gametocytes,” which infect mosquitoes when they take a blood meal. In the mosquito, the gametocytes multiply and develop into sporozoites, thus completing the parasite's life cycle. Malaria can be prevented by controlling the mosquitoes that spread the parasite and by avoiding mosquito bites. Effective treatment with antimalarial drugs also helps to reduce malaria transmission and is a key component of global efforts to control and eliminate malaria.

          Why Was This Study Done?

          Planning and evaluating malaria control and elimination efforts relies on having accurate and sensitive methods to measure parasite prevalence—the proportion of a population infected with parasites. It is particularly important to know how many people are carrying low-density infections because although these individuals have no symptoms, they contribute to malaria transmission. In the past, malaria was usually diagnosed by looking for parasites in blood using light microscopy, but molecular tests based on “quantitative polymerase chain reactions” (qPCRs) are now available that detect much lower parasite densities in blood (submicroscopic infections). qPCRs detect parasite-specific DNA sequences in patient blood samples, but reliable detection of low-density infections remains imperfect because the abundance of target sequences in patient samples limits the sensitivity of current qPCR methods. Here, the researchers investigate whether the sensitivity of P. falciparum detection using qPCR can be improved by targeting multi-copy genomic sequences—DNA sequences that are repeated many times in the parasite's genetic blueprint.

          What Did the Researchers Do and Find?

          The researchers developed two new qPCRs for P. falciparum by using the telomere-associated repetitive element 2 (TARE-2; 250 copies/genome) and the var gene acidic terminal sequence ( varATS; 59 copies/genome) as target sequences. Direct comparison of these qPCRs with the standard 18S rRNA qPCR for P. falciparum, which targets a gene present at 5–8 copies/genome, indicated that the new assays were ten times more sensitive than the standard assay and could detect as few as 0.03–0.15 parasites/μl blood. Next, the researchers used light microscopy, 18S rRNA qPCR, and the two new qPCRs to look for P. falciparum parasites in 498 samples randomly selected from a malaria survey undertaken in Tanzania. Parasite prevalences were 25% by light microscopy, 50% by 18S rRNA qPCR, and 58% by TARE-2 or varATS qPCR. Compared to TARE-2 or varATS qPCR, 18S rRNA qPCR failed to identify 48 infections (16% of infections). Moreover, 40% of the positive samples missed by 18S rRNA qPCR contained gametocytes (detected by a different PCR-based assay) and therefore came from individuals capable of transmitting malaria parasites to mosquitoes. Finally, to test the suitability of the new ultra-sensitive assays for use in high-throughput screens, the researchers tested performance of the assays on sample pools. Both tests correctly identified all pools containing one low-density P. falciparum–positive sample among nine negative samples, whereas 18S rRNA qPCR identified none of these pools.

          What Do These Findings Mean?

          These findings provide evidence of low-density malaria infections in individuals previously thought to be parasite-free, even after testing with a molecular diagnostic. Notably, in the population considered in this study, the standard 18S rRNA qPCR underestimated parasite prevalence by nearly 10%. The assays developed in this study have some important limitations, however. First, they detect only P. falciparum, and malaria control programs ideally need assays that detect all the Plasmodium species that cause malaria. Second, because the TARE-2 and varATS qPCRs require advanced laboratory infrastructure, they cannot be used in remote field settings. Nevertheless, because low-density infections are likely to become increasingly common as countries improve malaria control, these findings highlight the need for ultra-sensitive tools such as the TARE-2 and varATS qPCRs for community surveillance and for monitoring the progress of malaria control and elimination programs.

          Additional Information

          Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001788.

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          Most cited references60

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          A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies.

          A nested polymerase chain reaction (PCR) assay that uses Plasmodium genus-specific primers for the initial PCR (nest 1) amplification and either genus- or species-specific primers for the nest 2 amplifications was tested on laboratory and field samples. With in vitro cultured Plasmodium falciparum-infected blood samples, it was capable of detecting six parasites/microl of blood using DNA prepared from 25-microl blood spots on filter paper. The assay was evaluated on fingerprick blood samples collected on filter paper from 129 individuals living in a malaria-endemic area in Malaysia. Malaria prevalence by genus-specific nested PCR was 35.6% (46 of 129) compared with 28.7% (37 of 129) by microscopy. The nested PCR detected seven more malaria samples than microscopy in the first round of microscopic examination, malaria in three microscopically negative samples, six double infections identified as single infections by microscopy and one triple infection identified as a double infection by microscopy. The nested PCR assay described is a sensitive technique for collecting accurate malaria epidemiologic data. When coupled with simple blood spot sampling, it is particularly useful for screening communities in remote regions of the world.
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            The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes.

            The human malaria parasite Plasmodium falciparum evades host immunity by varying the antigenic and adhesive character of infected erythrocytes. We describe a large and extremely diverse family of P. falciparum genes (var) that encode 200-350 kDa proteins having the expected properties of antigenically variant adhesion molecules. Predicted amino acid sequences of var genes show a variable extracellular segment with domains having receptor-binding features, a transmembrane sequence, and a terminal segment that is a probable submembrane anchor. There are 50-150 var genes on multiple parasite chromosomes, and some are in clustered arrangements. var probes detect two classes of transcripts in steady-state RNA: 7-9 kb var transcripts, and an unusual family of 1.8-2.4 kb transcripts that may be involved in expression or rearrangements of var genes.
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              Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and species-specific real-time PCR assays.

              There have been reports of increasing numbers of cases of malaria among migrants and travelers. Although microscopic examination of blood smears remains the "gold standard" in diagnosis, this method suffers from insufficient sensitivity and requires considerable expertise. To improve diagnosis, a multiplex real-time PCR was developed. One set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus Plasmodium was designed; the primer set was polymorphic enough internally to design four species-specific probes for P. falciparum, P. vivax, P. malarie, and P. ovale. Real-time PCR with species-specific probes detected one plasmid copy of P. falciparum, P. vivax, P. malariae, and P. ovale specifically. The same sensitivity was achieved for all species with real-time PCR with the 18S screening probe. Ninety-seven blood samples were investigated. For 66 of them (60 patients), microscopy and real-time PCR results were compared and had a crude agreement of 86% for the detection of plasmodia. Discordant results were reevaluated with clinical, molecular, and sequencing data to resolve them. All nine discordances between 18S screening PCR and microscopy were resolved in favor of the molecular method, as were eight of nine discordances at the species level for the species-specific PCR among the 31 samples positive by both methods. The other 31 blood samples were tested to monitor the antimalaria treatment in seven patients. The number of parasites measured by real-time PCR fell rapidly for six out of seven patients in parallel to parasitemia determined microscopically. This suggests a role of quantitative PCR for the monitoring of patients receiving antimalaria therapy.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS Med
                PLoS Med
                plos
                plosmed
                PLoS Medicine
                Public Library of Science (San Francisco, CA USA )
                1549-1277
                1549-1676
                3 March 2015
                March 2015
                : 12
                : 3
                : e1001788
                Affiliations
                [1 ]Swiss Tropical and Public Health Institute, Basel, Switzerland
                [2 ]University of Basel, Basel, Switzerland
                [3 ]Biological Sciences Department, Dar es Salaam University College of Education, Dar es Salaam, Tanzania
                [4 ]Ifakara Health Institute, Bagamoyo, Tanzania
                [5 ]Papua New Guinea Institute of Medical Research, Madang and Maprik, Papua New Guinea
                [6 ]Walter and Eliza Hall Institute, Parkville, Victoria, Australia
                [7 ]Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia
                [8 ]Centre de Recerca en Salut Internacional de Barcelona, Barcelona, Spain
                Mahidol-Oxford Tropical Medicine Research Unit, THAILAND
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: IF. Performed the experiments: NH FM. Analyzed the data: NH IF. Wrote the paper: NH IF. Enrolled patients: FM SS LJR IM. Agree with manuscript results and conclusions: NH FM SS LJR IM IF. ICMJE criteria for authorship read and met: NH FM SS LJR IM IF.

                Article
                PMEDICINE-D-14-02316
                10.1371/journal.pmed.1001788
                4348198
                25734259
                9ac3bb76-78f9-487d-beab-627d58808fe4
                Copyright @ 2015

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 18 July 2014
                : 8 January 2015
                Page count
                Figures: 4, Tables: 2, Pages: 21
                Funding
                This work was supported by Swiss National Science Foundation [grant number 310030_134889], International Centers of Excellence in Malaria Research [grant number U19 AI089686) and Bill and Melinda Gates Foundation [grant number OPP1034577]. FM received funding from the Science and Technology Higher Education Project (STHEP) through the Dar-Es-Salaam University College of Education (DULE) and the Stipendienkommission Basel Stadt. IM is supported by an NHMRC Senior Research Fellowship (GNT1043345). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

                Medicine
                Medicine

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