A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi.
ABSTRACT: BACKGROUND:The misdiagnosis of Plasmodium knowlesi by microscopy has prompted a re-evaluation of the geographic distribution, prevalence and pathogenesis of this species using molecular diagnostic tools. In this report, a specific probe for P. knowlesi, that can be used in a previously described TaqMan real-time PCR assay for detection of Plasmodium spp., and Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale, was designed and validated against clinical samples. METHODS:A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within the P. knowlesi small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were determined using plasmids containing P. knowlesi DNA and genomic DNA of P. falciparum, P. knowlesi, P. malariae, P. ovale and P. vivax isolated from clinical samples. DNA samples of the simian malaria parasites Plasmodium cynomolgi and Plasmodium inui that can infect humans under experimental conditions were also examined together with human DNA samples. RESULTS:Analytical sensitivity of the P. knowlesi-specific assay was 10 copies/μL and quantitation was linear over a range of 10-106 copies. The sensitivity of the assay is equivalent to nested PCR and P. knowlesi DNA was detected from all 40 clinical P. knowlesi specimens, including one from a patient with a parasitaemia of three parasites/μL of blood. No cross-reactivity was observed with 67 Plasmodium DNA samples (31 P. falciparum, 23 P. vivax, six P. ovale, three P. malariae, one P. malariae/P. ovale, one P. falciparum/P. malariae, one P. inui and one P. cynomolgi) and four samples of human DNA. CONCLUSIONS:This test demonstrated excellent sensitivity and specificity, and adds P. knowlesi to the repertoire of Plasmodium targets for the clinical diagnosis of malaria by real-time PCR assays. Furthermore, quantitation of DNA copy number provides a useful advantage over other molecular assays to investigate the correlation between levels of infection and the spectrum of disease.
Project description:18S ribosomal RNA gene sequences of different species of Plasmodium were aligned and analyzed to determine the molecular diversity among different species of Plasmodium. AT content of P. cynomolgi, P. ovale, P. falciparum, P. vivax and P. malariae ranged from 62.30 to 63.15, 63.90 to 65.29, 66.67 to 68.40, 61.66 to 63.25 and 64.09 to 76.36 in case respectively. GC content of P. cynomolgi, P. ovale, P. falciparum, P. vivaxand P. malariae ranged from 36.85 to 37.70, 34.71 to 36.43, 31.60 to 33.27, 23.64 to 35.91 and 36.75 to 38.34 respectively. Molecular weight (Da) of P. cynomolgi, P. ovale, P. falciparum, P. vivax and P. malariae ranged from 1,160,127.00 to 1,400,332.00, 437,273.00 to 481,438.00, 668,021.00 to 685,243.00, 540,378.00 to 1,176,962.00 and 132,788.00 to 305,211.00 respectively. Relative melting temperature (Tm) of P. cynomolgi and P. ovale varied from 0.36799 to 0.36837 and 0.36712 to 0.36814 respectively. In P. falciparum and P. vivax relative Tm ranged from 0.36545 to 0.36626 and 0.36802 to 0.36866 respectively. Relative Tm of P. malariae varied from 0.36174 to 0.36790.
Project description:BACKGROUND:Artemisinin combination therapy (ACT) is recommended as first-line treatment for uncomplicated Plasmodium falciparum malaria, whereas chloroquine is still commonly used for the treatment of non-falciparum species (Plasmodium vivax, Plasmodium ovale and Plasmodium malariae). A more simplified, more uniform treatment approach across all malaria species is worthwhile to be considered both in endemic areas and for malaria as an imported condition alike. METHODS:A PROSPERO-registered systematic review to determine the efficacy and safety of ACT for the treatment of non-falciparum malaria was conducted, following PRISMA guidelines. Without language restrictions, Medline/PubMed, Embase, Cochrane Central Register of Controlled Trials, Web of Science, LILACS, Biosis Previews and the African Index Medicus were searched for studies published up to November 2014. RESULTS:The literature search identified 986 reports; 40 publications were found eligible for inclusion, all of them on non-falciparum malaria in endemic areas. Most evidence was available for P. vivax (n?=?35). Five clinical trials in total were identified evaluating ACT for P. ovale, P. malariae and Plasmodium knowlesi. Most ACT presentations have high efficacy against P. vivax parasites; artemisinin-based combinations have shorter parasite and fever clearance times compared to chloroquine. ACT is as effective as chloroquine in preventing recurrent parasitaemia before day 28. Artemisinin-based combinations with long half-lives show significantly fewer recurrent parasitaemia up to day 63. The limited evidence available supports both the use of chloroquine and an ACT for P. ovale and P. malariae. ACT seems to be preferable for optimal treatment of P. knowlesi. CONCLUSION:ACT is at least equivalent to chloroquine in effectively treating non-falciparum malaria. These findings may facilitate development of simplified protocols for treating all forms of malaria with ACT, including returning travellers. Obtaining comprehensive efficacy and safety data on ACT use for non-falciparum species particularly for P. ovale, P. malariae and P. knowlesi should be a research priority. TRIAL REGISTRATION:CRD42014009103.
Project description:In spite of the high prevalence of malaria in Southeastern Bangladesh, there remains a significant shortage of information regarding the presence of three of five human malaria parasites: Plasmodium ovale, P. malariae, and P. knowlesi. The presence of P. ovale and P. knowlesi has previously never been reported from Bangladesh. We used a genus- and species-specific nested polymerase chain reaction, targeting highly conserved regions of the small subunit ribosomal RNA (SSU rRNA) gene, to investigate the presence of malaria parasites in a total number of 379 patient samples in a survey of patients with febrile illnesses in the Chittagong Hill Tracts in Southeastern Bangladesh. We identified the first cases of P. ovale in Bangladesh. They were confirmed by sequence analysis; 189 of 379 samples (49.9%; 95% confidence interval = 44.9-54.9%) were positive for Plasmodium sp. by PCR. P. falciparum monoinfections accounted for 68.3% (61.3-74.5%), followed by P. vivax (15.3%; 10.9-21.2%), P. malariae (1.6%; 0.5-4.6%), P. ovale (1.6%; 0.5-4.6%), and mixed infections (13.2%; 9.1-18.8%). We found no evidence of P. knowlesi in this region.
Project description:Malaria is the leading identifiable cause of fever in returning travelers. Accurate Plasmodium species identification has therapy implications for P. vivax and P. ovale, which have dormant liver stages requiring primaquine. Compared to microscopy, nucleic acid tests have improved specificity for species identification and higher sensitivity for mixed infections. Here, we describe a SYBR green-based real-time PCR assay for Plasmodium species identification from whole blood, which uses a panel of reactions to detect species-specific non-18S rRNA gene targets. A pan-Plasmodium 18S rRNA target is also amplified to allow species identification or confirmation by sequencing if necessary. An evaluation of assay accuracy, performed on 76 clinical samples (56 positives using thin smear microscopy as the reference method and 20 negatives), demonstrated clinical sensitivities of 95.2% for P. falciparum (20/21 positives detected) and 100% for the Plasmodium genus (52/52), P. vivax (20/20), P. ovale (9/9), and P. malariae (6/6). The sensitivity of the P. knowlesi-specific PCR was evaluated using spiked whole blood samples (100% [10/10 detected]). The specificities of the real-time PCR primers were 94.2% for P. vivax (49/52) and 100% for P. falciparum (51/51), P. ovale (62/62), P. malariae (69/69), and P. knowlesi (52/52). Thirty-three specimens were used to test species identification by sequencing the pan-Plasmodium 18S rRNA PCR product, with correct identification in all cases. The real-time PCR assay also identified two samples with mixed P. falciparum and P. ovale infection, which was confirmed by sequencing. The assay described here can be integrated into a malaria testing algorithm in low-prevalence areas, allowing definitive Plasmodium species identification shortly after malaria diagnosis by microscopy.
Project description:Microsatellites can be utilized to explore genotypes, population structure, and other genomic features of eukaryotes. Systematic characterization of microsatellites has not been a focus for several species of Plasmodium, including P. malariae and P. ovale, as the majority of malaria elimination programs are focused on P. falciparum and to a lesser extent P. vivax. Here, five human malaria species (P. falciparum, P. vivax, P. malariae, P. ovale curtisi, and P. knowlesi) were investigated with the aim of conducting in-depth categorization of microsatellites for P. malariae and P. ovale curtisi. Investigation of reference genomes for microsatellites with unit motifs of 1-10 base pairs indicates high diversity among the five Plasmodium species. Plasmodium malariae, with the largest genome size, displays the second highest microsatellite density (1421 No./Mbp; 5% coverage) next to P. falciparum (3634 No./Mbp; 12% coverage). The lowest microsatellite density was observed in P. vivax (773 No./Mbp; 2% coverage). A, AT, and AAT are the most commonly repeated motifs in the Plasmodium species. For P. malariae and P. ovale curtisi, microsatellite-related sequences are observed in approximately 18-29% of coding sequences (CDS). Lysine, asparagine, and glutamic acids are most frequently coded by microsatellite-related CDS. The majority of these CDS could be related to the gene ontology terms "cell parts," "binding," "developmental processes," and "metabolic processes." The present study provides a comprehensive overview of microsatellite distribution and can assist in the planning and development of potentially useful genetic tools for further investigation of P. malariae and P. ovale curtisi epidemiology.
Project description:Since 1960, a total of seven species of monkey malaria have been reported as transmissible to man by mosquito bite: Plasmodium cynomolgi, Plasmodium brasilianum, Plasmodium eylesi, Plasmodium knowlesi, Plasmodium inui, Plasmodium schwetzi and Plasmodium simium. With the exception of P. knowlesi, none of the other species has been found to infect humans in nature. In this report, it is described the first known case of a naturally acquired P. cynomolgi malaria in humans.The patient was a 39-year-old woman from a malaria-free area with no previous history of malaria or travel to endemic areas. Initially, malaria was diagnosed and identified as Plasmodium malariae/P. knowlesi by microscopy in the Terengganu State Health Department. Thick and thin blood films stained with 10% Giemsa were performed for microscopy examination. Molecular species identification was performed at the Institute for Medical Research (IMR, Malaysia) and in the Malaria & Emerging Parasitic Diseases Laboratory (MAPELAB, Spain) using different nested PCR methods.Microscopic re-examination in the IMR showed characteristics of Plasmodium vivax and was confirmed by a nested PCR assay developed by Snounou et al. Instead, a different PCR assay plus sequencing performed at the MAPELAB confirmed that the patient was infected with P. cynomolgi and not with P. vivax.This is the first report of human P. cynomolgi infection acquired in a natural way, but there might be more undiagnosed or misdiagnosed cases, since P. cynomolgi is morphologically indistinguishable from P. vivax, and one of the most used PCR methods for malaria infection detection may identify a P. cynomolgi infection as P. vivax.Simian Plasmodium species may routinely infect humans in Southeast Asia. New diagnostic methods are necessary to distinguish between the human and monkey malaria species. Further epidemiological studies, incriminating also the mosquito vector(s), must be performed to know the relevance of cynomolgi malaria and its implication on human public health and in the control of human malaria.The zoonotic malaria cannot be ignored in view of increasing interactions between man and wild animals in the process of urbanization.
Project description:Molecular diagnosis of malaria offers many potential advantages over microscopy, including identification of malaria to the species level in an era with few experienced microscopists. We developed high-throughput multiplex 5' nuclease quantitative PCR (qPCR) assays, with the potential to support large studies, to specifically identify Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. We compared qPCR to microscopy and confirmed discordant results with an alternative target PCR assay. The assays specifically detected 1 to 6 parasites/?l of blood. The clinical sensitivities (95% confidence intervals [CIs]) of the 4-plex assay to detect microscopically confirmed malaria were 95.8% (88.3 to 99.1%) for P. falciparum, 89.5% (75.2 to 97.1%) for P. vivax, 94.1% (71.3 to 99.9%) for P. ovale, and 100% (66.4 to 100%) for P. malariae. The specificities (95% CIs) were 98.6% (92.4 to 100%) for P. falciparum, 99% (84.8 to 100%) for P. vivax, 98.4% (94.4 to 99.8%) for P. ovale, and 99.3% (95.9 to 100%) for P. malariae. The clinical specificity for samples without malaria was 100%. The clinical sensitivity of the 5-plex assay for confirmed P. knowlesi malaria was 100% (95% CI, 69.2 to 100%), and the clinical specificity was 100% (95% CI, 87.2 to 100%). Coded retesting and testing with an alternative target PCR assay showed improved sensitivity and specificity of multiplex qPCR versus microscopy. Additionally, 91.7% (11/12) of the samples with uncertain species by microscopy were identified to the species level identically by both our multiplex qPCR assay and the alternative target PCR assay, including 9 P. falciparum infections. Multiplex qPCR can rapidly and simultaneously identify all 5 Plasmodium species known to cause malaria in humans, and it offers an alternative or adjunct to microscopy for clinical diagnosis as well as a needed high-throughput tool for research.
Project description:BACKGROUND:Malaria remains a global health problem and accurate surveillance of Plasmodium parasites that are responsible for this disease is required to guide the most effective distribution of control measures. Serological surveillance will be particularly important in areas of low or periodic transmission because patient antibody responses can provide a measure of historical exposure. While methods for detecting host antibody responses to Plasmodium falciparum and Plasmodium vivax are well established, development of serological assays for Plasmodium knowlesi, Plasmodium ovale and Plasmodium malariae have been inhibited by a lack of immunodiagnostic candidates due to the limited availability of genomic information. METHODS:Using the recently completed genome sequences from P. malariae, P. ovale and P. knowlesi, a set of 33 candidate cell surface and secreted blood-stage antigens was selected and expressed in a recombinant form using a mammalian expression system. These proteins were added to an existing panel of antigens from P. falciparum and P. vivax and the immunoreactivity of IgG, IgM and IgA immunoglobulins from individuals diagnosed with infections to each of the five different Plasmodium species was evaluated by ELISA. Logistic regression modelling was used to quantify the ability of the responses to determine prior exposure to the different Plasmodium species. RESULTS:Using sera from European travellers with diagnosed Plasmodium infections, antigens showing species-specific immunoreactivity were identified to select a panel of 22 proteins from five Plasmodium species for serological profiling. The immunoreactivity to the antigens in the panel of sera taken from travellers and individuals living in malaria-endemic regions with diagnosed infections showed moderate power to predict infections by each species, including P. ovale, P. malariae and P. knowlesi. Using a larger set of patient samples and logistic regression modelling it was shown that exposure to P. knowlesi could be accurately detected (AUC?=?91%) using an antigen panel consisting of the P. knowlesi orthologues of MSP10, P12 and P38. CONCLUSIONS:Using the recent availability of genome sequences to all human-infective Plasmodium spp. parasites and a method of expressing Plasmodium proteins in a secreted functional form, an antigen panel has been compiled that will be useful to determine exposure to these parasites.
Project description:BACKGROUND: While Malaysia has had great success in controlling Plasmodium falciparum and Plasmodium vivax, notifications of Plasmodium malariae and the microscopically near-identical Plasmodium knowlesi increased substantially over the past decade. However, whether this represents microscopic misdiagnosis or increased recognition of P. knowlesi has remained uncertain. METHODS: To describe the changing epidemiology of malaria in Sabah, in particular the increasing incidence of P. knowlesi, a retrospective descriptive study was undertaken involving a review of Department of Health malaria notification data from 2012-2013, extending a previous review of these data from 1992-2011. In addition, malaria PCR and microscopy data from the State Public Health Laboratory were reviewed to estimate the accuracy of the microscopy-based notification data. RESULTS: Notifications of P. malariae/P. knowlesi increased from 703 in 2011 to 815 in 2012 and 996 in 2013. Notifications of P. vivax and P. falciparum decreased from 605 and 628, respectively, in 2011, to 297 and 263 in 2013. In 2013, P. malariae/P. knowlesi accounted for 62% of all malaria notifications compared to 35% in 2011. Among 1,082 P. malariae/P. knowlesi blood slides referred for PCR testing during 2011-2013, there were 924 (85%) P. knowlesi mono-infections, 30 (2.8%) P. falciparum, 43 (4.0%) P. vivax, seven (0.6%) P. malariae, six (0.6%) mixed infections, 31 (2.9%) positive only for Plasmodium genus, and 41 (3.8%) Plasmodium-negative. Plasmodium knowlesi mono-infection accounted for 32/156 (21%) and 33/87 (38%) blood slides diagnosed by microscopy as P. falciparum and P. vivax, respectively. Twenty-six malaria deaths were reported during 2010-2013, including 12 with 'P. malariae/P. knowlesi' (all adults), 12 with P. falciparum (seven adults), and two adults with P. vivax. CONCLUSIONS: Notifications of P. malariae/P. knowlesi in Sabah are increasing, with this trend likely reflecting a true increase in incidence of P. knowlesi and presenting a major threat to malaria control and elimination in Malaysia. With the decline of P. falciparum and P. vivax, control programmes need to incorporate measures to protect against P. knowlesi, with further research required to determine effective interventions.
Project description:The merozoite surface protein-9 (MSP-9) has been considered a target for an anti-malarial vaccine since it is one of many proteins involved in the erythrocyte invasion, a critical step in the parasite life cycle. Orthologs encoding this antigen have been found in all known species of Plasmodium parasitic to primates. In order to characterize and investigate the extent and maintenance of MSP-9 genetic diversity, we analyzed DNA sequences of the following malaria parasite species: Plasmodium falciparum, Plasmodium reichenowi, Plasmodium chabaudi, Plasmodium yoelii, Plasmodium berghei, Plasmodium coatneyi, Plasmodium gonderi, Plasmodium knowlesi, Plasmodium inui, Plasmodium simiovale, Plasmodium fieldi, Plasmodium cynomolgi and Plasmodium vivax and evaluated the signature of natural selection in all MSP-9 orthologs. Our findings suggest that the gene encoding MSP-9 is under purifying selection in P. vivax and closely related species. We further explored how selection affected different regions of MSP-9 by comparing the polymorphisms in P. vivax and P. falciparum, and found contrasting patterns between these two species that suggest differences in functional constraints. This observation implies that the MSP-9 orthologs in human parasites may interact differently with the host immune response. Thus, studies carried out in one species cannot be directly translated into the other.