Publications by authors named "Nicolas Veland"

19 Publications

  • Page 1 of 1

Identifying proteins bound to native mitotic ESC chromosomes reveals chromatin repressors are important for compaction.

Nat Commun 2020 08 17;11(1):4118. Epub 2020 Aug 17.

Lymphocyte Development Group, MRC London Institute of Medical Sciences, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.

Epigenetic information is transmitted from mother to daughter cells through mitosis. Here, to identify factors that might play a role in conveying epigenetic memory through cell division, we report on the isolation of unfixed, native chromosomes from metaphase-arrested cells using flow cytometry and perform LC-MS/MS to identify chromosome-bound proteins. A quantitative proteomic comparison between metaphase-arrested cell lysates and chromosome-sorted samples reveals a cohort of proteins that were significantly enriched on mitotic ESC chromosomes. These include pluripotency-associated transcription factors, repressive chromatin-modifiers such as PRC2 and DNA methyl-transferases, and proteins governing chromosome architecture. Deletion of PRC2, Dnmt1/3a/3b or Mecp2 in ESCs leads to an increase in the size of individual mitotic chromosomes, consistent with de-condensation. Similar results were obtained by the experimental cleavage of cohesin. Thus, we identify chromosome-bound factors in pluripotent stem cells during mitosis and reveal that PRC2, DNA methylation and Mecp2 are required to maintain chromosome compaction.
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http://dx.doi.org/10.1038/s41467-020-17823-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431861PMC
August 2020

The ZBTB24-CDCA7 axis regulates HELLS enrichment at centromeric satellite repeats to facilitate DNA methylation.

Protein Cell 2020 03;11(3):214-218

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, 78957, USA.

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http://dx.doi.org/10.1007/s13238-019-00682-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026229PMC
March 2020

DNMT3L facilitates DNA methylation partly by maintaining DNMT3A stability in mouse embryonic stem cells.

Nucleic Acids Res 2019 01;47(1):152-167

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA.

DNMT3L (DNMT3-like), a member of the DNMT3 family, has no DNA methyltransferase activity but regulates de novo DNA methylation. While biochemical studies show that DNMT3L is capable of interacting with both DNMT3A and DNMT3B and stimulating their enzymatic activities, genetic evidence suggests that DNMT3L is essential for DNMT3A-mediated de novo methylation in germ cells but is dispensable for de novo methylation during embryogenesis, which is mainly mediated by DNMT3B. How DNMT3L regulates DNA methylation and what determines its functional specificity are not well understood. Here we show that DNMT3L-deficient mouse embryonic stem cells (mESCs) exhibit downregulation of DNMT3A, especially DNMT3A2, the predominant DNMT3A isoform in mESCs. DNA methylation analysis of DNMT3L-deficient mESCs reveals hypomethylation at many DNMT3A target regions. These results confirm that DNMT3L is a positive regulator of DNA methylation, contrary to a previous report that, in mESCs, DNMT3L regulates DNA methylation positively or negatively, depending on genomic regions. Mechanistically, DNMT3L forms a complex with DNMT3A2 and prevents DNMT3A2 from being degraded. Restoring the DNMT3A protein level in DNMT3L-deficient mESCs partially recovers DNA methylation. Thus, our work uncovers a role for DNMT3L in maintaining DNMT3A stability, which contributes to the effect of DNMT3L on DNMT3A-dependent DNA methylation.
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http://dx.doi.org/10.1093/nar/gky947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326784PMC
January 2019

Identification of Rpl29 as a major substrate of the lysine methyltransferase Set7/9.

J Biol Chem 2018 08 29;293(33):12770-12780. Epub 2018 Jun 29.

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, Texas 78957; Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Smithville, Texas 78957; Program in Genetics and Epigenetics, The University of Texas MD Anderson Cancer Center UT Health Graduate School of Biomedical Sciences, Houston, Texas 77030. Electronic address:

Set7/9 (also known as Set7, Set9, Setd7, and Kmt7) is a lysine methyltransferase that catalyzes the methylation of multiple substrates, including histone H3 and non-histone proteins. Although not essential for normal development and physiology, Set7/9-mediated methylation events play important roles in regulating cellular pathways involved in various human diseases, making Set7/9 a promising therapeutic target. Multiple Set7/9 inhibitors have been developed, which exhibit varying degrees of potency and selectivity However, validation of these compounds has been hampered by the lack of a reliable cellular biomarker for Set7/9 activity. Here, we report the identification of Rpl29, a ribosomal protein abundantly expressed in all cell types, as a major substrate of Set7/9. We show that Rpl29 lysine 5 (Rpl29K5) is methylated exclusively by Set7/9 and can be demethylated by Lsd1 (also known as Kdm1a). Rpl29 is not a core component of the ribosome translational machinery and plays a regulatory role in translation efficiency. Our results indicate that Rpl29 methylation has no effect on global protein synthesis but affects Rpl29 subcellular localization. Using an Rpl29 methylation-specific antibody, we demonstrate that Rpl29K5 methylation is present ubiquitously and validate that ()-PFI-2, a Set7/9 inhibitor, efficiently reduces Rpl29K5 methylation in cell lines. Thus, Rpl29 methylation can serve as a specific cellular biomarker for measuring Set7/9 activity.
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http://dx.doi.org/10.1074/jbc.RA118.002890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102145PMC
August 2018

The Arginine Methyltransferase PRMT6 Regulates DNA Methylation and Contributes to Global DNA Hypomethylation in Cancer.

Cell Rep 2017 Dec;21(12):3390-3397

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA; Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA; Program in Genetics and Epigenetics, The University of Texas MD Anderson Cancer Center, UTHealth Graduate School of Biomedical Sciences, Houston, TX 77030, USA. Electronic address:

DNA methylation plays crucial roles in chromatin structure and gene expression. Aberrant DNA methylation patterns, including global hypomethylation and regional hypermethylation, are associated with cancer and implicated in oncogenic events. How DNA methylation is regulated in developmental and cellular processes and dysregulated in cancer is poorly understood. Here, we show that PRMT6, a protein arginine methyltransferase responsible for asymmetric dimethylation of histone H3 arginine 2 (H3R2me2a), negatively regulates DNA methylation and that PRMT6 upregulation contributes to global DNA hypomethylation in cancer. Mechanistically, PRMT6 overexpression impairs chromatin association of UHRF1, an accessory factor of DNMT1, resulting in passive DNA demethylation. The effect is likely due to elevated H3R2me2a, which inhibits the interaction between UHRF1 and histone H3. Our work identifies a mechanistic link between protein arginine methylation and DNA methylation, which is disrupted in cancer.
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http://dx.doi.org/10.1016/j.celrep.2017.11.082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753604PMC
December 2017

Zscan4 Inhibits Maintenance DNA Methylation to Facilitate Telomere Elongation in Mouse Embryonic Stem Cells.

Cell Rep 2017 Aug;20(8):1936-1949

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA; Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA; The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX 77030, USA. Electronic address:

Proper telomere length is essential for embryonic stem cell (ESC) self-renewal and pluripotency. Mouse ESCs (mESCs) sporadically convert to a transient totipotent state similar to that of two-cell (2C) embryos to recover shortened telomeres. Zscan4, which exhibits a burst of expression in 2C-like mESCs, is required for telomere extension in these cells. However, the mechanism by which Zscan4 extends telomeres remains elusive. Here, we show that Zscan4 facilitates telomere elongation by inducing global DNA demethylation through downregulation of Uhrf1 and Dnmt1, major components of the maintenance DNA methylation machinery. Mechanistically, Zscan4 recruits Uhrf1 and Dnmt1 and promotes their degradation, which depends on the E3 ubiquitin ligase activity of Uhrf1. Blocking DNA demethylation prevents telomere elongation associated with Zscan4 expression, suggesting that DNA demethylation mediates the effect of Zscan4. Our results define a molecular pathway that contributes to the maintenance of telomere length homeostasis in mESCs.
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http://dx.doi.org/10.1016/j.celrep.2017.07.070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595351PMC
August 2017

PRMT5 C-terminal Phosphorylation Modulates a 14-3-3/PDZ Interaction Switch.

J Biol Chem 2017 02 28;292(6):2255-2265. Epub 2016 Dec 28.

From the Department of Epigenetics and Molecular Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Smithville, Texas 78957,

PRMT5 is the primary enzyme responsible for the deposition of the symmetric dimethylarginine in mammalian cells. In an effort to understand how PRMT5 is regulated, we identified a threonine phosphorylation site within a C-terminal tail motif, which is targeted by the Akt/serum- and glucocorticoid-inducible kinases. While investigating the function of this posttranslational modification, we serendipitously discovered that its free C-terminal tail binds PDZ domains (when unphosphorylated) and 14-3-3 proteins (when phosphorylated). In essence, a phosphorylation event within the last few residues of the C-terminal tail generates a posttranslational modification-dependent PDZ/14-3-3 interaction "switch." The C-terminal motif of PRMT5 is required for plasma membrane association, and loss of this switching capacity is not compatible with life. This signaling phenomenon was recently reported for the HPV E6 oncoprotein but has not yet been observed for mammalian proteins. To investigate the prevalence of PDZ/14-3-3 switching in signal transduction, we built a protein domain microarray that harbors PDZ domains and 14-3-3 proteins. We have used this microarray to interrogate the C-terminal tails of a small group of candidate proteins and identified ERBB4, PGHS2, and IRK1 (as well as E6 and PRMT5) as conforming to this signaling mode, suggesting that PDZ/14-3-3 switching may be a broad biological paradigm.
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http://dx.doi.org/10.1074/jbc.M116.760330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5313098PMC
February 2017

An approach for interlaboratory comparison of conventional and real-time PCR assays for diagnosis of human leishmaniasis.

Exp Parasitol 2013 Jul 3;134(3):281-9. Epub 2013 Apr 3.

WHO Collaborating Center for Leishmaniasis, Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo, Km2, 28220 Madrid, Spain.

Protozoa of the Leishmania genus are transmitted to humans by the bite of infected sandflies, and are the causative agents of leishmaniasis which ranges from cutaneous to visceral clinical forms. The definitive diagnosis of leishmaniasis has relied traditionally on parasite demonstration, either by microscopy or culture; in the last years, diagnosis based on PCR methods has overcome some drawbacks of traditional methods, increasing sensitivity and allowing using less invasive sampling for diagnosis. However, there are not defined protocols and almost each laboratory applies its own in-house method. Although there are several studies comparing the performance of different methods within the same laboratory, those addressing interlaboratory comparison are scarce, in spite of the growing number of collaborative projects between partners from different leishmaniasis endemic and non-endemic countries. In this work we propose a protocol for interlaboratory comparison of conventional and real-time PCR methods involving four participant laboratories from four different endemic regions in four continents; the protocol includes a quality control step and reduces the variability among the samples tested by each participant. A panel of 77 samples from human origin and 9 from different parasite strains was blindly tested by the participants, aiming to assess the sensitivity of the different methods as well as their usefulness for species identification. Real-time PCR methods targeting the kDNA minicircles returned the highest sensitivity, while both PCR targeting ITS-1 and further HaeIII digestion and a combined algorithm including hsp70 PCR and restriction fragment length polymorphism analysis were the most appropriate approaches for species identification.
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http://dx.doi.org/10.1016/j.exppara.2013.03.026DOI Listing
July 2013

Simultaneous infection with Leishmania (Viannia) braziliensis and L. (V.) lainsoni in a Peruvian patient with cutaneous leishmaniasis.

Am J Trop Med Hyg 2013 Apr 4;88(4):774-7. Epub 2013 Feb 4.

Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru.

Conventional understanding suggests that simultaneous infection with more than one species of Leishmania is unlikely. In Peru, co-infections are clinically relevant because causative species dictates prognosis, treatment response, and follow-up. We describe a case of Leishmania (Viannia) braziliensis and L. (V.) lainsoni co-infection in a Peruvian patient with cutaneous leishmaniasis.
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http://dx.doi.org/10.4269/ajtmh.12-0594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3617868PMC
April 2013

Non-invasive cytology brush PCR for the diagnosis and causative species identification of American cutaneous leishmaniasis in Peru.

PLoS One 2012 21;7(11):e49738. Epub 2012 Nov 21.

Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia (UPCH), Lima, Peru.

Background: Traditional methods of detecting Leishmania from cutaneous lesions involve invasive diagnostic procedures, such as scrapings, which cause discomfort, require technical expertise, and carry risks of invasive procedures. We compared the performance of 2 novel, molecular-based non-invasive methods for the diagnosis of cutaneous leishmaniasis (CL).

Methods: Consecutive patients presenting to the Leishmania Clinic at the Hospital Nacional Cayetano Heredia were enrolled. PCR was performed on filter paper lesion impressions (FPLIs), cytology brushes, and lancets for detection of Leishmania DNA. Smears from lesion scrapings and leishmanin skin test were also performed. Outcome measures were sensitivity and specificity. Composite reference standard was any 2 of 5 tests positive. Species identification was performed by PCR assays of positive specimens.

Results: Ninety patients with 129 lesions were enrolled, 117 of which fulfilled reference criteria for a diagnosis of CL. Of these 117 lesions, 113 were positive by PCR of lancets used for lesion scrapings versus 116 by PCR of FPLIs (p=0.930) or 116 by PCR of cytology brushes (p=0.930). Sensitivity and specificity of PCR on lancets were 96.6% [95% CI 93.3-99.9%] and 100%, respectively. Sensitivity and specificity of FPLI PCR were 99.1% [95% CI 97.4-100%] and 100%, respectively. Sensitivity and specificity of cytology brush PCR were 99.1% [95% CI 97.4-100%] and 100%, respectively. Giemsa-stained lesion smear and leishmanin skin test had inferior sensitivities at 47.9% [95% CI 38.9-57.0%] and 82.3% [95% CI 73.9-90.7%], respectively, compared to PCR of invasive or non-invasive specimens (p<0.001).

Conclusions: Cytology brush PCR constitutes a sensitive and specific alternative to traditional diagnostic assays performed on invasive specimens such as lesion scrapings. It performs comparatively to non-invasive FPLI PCR. This novel, rapid, and well-tolerated method has the potential for widespread use in the field and in pediatric populations where traditional specimen collection is difficult.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0049738PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3504088PMC
May 2013

Accurate and rapid species typing from cutaneous and mucocutaneous leishmaniasis lesions of the New World.

Diagn Microbiol Infect Dis 2012 Oct 21;74(2):142-50. Epub 2012 Jul 21.

Institute of Tropical Medicine Pedro Kourí, 13600 Havana, Cuba.

The heat-shock protein 70 gene (hsp70) has been exploited for Leishmania species identification in the Old and New World, using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis. Three new Leishmania-specific hsp70 PCRs were recently described, and we applied 2 of these on 89 clinical samples from a total of 73 Peruvian patients with either cutaneous or mucocutaneous leishmaniasis. The new PCRs on average showed a 2- to 3-fold improved sensitivity in the tested sample types (lesion biopsies, aspirates, and scrapings), for both genus detection and species typing, and were most successful in biopsies. Leishmania braziliensis, L. peruviana, and L. guyanensis were encountered. About one third of the L. braziliensis parasites contained 2 hsp70 alleles. This study is a paradigm for the implementation of a globally applicable upgraded tool for the identification of Leishmania directly on human specimens from cutaneous and mucocutaneous lesions in the New World.
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http://dx.doi.org/10.1016/j.diagmicrobio.2012.06.010DOI Listing
October 2012

Evolution of the Leishmania braziliensis species complex from amplified fragment length polymorphisms, and clinical implications.

Infect Genet Evol 2012 Dec 10;12(8):1994-2002. Epub 2012 Apr 10.

Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

In order to get more insight into its evolution and geographical distribution, we investigated the Leishmania (Viannia) braziliensis species complex using amplified fragment length polymorphisms and sequencing of a heat-shock protein 70 gene fragment. Previously, several assays had alluded to the high genetic diversity of the group, and single-locus assays typically identified two species, i.e. L. braziliensis and Leishmania peruviana, with occasional genetic signatures of both in the same strain. By analysis of 53 parasite isolates from Peru, and eight additional ones from other countries, we identified an atypical L. braziliensis cluster, and confirmed the origin of L. peruviana from the L. braziliensis cluster during the colonization of the western Andean coastal valleys. We discuss the clinical and taxonomical implications of our findings in relation to currently used species typing assays.
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http://dx.doi.org/10.1016/j.meegid.2012.03.028DOI Listing
December 2012

Leishmania (Viannia) species identification on clinical samples from cutaneous leishmaniasis patients in Peru: assessment of a molecular stepwise approach.

J Clin Microbiol 2012 Feb 23;50(2):495-8. Epub 2011 Nov 23.

Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru.

We present an algorithm based on three PCR assays for Leishmania (Viannia) species identification and assessed its performance using 70 specimens from Peruvian patients. The succession of the assayed targets can be ordered according to species prevalence. Sequential progression through the algorithm reduced the number of samples here studied by approximately 30% after each step.
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http://dx.doi.org/10.1128/JCM.05061-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3264178PMC
February 2012

Non-invasive cytology brush PCR diagnostic testing in mucosal leishmaniasis: superior performance to conventional biopsy with histopathology.

PLoS One 2011 27;6(10):e26395. Epub 2011 Oct 27.

Tropical Disease Unit, Division of Infectious Diseases, Toronto General Hospital, University of Toronto, Toronto, Canada.

Background: Traditional methods of diagnosing mucosal leishmaniasis (ML), such as biopsy with histopathology, are insensitive and require collection of an invasive diagnostic specimen.

Methods: We compared standard invasive procedures including biopsy histopathology, biopsy PCR, and leishmanin skin test (LST) to a novel, non-invasive, cytology-brush based PCR for the diagnosis of ML in Lima, Peru. Consensus reference standard was 2/4 tests positive, and outcome measures were sensitivity and specificity. Leishmania species identification was performed by PCR-based assays of positive specimens.

Results: Twenty-eight patients were enrolled, 23 of whom fulfilled criteria for a diagnosis of ML. Sensitivity and specificity of biopsy with histopathology were 21.7% [95% CI 4.9-38.5%] and 100%; 69.6% [95% CI 50.8-88.4%] and 100% for LST; 95.7% [95% CI 87.4-100%] and 100% for biopsy PCR; and 95.7% [95% CI 87.4-100%] and 90% [95% CI 71.4-100%] for cytology brush PCR using both Cervisoft® and Histobrush® cervical cytology brushes. Represented species identified by PCR-RFLP included: L. (V). braziliensis (n = 4), and L. (V). peruviana (n = 3).

Conclusions: Use of commercial grade cytology brush PCR for diagnosis of ML is sensitive, rapid, well tolerated, and carries none of the risks of invasive diagnostic procedures such as biopsy. Further optimization is required for adequate species identification. Further evaluation of this method in field and other settings is warranted.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026395PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3203107PMC
March 2012

Polymerase chain reaction detection of Leishmania kDNA from the urine of Peruvian patients with cutaneous and mucocutaneous leishmaniasis.

Am J Trop Med Hyg 2011 Apr;84(4):556-61

Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru.

We hypothesized that Leishmania kDNA may be present in urine of patients with cutaneous leishmaniasis (CL). Urine samples and standard diagnostic specimens were collected from patients with skin lesions. kDNA polymerase chain reaction (PCR) was performed on samples from patients and 10 healthy volunteers from non-endemic areas. Eighty-six of 108 patients were diagnosed with CL and 18 (21%) had detectable Leishmania Viannia kDNA in the urine. Sensitivity and specificity were 20.9% (95% confidence interval [CI] 12.3-29.5%) and 100%. Six of 8 patients with mucocutaneous involvement had detectable kDNA in urine versus 12 of 78 patients with isolated cutaneous disease (P < 0.001). L. (V.) braziliensis (N = 3), L. (V.) guyanensis (N = 6), and L. (V.) peruviana (N = 3) were identified from urine. No healthy volunteer or patient with an alternate diagnosis had detectable kDNA in urine. Sensitivity of urine PCR is sub-optimal for diagnosis. On the basis of these preliminary data in a small number of patients, detectable kDNA in urine may identify less localized forms of infection and inform treatment decisions.
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http://dx.doi.org/10.4269/ajtmh.2011.10-0556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3062448PMC
April 2011

Diagnostic performance of filter paper lesion impression PCR for secondarily infected ulcers and nonulcerative lesions caused by cutaneous leishmaniasis.

J Clin Microbiol 2011 Mar 22;49(3):1097-100. Epub 2010 Dec 22.

Tropical Disease Unit, Toronto General Hospital, 200 Elizabeth Street, 13th Floor, North Wing, Room 1350, Toronto, ON M5G 1C4, Canada.

We compared traditional cutaneous leishmaniasis diagnostic methods to filter paper lesion impression (FPLI) PCR for secondarily infected ulcers and nonulcerative lesions. The sensitivity and specificity of FPLI PCR for secondarily infected lesions (n = 8) were 100%. In primarily nonulcerative lesions (n = 15), the sensitivity of FPLI PCR was inferior to that of pooled-invasive-specimen PCR (72.7% versus 100%) (P = 0.10). FPLI PCR is sensitive, specific, and unlike invasive procedures, can be used in secondarily infected ulcers. Invasive specimen collection is superior in nonulcerative lesions.
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http://dx.doi.org/10.1128/JCM.02457-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067679PMC
March 2011

Clinical and demographic stratification of test performance: a pooled analysis of five laboratory diagnostic methods for American cutaneous leishmaniasis.

Am J Trop Med Hyg 2010 Aug;83(2):345-50

Division of Infectious Diseases, University Health Network-Toronto General Hospital, Toronto, Ontario, Canada.

We evaluated performance characteristics of five diagnostic methods for cutaneous leishmaniasis. Patients who came to the Leishmania Clinic of Hospital Nacional Cayetano Heredia in Lima, Peru, were enrolled in the study. Lesion smears, culture, microculture, polymerase chain reaction (PCR), and leishmanin skin test (LST) were performed. A total of 145 patients with 202 lesions were enrolled: 114 patients with 161 lesions fulfilled criteria for cutaneous leishmaniasis. Sensitivity and specificity were 57.8% (95% confidence interval [CI] = 50.2-65.4%) and 100.0% for culture, 78.3% (95% CI = 71.9-84.7%) and 100.0% for microculture, 71.4% (95% CI = 64.4-78.4%) and 100.0% for smears, 78.2% (95% CI = 70.6-85.8%) and 77.4% (95% CI = 62.7-92.1%) for LST, and 96.9% (95% CI = 94.2-99.6%) and 65.9% (95% CI = 51.4-80.4%) for PCR. PCR was more sensitive than the other assays (P < 0.001). Sensitivities of culture, smears, and LST varied by lesion duration and appearance. PCR offers performance advantages over other assays, irrespective of patient age, sex, lesion duration, or appearance. That clinical factors influence performance of non-molecular assays offers clinicians a patient-focused approach to diagnostic test selection.
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http://dx.doi.org/10.4269/ajtmh.2010.09-0414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911183PMC
August 2010

Detection and species identification of Leishmania DNA from filter paper lesion impressions for patients with American cutaneous leishmaniasis.

Clin Infect Dis 2010 Jan;50(1):e1-6

Tropical Disease Unit, Division of Infectious Diseases, Toronto General Hospital, Toronto, Canada.

Background: Traditional detection of Leishmania from ulcers involves collection of invasive specimens that cause discomfort, require technical expertise, and carry risks of invasive procedures. We compared traditional diagnostic methods with a molecular noninvasive filter paper-based method for the diagnosis of cutaneous leishmaniasis.

Methods: Consecutive patients presenting to the Leishmania Clinic at Hospital Nacional Cayetano Heredia were enrolled. Polymerase chain reaction (PCR) was performed on lesion scrapings, aspirates, and filter paper impressions. The reference standard was any 2 of 5 tests positive: smear, aspirate culture, invasive-specimen PCR (scrapings and aspirates), filter paper PCR, and leishmanin skin test. Outcome measures were sensitivity and specificity. Leishmania speciation was performed by PCR-restriction fragment length polymorphism (RFLP) of positive specimens.

Results: Forty-five patients with 66 lesions were enrolled. Of 52 lesions diagnosed as cutaneous leishmaniasis, 50 were positive by PCR of invasive specimens versus 48 by PCR of filter papers (P=.930). Sensitivity and specificity of PCR on invasively obtained specimens were 94.2% (95% confidence interval [CI], 87.9%-100%) and 92.9% (95% CI, 79.4%-100%). Sensitivity and specificity of filter paper PCR were 92.3% (95% CI, 85.1%-99.5%) and 100%. Culture, smear, and leishmanin skin test all had inferior sensitivities, compared with PCR of invasive or noninvasive specimens (P<.001). Of 50 specimens positive by PCR, 19 had sufficient DNA for PCR-RFLP analysis.

Conclusions: Filter paper PCR constitutes a sensitive and specific alternative to traditional diagnostic assays. This novel, rapid, well-tolerated method has the potential for widespread use in the field and in pediatric populations where traditional specimen collection is most difficult to perform, and can potentially be used for rapid species identification.
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http://dx.doi.org/10.1086/648730DOI Listing
January 2010

Isolation and molecular identification of Leishmania (Viannia) peruviana from naturally infected Lutzomyia peruensis (Diptera: Psychodidae) in the Peruvian Andes.

Mem Inst Oswaldo Cruz 2007 Aug;102(5):655-8

Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Perú.

Leishmania (Viannia) peruviana was isolated from 1/75 Lutzomyia peruensis captured during May 2006 in an endemic cutaneous leishmaniasis region of the Peruvian Andes (Chaute, Huarochiri, Lima, Peru). Sand fly gut with promastigotes was inoculated into a hamster and the remaining body was fixed in ethanol. L. (Viannia) sp. was determined by polymerase chain reaction (PCR), and Leishmania species through molecular genotyping by PCR-restriction fragment length polymorphism analyses targeting the genes cpb and hsp70, resulting L. (V.) peruviana. The infected sand fly appeared 15 days after the rains finished, time expected and useful real time data for interventions when transmission is occurring.
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http://dx.doi.org/10.1590/s0074-02762007005000077DOI Listing
August 2007