Publications by authors named "Mara Leimanis"

20 Publications

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Gene expression signatures identify paediatric patients with multiple organ dysfunction who require advanced life support in the intensive care unit.

EBioMedicine 2020 Dec 25;62:103122. Epub 2020 Nov 25.

Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, MI 49503, USA; Pediatric Intensive Care Unit, Helen DeVos Children's Hospital, 100 Michigan Street NE, Grand Rapids, MI 49503, USA; Office of Research, Spectrum Health, 15 Michigan Street NE, Grand Rapids, MI 49503, USA. Electronic address:

Background: Multiple organ dysfunction syndrome (MODS) occurs in the setting of a variety of pathologies including infection and trauma. Some patients decompensate and require Veno-Arterial extra corporeal membrane oxygenation (ECMO) as a palliating manoeuvre for recovery of cardiopulmonary function. The molecular mechanisms driving progression from MODS to cardiopulmonary collapse remain incompletely understood, and no biomarkers have been defined to identify those MODS patients at highest risk for progression to requiring ECMO support.

Methods: Whole blood RNA-seq profiling was performed for 23 MODS patients at three time points during their ICU stay (at diagnosis of MODS, 72 hours after, and 8 days later), as well as four healthy controls undergoing routine sedation. Of the 23 MODS patients, six required ECMO support (ECMO patients). The predictive power of conventional demographic and clinical features was quantified for differentiating the MODS and ECMO patients. We then compared the performance of markers derived from transcriptomic profiling including [1] transcriptomically imputed leukocyte subtype distribution, [2] relevant published gene signatures and [3] a novel differential gene expression signature computed from our data set. The predictive power of our novel gene expression signature was then validated using independently published datasets.

Finding: None of the five demographic characteristics and 14 clinical features, including The Paediatric Logistic Organ Dysfunction (PELOD) score, could predict deterioration of MODS to ECMO at baseline. From previously published sepsis signatures, only the signatures positively associated with patient's mortality could differentiate ECMO patients from MODS patients, when applied to our transcriptomic dataset (P-value ranges from 0.01 to 0.04, Student's test). Deconvolution of bulk RNA-Seq samples suggested that lower neutrophil counts were associated with increased risk of progression from MODS to ECMO (P-value = 0.03, logistic regression, OR=2.82 [95% CI 0.63 - 12.45]). A total of 30 genes were differentially expressed between ECMO and MODS patients at baseline (log2 fold change ≥ 1 or ≤ -1 with false discovery rate ≤ 0.01). These genes are involved in protein maintenance and epigenetic-related processes. Further univariate analysis of these 30 genes suggested a signature of seven DE genes associated with ECMO (OR > 3.0, P-value ≤ 0.05, logistic regression). Notably, this contains a set of histone marker genes, including H1F0, HIST2H3C, HIST1H2AI, HIST1H4, HIST1H2BL and HIST1H1B, that were highly expressed in ECMO. A risk score derived from expression of these genes differentiated ECMO and MODS patients in our dataset (AUC = 0.91, 95% CI 0.79-1.00, P-value = 7e-04, logistic regression) as well as validation dataset (AUC= 0.73, 95% CI 0.53-0.93, P-value = 2e-02, logistic regression).

Interpretation: This study demonstrates that transcriptomic features can serve as indicators of severity that could be superior to traditional methods of ascertaining acuity in MODS patients. Analysis of expression of signatures identified in this study could help clinicians in the diagnosis and prognostication of MODS patients after arrival to the Hospital.
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http://dx.doi.org/10.1016/j.ebiom.2020.103122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7704404PMC
December 2020

Virus-induced genetics revealed by multidimensional precision medicine transcriptional workflow applicable to COVID-19.

Physiol Genomics 2020 06 21;52(6):255-268. Epub 2020 May 21.

Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, Michigan.

Precision medicine requires the translation of basic biological understanding to medical insights, mainly applied to characterization of each unique patient. In many clinical settings, this requires tools that can be broadly used to identify pathology and risks. Patients often present to the intensive care unit with broad phenotypes, including multiple organ dysfunction syndrome (MODS) resulting from infection, trauma, or other disease processes. Etiology and outcomes are unique to individuals, making it difficult to cohort patients with MODS, but presenting a prime target for testing/developing tools for precision medicine. Using multitime point whole blood (cellular/acellular) total transcriptomics in 27 patients, we highlight the promise of simultaneously mapping viral/bacterial load, cell composition, tissue damage biomarkers, balance between syndromic biology versus environmental response, and unique biological insights in each patient using a single platform measurement. Integration of a transcriptome workflow yielded unexpected insights into the complex interplay between host genetics and viral/bacterial specific mechanisms, highlighted by a unique case of virally induced genetics (VIG) within one of these 27 patients. The power of RNA-Seq to study unique patient biology while investigating environmental contributions can be a critical tool moving forward for translational sciences applied to precision medicine.
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http://dx.doi.org/10.1152/physiolgenomics.00045.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7303726PMC
June 2020

Outcomes after completion total gastrectomy for gastric remnant cancer: experience from a Canadian tertiary centre.

Can J Surg 2018 08;61(4):270-277

From the Division of Thoracic Surgery, McGill University Health Centre, Montréal, Que.

Background: There is controversy about the safety and outcomes of completion total gastrectomy (CTG) for gastric adenocarcinoma. We compared a cohort of patients who underwent CTG for gastric remnant cancer (GRC) after partial gastrectomy for benign disease with patients who underwent primary total gastrectomy (PTG) for sporadic gastric cancer.

Methods: We retrospectively reviewed a single-institution, prospectively maintained clinical database of patients who had undergone gastrectomy from 2005 to 2016 for demographic, surgical, clinical and tumour pathology data, as well as postoperative, pathologic and oncologic outcomes including complications, length of stay, disease-free survival and overall survival. We used the χ and Wilcoxon rank-sum tests to compare groups and performed the Mantel-Cox log-rank test for Kaplan-Meier survival estimates. We compared the CTG group to all patients in the PTG group and to a 5:1 propensity-matched PTG cohort.

Results: We analyzed data for 64 patients (9 CTG, 55 PTG). The groups were equivalent at baseline and had similar operative, perioperative treatment and pathologic characteristics. After propensity matching, the reoperation rate for complications was higher after CTG than PTG (22% v. 0%, = 0.03), but there was no significant difference in the overall complication rate or length of stay. At 5 years, there was no difference in disease-free survival (28% v. 58%, = 0.4) or overall survival (33% v. 44%, = 0.7).

Conclusion: Our findings suggest that CTG for gastric adenocarcinoma can be undertaken safely a priori with no additional risk of recurrence or death compared to PTG for sporadic gastric cancer.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6066381PMC
August 2018

Application of an individualized operative strategy for wedge resection of gastric gastrointestinal stromal tumors: Effectiveness for tumors in difficult locations.

Surgery 2016 10 30;160(4):1038-1048. Epub 2016 Jul 30.

Department of Surgery, Montreal General Hospital, McGill University Health Center, Montreal, Canada.

Background: There is some concern that wedge resection of gastric gastrointestinal stromal tumors is not feasible in certain anatomic locations, such as the cardia or antrum. We sought to review our experience with treatment of gastric gastrointestinal stromal tumors with a particular focus on nonanatomic wedge resections in these challenging locations.

Methods: Patients undergoing resection of gastrointestinal stromal tumors from 2000-2014 at the Montreal General Hospital were identified from a prospectively collected database, and outcomes were tabulated. An individualized operative strategy was used to guide resection based on tumor location, size, and characteristics. Disease-free survival and overall survival analyzed using the Kaplan-Meier method. Data are presented as median (range).

Results: We identified 59 patients who underwent operative resection for gastric gastrointestinal stromal tumors. Tumor location was fundus/body/greater curvature in 35 (59%) patients, lesser curvature in 8 (14%) patients, antrum in 8 (14%) patients, and cardia in 8 (14%) patients. Median tumor size was 4.5 cm (1.4-25 cm). The majority of cardia and antral lesions were removed with wedge resections (14/16, 87%). For cardial and antral tumors, on-table gastroscopy was used to guide the operative approach and prevent narrowing of the Gastroesophageal junction or pylorus in all patients undergoing wedge resection. Negative pathologic margins were achieved in all patients. The 5-year disease-free survival was 91% and 5-year overall survival was 95%.

Conclusion: When selected appropriately, and under the guidance of on-table gastroscopy, laparoscopic nonanatomic wedge resection can be performed successfully in the majority of cases, even for gastrointestinal stromal tumors near the GEJ or pylorus, with excellent oncologic outcomes.
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http://dx.doi.org/10.1016/j.surg.2016.06.004DOI Listing
October 2016

Evolving Management of Zenker's Diverticulum in the Endoscopic Era: A North American Experience.

World J Surg 2016 Jun;40(6):1390-6

Division of Thoracic, McGill University, Montreal General Hospital, Room L9-112, 1650 Cedar Avenue, Montreal, QC, H3A 1G4, Canada.

Background: Open surgical cricopharyngeal myotomy(CM) is considered standard of care for Zenker's diverticulum(ZD). Trans-oral CM has been described using a rigid stapling device for two decades; however, this remains problematic for severely kyphotic patients. This problem can be overcome with flexible endoscopy utilizing an electrosurgical needle knife. We sought to compare clinical outcomes between these techniques to stratify patient selection.

Methods: Patients undergoing ZD treatment from 1992 to 2015 were reviewed. Demographics, diverticulum size, post-operative complications, and length of stay (LOS) were compared between open cricopharyngeal myotomy (OpenCM), rigid trans-oral stapling myotomy (RigidCM), and flexible endoscopic myotomy (FlexCM). Dysphagia scores (DS, 0:best-4:worst) and pneumonia incidence were assessed pre-operatively and post-operatively.

Results: 62 patients underwent OpenCM (39/62(63 %)) or endoscopic CM (23/62(37 %) (8 RigidCM/15 FlexCM)). CM significantly reduced dysphagia for all approaches [OpenCM:2(2-3)-0(0-0); RigidCM:2(2-2)-0(0-0); FlexCM:3(3-3)-0(0-0)]. FlexCM patients had significantly worse pre-operative DS. Endoscopic CM was attempted and completed in 23/35(66 %) patients. Reasons for OpenCM conversion included inability to position the diverticular retractor due to patient body habitus (RigidCM), and the inability to position the overtube due to small ZD (FlexCM). Major post-operative complications were rare and similar in all groups. Medium-to-long-term post-myotomy pneumonia was comparable between groups. LOS (days) was reduced for FlexCM (1(1-2)) versus RigidCM (3(2-6)) and OpenCM (4(3-7)).

Conclusions: CM is highly effective for treating ZD. Open and endoscopic approaches offer comparable outcomes and dysphagia resolution. FlexCM is efficacious for large ZD and can be performed in most patients irrespective of body habitus. FlexCM represents an excellent approach for large ZD, while OpenCM should be reserved for small ZD for which an overtube cannot be positioned.
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http://dx.doi.org/10.1007/s00268-016-3442-0DOI Listing
June 2016

A 2-amino quinoline, 5-(3-(2-(7-chloroquinolin-2-yl)ethenyl)phenyl)-8-dimethylcarbamyl-4,6-dithiaoctanoic acid, interacts with PfMDR1 and inhibits its drug transport in Plasmodium falciparum.

Mol Biochem Parasitol 2014 Jun 8;195(1):34-42. Epub 2014 Jun 8.

Institute of Parasitology, McGill University, Ste. Anne de Bellevue, Montréal, Québec, Canada. Electronic address:

Malaria is a major disease in the tropics where chemotherapy remains the main mode of treatment and as such the rise and spread of drug-resistant malaria can lead to human tragedy. Two membrane transport proteins, PfMDR1 (Plasmodium falciparum multidrug resistance protein 1) and PfCRT (P. falciparum chloroquine resistance transporter), have been shown to cause resistance to several antimalarials. Both PfMDR1 and PfCRT are localized to the digestive vacuolar membrane and appear to regulate the transport of drugs and physiological metabolites. In this study we have used MK571, a 2-amino quinoline, to explore its interaction with PfMDR1 and PfCRT in chloroquine-sensitive and -resistant strains of P. falciparum. Our results show that chloroquine-resistant strains (e.g., K1, Dd2, and 7G8) are consistently more sensitive to MK571 than chloroquine-sensitive strains (e.g., 3D7, 106/1 and D10). This association, however, was not maintained with the chloroquine-resistant strain FCB which IC50 value was similar to chloroquine-sensitive strains. Moreover, the susceptibility of chloroquine-sensitive and -resistant strains to MK571 does not correlate with mutated PfCRT, nor is it reversible with verapamil; but correlates with mutations in PfMDR1. Furthermore, MK571 appears to target the parasite's digestive vacuole (DV), as demonstrated by the ability of MK571 to: (1) block the accumulation of the fluorescent dye Fluo-4 AM, a PfMDR1 substrate, into the digestive vacuole; (2) reduce the transvacuolar pH gradient; and (3) inhibit the formation of β-hematin in vitro. Moreover, the presence of non-toxic concentrations of MK571 sensitized both chloroquine-sensitive and -resistant parasites to mefloquine and halofantrine, likely by competing against PfMDR1-mediated sequestering of the drugs into the DV compartment and away from the drugs' cytosolic targets. Our data, nevertheless, found only a minimal decrease in MK571 IC50 value in FCB parasite which second pfmdr1 copy was inactivated via gene disruption. Taken together, the findings of this study suggest that MK571 interacts with native and mutant PfMDR1 and modulates the import of drugs or solutes into the parasite's DV and, as such, MK571 may be a useful tool in the characterization of PfMDR1 drug interactions and substrate specificity.
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http://dx.doi.org/10.1016/j.molbiopara.2014.05.006DOI Listing
June 2014

Treatment of suspected hyper-reactive malarial splenomegaly (HMS) in pregnancy with mefloquine.

Am J Trop Med Hyg 2014 Apr 3;90(4):609-611. Epub 2014 Mar 3.

Malaria infections in pregnancy are associated with adverse outcomes for both mother and child. There are few data on hyper-reactive malarial splenomegaly, an aberrant immunological response to chronic or recurrent malaria in pregnancy. This retrospective assessment reviewed the impact of mefloquine treatment on pregnant women with suspected hyper-reactive malarial splenomegaly in an area of low malaria transmission in the 1990s, showing significant reductions in spleen size and anemia and anti-malarial antibody titers without any notable negative effect on treated women or their newborns.
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http://dx.doi.org/10.4269/ajtmh.13-0706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3973501PMC
April 2014

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

Malar J 2012 Oct 8;11:339. Epub 2012 Oct 8.

Shoklo Malaria Research Unit, Mae Sot, Tak Province, Thailand.

Background: Blood samples collected in epidemiological and clinical investigations and then stored, often at room temperature, as blood spots dried on a filter paper have 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 decreases 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.
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http://dx.doi.org/10.1186/1475-2875-11-339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3507721PMC
October 2012

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

Malar J 2011 Oct 28;10:323. Epub 2011 Oct 28.

Shoklo Malaria Research Unit, Mae Sot, Thailand.

Background: 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 Plasmodium 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.

Methods: 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.

Results: 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.

Conclusions: 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.
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http://dx.doi.org/10.1186/1475-2875-10-323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3219859PMC
October 2011

Photoaffinity labeling of the multidrug resistance protein 2 (ABCC2/cMOAT) with a photoreactive analog of LTC(4).

Int J Biochem Mol Biol 2011 15;2(1):39-46. Epub 2010 Dec 15.

Institute of Parasitology, Macdonald Campus McGill University, Montreal Canada.

Several studies have shown that the multidrug resistant protein MRP2 mediates the transport of chemotherapeutic drugs and normal cell metabolites, including Leukotriene C (LTC(4)); however direct binding of the LTC(4) to MRP2 has not been demonstrated. In this study, a photoreactive analog of LTC(4) (IAALTC(4)) was used to demonstrate its direct binding to MRP2. Our results show specific photoaffinity labeling of MRP2 with IAALTC(4) in plasma membranes from MDCKII(MRP2) cells. The photoaffinity labeling signal of MRP2 with IAALTC(4) was much lower than that of MRP1, consistent with previous studies whereby the measured K(m) values of MRP1 and MRP2 for LTC(4) were 1 μM and 0.1 μM LTC(4), respectively. Competition of IAALTC(4) photoaffinity labeling to MRP2 with MK571, a well characterized inhibitor of MRP2 function, showed ~75% reduction in binding in the presence of 50 μM excess MK571. Interestingly, unmodified LTC(4) enhanced the photoaffinity labeling of IAALTC(4) to MRP2, whereas excess GSH and Quercetin had no significant effect. Mild tryptic digestion of photoaffinity labeled MRP2 revealed several photoaffinity labeled peptides that localized the IAALTC(4) binding to a 15 kDa amino acid sequence that contains transmembrane 16 and 17. Together these results provide the first demonstration of direct LTC(4) binding to MRP2.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3180033PMC
December 2010

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

Malar J 2011 May 5;10:113. Epub 2011 May 5.

Shoklo Malaria Research Unit, PO Box 46 Mae Sot, Tak 63110, 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.
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http://dx.doi.org/10.1186/1475-2875-10-113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3112451PMC
May 2011

Methotrexate is highly potent against pyrimethamine-resistant Plasmodium vivax.

J Infect Dis 2011 Jan 9;203(2):207-10. Epub 2010 Dec 9.

Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok.

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.
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http://dx.doi.org/10.1093/infdis/jiq024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3071051PMC
January 2011

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

Malar J 2009 Oct 27;8:241. Epub 2009 Oct 27.

Epicentre, Paris, France.

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.
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http://dx.doi.org/10.1186/1475-2875-8-241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2774865PMC
October 2009

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

Malar J 2009 Jun 2;8:115. Epub 2009 Jun 2.

Singapore Immunology Network, BIOPOLIS, Singapore.

Background: Investigations of Plasmodium vivax are restricted to samples collected from infected persons or primates, because this parasite cannot be maintained in in vitro cultures. Contamination of P. vivax isolates with host leukocytes and platelets is detrimental to a range of ex vivo 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 P. vivax IRBCs by CF11 cellulose filtration.

Methods And Results: 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 x 103 per microl [95%CI 5.2-13.5] to 0.01 x 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 x 103 per microl [95%CI 107.5-315.7] to 0.8 x 103 per microl [95%CI -0.7-2.2]. Analysis of 30 P. vivax blood samples before and after CF11 filtration showed only a minor loss in parasitaemia (
Conclusion: CF11 filtration is the most cost and time efficient method for the production of leukocyte- and platelet-free P. vivax-infected erythrocytes from field isolates.
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http://dx.doi.org/10.1186/1475-2875-8-115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2694833PMC
June 2009

Autofluorescence of condensed heme aggregates in malaria pigment and its synthetic equivalent hematin anhydride (beta-hematin).

J Phys Chem B 2009 Jun;113(24):8391-401

Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec, Canada H3A 2K6.

The condensed crystalline phase of iron(III) protoporphyrin IX either isolated from parasite culture as malaria pigment (hemozoin) or synthetic equivalent hematin anhydride exhibits a solid-state autofluorescence characterized by an excitation maximum of 555 nm and an emission maximum of 577 nm. The excitation spectrum maximum at 555 nm corresponds to the Q(0,0) band in the absorption spectrum which represents the lowest singlet of the material. This suggests that the fluorescent emission is due to the heme condensed phase. The photoluminescence lifetime of tau(f) = 2.7 +/- 0.8 ns as measured at four wavelengths between 550 and 600 nm is in the range of Frankel exciton in porphyrinic condensed phases. The material is shown to have an optical band gap of 2.04 eV characteristic of a semiconductor. Luminescence is markedly dependent upon the degree of hydration and the emission does not seem to be caused by presence of zinc(II) protoporphyrin IX or free-base protoporphyrin IX in the lattice. The autofluorescence can be used for in vivo tracking of hemozoin, for determination of parasitemia levels, and for infection monitoring and possibly for drug screening studies.
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http://dx.doi.org/10.1021/jp8104375DOI Listing
June 2009

Sensitive detection of malaria infection by third harmonic generation imaging.

Biophys J 2008 Feb 7;94(4):L26-8. Epub 2007 Dec 7.

Malaria remains a major health concern worldwide, with 350-500 million cases reported annually in endemic countries. In this study, we report a novel and highly sensitive optical-based detection of malaria-infected blood cells by third harmonic generation (THG) imaging of hemozoin pigment that is naturally deposited by the parasite during its lifecycle. The THG signal from the hemozoin was greater than we have observed in any cell type with signal/noise ratios that reach 1000:1. This method allows a rapid and robust detection of early stage infections of blood cells. The immense nonlinear response of the intrinsic parasitic by-product pigments suggests that automated optical detection by THG could be used for sensitive and rapid screening of parasite infection in blood samples.
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http://dx.doi.org/10.1529/biophysj.107.125443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2212690PMC
February 2008

Modulation of GSH levels in ABCC1 expressing tumor cells triggers apoptosis through oxidative stress.

Biochem Pharmacol 2007 Jun 15;73(11):1727-37. Epub 2007 Feb 15.

Institute of Parasitology, Macdonald Campus, McGill University, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9.

The over-expression of ABCC1 transmembrane protein has been shown to cause multidrug resistance in tumor cell lines. ABCC1 is a member of the ABC transmembrane proteins that function as efflux pumps with diverse substrate specificity. Several endogenous cell metabolites, including the leukotriene C4 (LTC(4)) and glutathione (GSH) are substrates for ABCC1 protein. ABCC1 expression in certain tumor cells was demonstrated to confer hypersensitivity to glutathione modulating agents. In this report we have investigated the mechanism of collateral sensitivity seen in tumor cells over-expressing ABCC1 protein. The results of this study show that ABCC1 expression in tumor cells correlates with their hypersensitivity to various glutathione modulating agents, as demonstrated in H69AR-drug selected and HeLa/ABCC1-transfectant cells. This effect was triggered either through inhibition of GSH synthesis with BSO or by increasing ABCC1-mediated GSH transport with verapamil or apigenin. In addition, our results show that the hypersensitivity of ABCC1-expressing cells to BSO, verapamil or apigenin was preceded by an increase in reactive oxygen species (or ROS). A decrease in GSH level is also observed prior the increase in ROS. In addition, we show that hypersensitivity to the BSO, verapamil or apigenin leads to tumor cell death by apoptosis. Together, the results of this study demonstrate that ABCC1 potentiates oxidative stress in tumor cells through reductions in cellular GSH levels.
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http://dx.doi.org/10.1016/j.bcp.2007.02.005DOI Listing
June 2007

ABCG2 membrane transporter in mature human erythrocytes is exclusively homodimer.

Biochem Biophys Res Commun 2007 Mar 9;354(2):345-50. Epub 2007 Jan 9.

Institute of Parasitology, McGill University, Ste. Anne de Bellevue (Montreal), Que., Canada H9X-3V9.

The human ABCG2 protein, a member of ABC transporter family, was shown to transport anti-cancer drugs and normal cell metabolites. Earlier studies have demonstrated the expression of ABCG2 in hematopoietic stem cells and erythroid cells; however little is known about the expression and activity of ABCG2 in mature erythrocytes. In this report, we show that ABCG2 in mature human erythrocytes migrates with an apparent molecular mass of 140 kDa, under reducing conditions, on Fairbanks SDS gel system. In contrast, tumor cells expressing higher levels of ABCG2 show no detectable homodimers, when resolved under identical reducing conditions. Analysis of the same membrane extracts from tumor cells and human erythrocytes on Laemmli SDS gel system, where samples are boiled in the presence of increasing concentrations of disulfide reducing conditions and then analyzed, migrate with an apparent molecular mass of 70 kDa or a monomer. Drug transport studies using Pheophorbide A, a substrate of ABCG2, show the protein to be active in erythrocytes. Furthermore, Fumitremorgin C, a specific inhibitor of ABCG2 increases the accumulation of Pheophorbide A in erythrocytes and drug-resistant cells but not in the parental drug-sensitive cells. Given the ability of ABCG2 to transport protoprophyrin IX or heme, these findings may have implications on the normal function of erythrocytes.
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http://dx.doi.org/10.1016/j.bbrc.2006.12.219DOI Listing
March 2007

Lupeol long-chain fatty acid esters with antimalarial activity from Holarrhena floribunda.

J Nat Prod 2006 Jan;69(1):62-7

Laboratory of Biological Chemistry, Department of Chemistry, McGill University, Otto Maass Chemistry Building # 230, Sherbrooke Street West, Montreal, Quebec H3A 2K6, Canada.

An ethnopharmacological investigation was conducted among the Baka pygmies of Dja biosphere reserve (Cameroon) to collect information on the antimalarial plants used in their daily life. Holarrhena floribunda is one of those plants. Extracts of the stem barks of H. floribunda showed remarkable inhibitory activity against drug-resistant strains of Plasmodium falciparum at doses of 1.02-18.53 microg/mL when tested in vitro against two parasite clones designated as Indochina (W-2) and Sierra Leone (D-6). The aqueous extract was the most active against Indochina (W-2), with IC50 values of 1.02 microg/mL, while the ethanolic extract appeared to be the most active against Sierra Leone (D-6), with an IC50 of 4.33 microg/mL. The bioassay-guided fractionation of the neutral fraction of the crude extract led to the isolation of lupeol (1) and its three new long-chain fatty acid ester derivatives, namely, 3-O-(3'-hydroxyeicosanoyl)lupeol (2), 3-O-[(2'-(tetracosyloxy)acetyl]lupeol (3), and 3-O-[(1' '-hydroxyoctadecyloxy)-2'-hydroxypropanoyl]lupeol (4). These new compounds displayed some in vitro inhibition activity against the chloroquine-resistant strain FCR-3 isolated from Gambia and the chloroquine-sensitive standard strain 3D7. The hydroxy group of the fatty acid side chain appears to decrease the observed activity.
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http://dx.doi.org/10.1021/np050315yDOI Listing
January 2006

The leucotriene C4 binding sites in multidrug resistance protein 1 (ABCC1) include the first membrane multiple spanning domain.

Biochemistry 2005 Jan;44(1):340-51

Institute of Parasitology and Department of Biochemistry, McGill University, Ste-Anne-de-Bellevue, Quebec H9X 3V9, Canada.

The multiple drug resistance protein 1 (MRP1 or ABCC1) transports anticancer drugs and normal cell metabolites. Leucotriene C(4) (LTC(4)) is one of the highest affinity substrates of MRP1. In this study, we have synthesized and characterized a novel photoreactive azido analogue of LTC(4) (AALTC(4)). The specificity of AALTC(4) binding to MRP1 was confirmed using an LTC(4)-specific monoclonal antibody. Moreover, binding with radioiodinated [(125)I]AALTC(4) (or IAALTC(4)) to MRP1 was dramatically competed with unmodified LTC(4) and to a lesser degree by glutathione (GSH). Oxidized glutathione (GSSG) slightly increased IAALTC(4) binding to MRP1, while MK571, verapamil, and vincristine inhibited IAALTC(4) binding to MRP1. Using AALTC(4) together with a panel of epitope-specific and LTC(4)-specific monoclonal antibodies, we identified LTC(4) binding sites in MRP1. Western blotting of large tryptic fragments of MRP1 with three well-characterized epitope-specific mAbs (MRPr1, QCRL1, and MRPm6) showed LTC(4) binding in both the N- and C-terminal halves of MRP1. Furthermore, a peptide corresponding to the N-terminal membrane-spanning domain of MRP1 (MSD0) was photoaffinity labeled by AALTC(4), indicating that MSD0 contains an LTC(4) binding site. Higher resolution mapping of additional LTC(4) binding sites was obtained using eight MRP1 variants with each containing hemaglutanin A (HA) epitopes at different sites (at amino acid 4, 163, 271, 574, 653, 938, 1001, or 1222). MRP1 variants were photoaffinity labeled with IAALTC(4) and digested with trypsin to isolate specific regions of MRP1 that interact with LTC(4). These results confirmed that sequences in MSD0 interact with IAALTC(4). Other regions that were photoaffinity labeled by IAALTC(4) include TM 10-11, TM 16-17, and TM 12, shown previously to encode MRP1 drug binding site(s). Together, our results show a high-resolution map of LTC(4) binding domains in MRP1 and provide the first direct evidence for LTC(4) binding within MSD0.
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http://dx.doi.org/10.1021/bi048853hDOI Listing
January 2005