Considerations on the use of nucleic acid-based amplification for malaria parasite detection

<p>Abstract</p> <p>Background</p> <p>Nucleic acid amplification provides the most sensitive and accurate method to detect and identify pathogens. This is primarily useful for epidemiological investigations of malaria because the infections, often with two or more <it>Plasmodium </it>species present simultaneously, are frequently associated with microscopically sub-patent parasite levels and cryptic mixed infections. Numerous distinct equally adequate amplification-based protocols have been described, but it is unclear which to select for epidemiological surveys. Few comparative studies are available, and none that addresses the issue of inter-laboratory variability.</p> <p>Methods</p> <p>Blood samples were collected from patients attending malaria clinics on the Thai-Myanmar border. Frozen aliquots from 413 samples were tested independently in two laboratories by nested PCR assay. Dried blood spots on filter papers from the same patients were also tested by the nested PCR assay in one laboratory and by a multiplex PCR assay in another. The aim was to determine which protocol best detected parasites below the sensitivity level of microscopic examination.</p> <p>Results</p> <p>As expected PCR-based assays detected a substantial number of infected samples, or mixed infections, missed by microscopy (27 and 42 for the most sensitive assay, respectively). The protocol that was most effective at detecting these, in particular mixed infections, was a nested PCR assay with individual secondary reactions for each of the species initiated with a template directly purified from the blood sample. However, a lesser sensitivity in detection was observed when the same protocol was conducted in another laboratory, and this significantly altered the data obtained on the parasite species distribution.</p> <p>Conclusions</p> <p>The sensitivity of a given PCR assay varies between laboratories. Although, the variations are relatively minor, they primarily diminish the ability to detect low-level and mixed infections and are sufficient to obviate the main rationale to use PCR assays rather than microscopy or rapid diagnostic tests. The optimal approach to standardise methodologies is to provide PCR template standards. These will help researchers in different settings to ensure that the nucleic acid amplification protocols they wish to use provide the requisite level of sensitivity, and will permit comparison between sites.</p

Effective and cheap removal of leukocytes and platelets from Plasmodium vivax infected blood

<p>Abstract</p> <p>Background</p> <p>Investigations of <it>Plasmodium vivax </it>are restricted to samples collected from infected persons or primates, because this parasite cannot be maintained in <it>in vitro </it>cultures. Contamination of <it>P. vivax </it>isolates with host leukocytes and platelets is detrimental to a range of <it>ex vivo </it>and molecular investigations. Easy-to-produce CF11 cellulose filters have recently provided us with an inexpensive method for the removal of leukocytes and platelets. This contrasted with previous reports of unacceptably high levels of infected red blood cell (IRBC) retention by CF11. The aims of this study were to compare the ability of CF11 cellulose filters and the commercial filter Plasmodipur at removing leukocyte and platelet, and to investigate the retention of <it>P. vivax </it>IRBCs by CF11 cellulose filtration.</p> <p>Methods and Results</p> <p>Side-by-side comparison of six leukocyte removal methods using blood samples from five healthy donor showed that CF11 filtration reduced the mean initial leukocyte counts from 9.4 × 10³per μl [95%CI 5.2–13.5] to 0.01 × 10³[95%CI 0.01–0.03]. The CF11 was particularly effective at removing neutrophils. CF11 treatment also reduced initial platelet counts from 211.6 × 10³per μl [95%CI 107.5–315.7] to 0.8 × 10³per μl [95%CI -0.7–2.2]. Analysis of 30 <it>P. vivax </it>blood samples before and after CF11 filtration showed only a minor loss in parasitaemia (≤ 7.1% of initial counts). Stage specific retention of <it>P. vivax </it>IRBCs was not observed.</p> <p>Conclusion</p> <p>CF11 filtration is the most cost and time efficient method for the production of leukocyte- and platelet-free <it>P. vivax</it>-infected erythrocytes from field isolates.</p

Chloroquine resistant vivax malaria in a pregnant woman on the western border of Thailand

Chloroquine (CQ) resistant vivax malaria is spreading. In this case, Plasmodium vivax infections during pregnancy and in the postpartum period were not satisfactorily cleared by CQ, despite adequate drug concentrations. A growth restricted infant was delivered. Poor susceptibility to CQ was confirmed in-vitro and molecular genotyping was strongly suggestive of true recrudescence of P. vivax. This is the first clinically and laboratory confirmed case of two high-grade CQ resistant vivax parasite strains from Thailand

Methotrexate Is Highly Potent Against Pyrimethamine-Resistant Plasmodium vivax

Resistance of vivax malaria to treatment with antifolates, such as pyrimethamine (Pyr), is spreading as mutations in the dihydrofolatereductase (dhfr) genes are selected and disseminated. We tested the antitumor drug methotrexate (MTX), a potent competitive inhibitor of dhfr, against 11 Plasmodium vivax isolates ex vivo, 10 of which had multiple dhfr mutations associated with Pyr resistance. Despite high-grade resistance to Pyr (median 50% inhibitory concentration [IC50], 13,345 nM), these parasites were all highly susceptible to MTX (median IC50, 2.6 nM). Given its potency against Pyr-resistant P. vivax, the antimalarial potential of MTX deserves further investigation

Evaluation of three parasite lactate dehydrogenase-based rapid diagnostic tests for the diagnosis of falciparum and vivax malaria

BACKGROUND: In areas where non-falciparum malaria is common rapid diagnostic tests (RDTs) capable of distinguishing malaria species reliably are needed. Such tests are often based on the detection of parasite lactate dehydrogenase (pLDH). METHODS: In Dawei, southern Myanmar, three pLDH based RDTs (CareStart Malaria pLDH (Pan), CareStart Malaria pLDH (Pan, Pf) and OptiMAL-IT)were evaluated in patients presenting with clinically suspected malaria. Each RDT was read independently by two readers. A subset of patients with microscopically confirmed malaria had their RDTs repeated on days 2, 7 and then weekly until negative. At the end of the study, samples of study batches were sent for heat stability testing. RESULTS: Between August and November 2007, 1004 patients aged between 1 and 93 years were enrolled in the study. Slide microscopy (the reference standard) diagnosed 213 Plasmodium vivax (Pv) monoinfections, 98 Plasmodium falciparum (Pf) mono-infections and no malaria in 650 cases. The sensitivities (sens) and specificities (spec), of the RDTs for the detection of malaria were- CareStart Malaria pLDH (Pan) test: sens 89.1% [CI95 84.2-92.6], spec 97.6% [CI95 96.5-98.4]. OptiMal-IT: Pf+/- other species detection: sens 95.2% [CI95 87.5-98.2], spec 94.7% [CI95 93.3-95.8]; non-Pf detection alone: sens 89.6% [CI95 83.6-93.6], spec 96.5% [CI95 94.8-97.7]. CareStart Malaria pLDH (Pan, Pf): Pf+/- other species: sens 93.5% [CI95 85.4-97.3], spec 97.4% [95.9-98.3]; non-Pf: sens 78.5% [CI95 71.1-84.4], spec 97.8% [CI95 96.3-98.7]. Inter-observer agreement was excellent for all tests (kappa > 0.9). The median time for the RDTs to become negative was two days for the CareStart Malaria tests and seven days for OptiMAL-IT. Tests were heat stable up to 90 days except for OptiMAL-IT (Pf specific pLDH stable to day 20 at 35 degrees C). CONCLUSION: None of the pLDH-based RDTs evaluated was able to detect non-falciparum malaria with high sensitivity, particularly at low parasitaemias. OptiMAL-IT performed best overall and would perform best in an area of high malaria prevalence among screened fever cases. However, heat stability was unacceptable and the number of steps to perform this test is a significant drawback in the field. A reliable, heat-stable, highly sensitive RDT, capable of diagnosing all Plasmodium species has yet to be identified

The Feasibility of Studying Metabolites in PICU Multi-Organ Dysfunction Syndrome Patients over an 8-Day Course Using an Untargeted Approach

Metabolites are generated from critical biological functions and metabolism. This pediatric study reviewed plasma metabolites in patients suffering from multi-organ dysfunction syndrome (MODS) in the pediatric intensive care unit (PICU) using an untargeted metabolomics approach. Patients meeting the criteria for MODS were screened for eligibility and consented (n = 24), and blood samples were collected at baseline, 72 h, and 8 days; control patients (n = 4) presented for routine sedation in an outpatient setting. A subset of MODS patients (n = 8) required additional support with veno-atrial extracorporeal membrane oxygenation (VA-ECMO) therapy. Metabolites from thawed blood plasma were determined from ion pairing reversed-phase liquid chromatography–mass spectrometry (LC-MS) analysis. Chromatographic peak alignment, identification, relative quantitation, and statistical and bioinformatics evaluation were performed using MAVEN and MetaboAnalyst 4.0. Metabolite analysis revealed 115 peaks per sample. From the partial least squares-discriminant analysis (PLS-DA) with variance of importance (VIP) scores above ≥2.0, 7 dynamic metabolites emerged over the three time points: tauro-chenodeoxycholic acid (TCDCA), hexose, p-hydroxybenzoate, hydroxyphenylacetic acid (HPLA), 2_3-dihydroxybenzoic acid, 2-keto-isovalerate, and deoxyribose phosphate. After Bonferroni adjustment for repeated measures, hexose and p-hydroxybenzoate were significant at one time point or more. Kendall’s tau-b test was used for internal validation of creatinine. Metabolites may be benign or significant in describing a patient’s pathophysiology and require operator interpretation

Characterization of ABC transporters in both mammalian cells (ABCG2, ABCC2)and «plasmodium falciparum (Pgh1)»

In the first part of the thesis two ABC transporters in mammalian cells are explored. Initially, the expression of members of the ABC family of transporters in erythrocytes was measured. It was found that ABCG2 (also known as the breast cancer resistance protein, BCRP, the mitoxantrone resistance protein, and ABC placenta) was expressed in mature human erythrocytes. This work concentrated on characterizing the oligomerization of ABCG2 in membrane extracts from tumour cells and human erythrocytes. Given the ability of ABCG2 to transport protoporphyrin IX or heme, these findings may shed some light on the normal function of erythrocytes. The second chapter of the thesis attempts to elucidate the drug binding characteristics of ABCC2 (MRP2). A radiolabelled photoreactive analogue of LTC4 (IAALTC4) was synthesized and used to carry out photoaffinity labelling experiments; a technique used to predict drug binding to a target protein. LTC4 is an endogenous substrate of ABCC2 since previous reports have shown LTC4 transport by ABCC2. Our binding studies revealed specific photoaffinity labelling of IAALTC4 to ABCC2 transfected cells. This work shows for the first time the direct binding of LTC4 to ABCC2, and further expands on the current biochemical knowledge of ABCC2. The long-standing drug of choice to treat malaria, chloroquine (CQ), is no longer effective due to increasing drug resistance. The lack of both new drug development and a clear understanding of the mechanism(s) of drug resistance have made achieving the global initiative to halve the malaria burden by the year 2010 more problematic; this aim now requires alternative methods of treatment to CQ. Therefore, the second main objective of this thesis was to address the growing problem of malaria drug resistance by exploring alternative therapies and a potential modulator of CQ resistance. In the third chapter, modulators of MRP1-mediated resistance were explored in chloroquine-sensitive and -resistaDans la première partie de la thèse, deux transporteurs ABC ont été explorés. Initialement, nous avons fait l'analyse des protéines ABC (ATP-binding cassette) dans le globules rouges, en examinant leur niveau d'expression au niveau de leur membrane. Nous avons observé que ABCG2 aussi appelé : "breast cancer resistant protein,-BCRP", "mitoxantrone resistant protein", et "ABC placental", était exprimé dans les globules rouges matures. Ce travaille s'est concentré sur la caractérisation de l'oligomérization de ABCG2 dans la membrane des cellules cancéreuses et dans les globules rouges humaines. Nous savons déjà que ABCG2 transporte la protoporphyrin IX ou hème, alors nous souhaitons que ces résultats ajoutent à la connaissance de la fonction normale des globules rouges. Dans le deuxième chapitre, nous avons exploré les caractéristiques de liaison de ABCC2 (MRP2) avec les substrats. En utilisant un analogue photoréactif de LTC4 (IAALTC4) marqué pour faire des études de photoaffinités, une technique fut utilisée pour prédire la liaison d'un composé sur une protéine. La LTC4 est un substrat naturel (endogène) de ABCC2 et des résultats établis antérieurement ont montré le transport de LTC4 par ABCC2. Nos études de liaisons en photoaffinité demontrent spécifiquement que IAALTC4 trace les cellules transfectées avec ABCC2. Ces résultats montrent pour la première fois, la liaison de LTC4 à ABCC2, tout en nous apportant plus d'information biochimique sur ABCC2. La Chloroquine (CQ) est l'agent chimiothérapeutique le plus rèpandu pour combattre et traiter la malaria; toutefois son efficacité est en constante décroissance due à une résistance du parasite rependue mondialement. Les organismes mondiaux de santé préconisent l'utilisation de type de traitements visant une réduction de 50% du paludisme pour l'année 2010. Toutefois, le manque de nouvelles molécules et l'absence d'une compréhension claire des mécanismes

Hexosylceramides and Glycerophosphatidylcholine GPC(36:1) Increase in Multi-Organ Dysfunction Syndrome Patients with Pediatric Intensive Care Unit Admission over 8-Day Hospitalization

Glycero- and sphingo-lipids are important in plasma membrane structure, caloric storage and signaling. An un-targeted lipidomics approach for a cohort of critically ill pediatric intensive care unit (PICU) patients undergoing multi-organ dysfunction syndrome (MODS) was compared to sedation controls. After IRB approval, patients meeting the criteria for MODS were screened, consented (n = 24), and blood samples were collected from the PICU at HDVCH, Michigan; eight patients needed veno-arterial extracorporeal membrane oxygenation (VA ECMO). Sedation controls were presenting for routine sedation (n = 4). Plasma lipid profiles were determined by nano-electrospray (nESI) direct infusion high resolution/accurate mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Biostatistics analysis was performed using R v 3.6.0. Sixty-one patient samples over three time points revealed a ceramide metabolite, hexosylceramide (Hex-Cer) was high across all time points (mean 1.63–3.19%; vs. controls 0.22%). Fourteen species statistically differentiated from sedation controls (p-value ≤ 0.05); sphingomyelin (SM) [SM(d18:1/23:0), SM(d18:1/22:0), SM(d18:1/23:1), SM(d18:1/21:0), SM(d18:1/24:0)]; and glycerophosphotidylcholine (GPC) [GPC(36:01), GPC(18:00), GPC(O:34:02), GPC(18:02), GPC(38:05), GPC(O:34:03), GPC(16:00), GPC(40:05), GPC(O:36:03)]. Hex-Cer has been shown to be involved in viral infection and may be at play during acute illness. GPC(36:01) was elevated in all MODS patients at all time points and is associated with inflammation and brain injury

Long-term storage limits PCR-based analyses of malaria parasites in archival dried blood spots.

International audienceABSTRACT: BACKGROUND: Blood samples collected in epidemiological and clinical investigations and then stored, often at room temperature, as blood spots dried on a filter paper has become one of the most popular source of material for further molecular analyses of malaria parasites. The dried blood spots are often archived so that they can be used for further retrospective investigations of parasite prevalence, or as new genetic markers come to the fore. However, the suitability of the template obtained from dried blood spots that have been stored for long periods for DNA amplification is not known. METHODS: DNA from 267 archived blood spots collected over a period of 12 years from persons with microscopically confirmed Plasmodium falciparum infection was purified by one of two methods, Chelex and Qiagen columns. These templates were subjected to highly sensitive nested PCR amplification targeting three parasite loci that differ in length and/or copy number. RESULTS: When a 1.6 kb fragment of the parasites' small subunit ribosomal RNA was targeted (primary amplification), the efficiency of P. falciparum detection decreased in samples archived for more than six years, reaching very low levels for those stored for more than 10 years. Positive amplification was generally obtained more often with Qiagen-extracted templates. P. falciparum could be detected in 32 of the 40 negative Qiagen-extracted templates when a microsatellite of about 180 bp was targeted. The remaining eight samples gave a positive amplification when a small region of 238 bp of the higher copy number (20 to 200) mitochondrial genome was targeted. CONCLUSIONS: The average length of DNA fragments that can be recovered from dried blood spots decrease with storage time. Recovery of the DNA is somewhat improved, especially in older samples, by the use of a commercial DNA purification column, but targets larger than 1.5 kb are unlikely to be present 10 years after the initial blood collection, when the average length of the DNA fragments present is likely to be around a few hundred bp. In conclusion, the utility of archived dried blood spots for molecular analyses decreases with storage time