Publications by authors named "Patrick Bastien"

71 Publications

Impact of pre-analytic step duration on molecular diagnosis of toxoplasmosis for five types of biological samples.

PLoS One 2021 17;16(2):e0246802. Epub 2021 Feb 17.

Centre National de Référence Toxoplasmose-Pôle Biologie Moléculaire, France.

Introduction: Toxoplasma-PCR is essential to diagnose ocular, cerebral, disseminated and congenital toxoplasmosis. This multicenter study evaluated the impact of sample storage duration at +4°C on PCR assay performances in order to propose guidelines for the storage of samples during shipment or/and before PCR.

Materials And Methods: Five matrices, amniotic (AF), cerebrospinal (CSF), and bronchoalveolar lavage fluids (BALF), whole blood (WB) and buffy coat (BC), were artificially spiked with different amounts of Toxoplasma gondii (20, 100, 500 tachyzoites per mL of sample) or with previously infected THP1 cells. DNA extractions were performed at day 0 and after 2, 4 and 7 days of storage at +4°C. Each extract was amplified at least twice by real-time PCR.

Results: A total of 252 spiked samples was studied. No increase of crossing point was observed and all samples were positive for AF, BALF, BC and infected THP1-spiked WB after up to 7 days at 4°C. For CSF spiked with 20 parasites/mL, only 50% of PCR reactions were positive at D7 (p<0.05). For WB spiked with type II parasites, all reactions remained positive at D7 but amplifications were significantly delayed from D2; and for WB spiked with RH strain, the proportion of positive reactions decreased at D7.

Conclusion: The storage of clinical samples at +4°C is compatible with the molecular detection of T. gondii parasites. Provided that PCR assays are performed in duplicate, storage of samples is possible up to 7 days. However, from the fifth day onwards, and for samples susceptible to contain low parasitic loads, we recommend to perform the PCR in multiplicate.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0246802PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888589PMC
February 2021

Gene Editing in Trypanosomatids: Tips and Tricks in the CRISPR-Cas9 Era.

Trends Parasitol 2020 09 20;36(9):745-760. Epub 2020 Jul 20.

MiVEGEC, Univ. Montpellier, CNRS, IRD, CHU, Montpellier, France. Electronic address:

Gene editing in trypanosomatids has long been proven difficult. The development of CRISPR-Cas9 has improved this issue, opening the way to a better understanding of biological processes and drug-resistance mechanisms, and screening of drug targets. Different strategies have now been developed: either PCR- or plasmid-based, differing mainly in the nature of the donor DNA and the single guide RNA transcription. Here we review the main genetic tools available for Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei for gene tagging, single-base editing, and deletion of nonessential and essential genes. We discuss the main advantages and challenges of different strategies and how to choose 'the right cut' depending on the importance of untranslated regions. These considerations allow selection of the most accurate gene editing approach for a given functional analysis.
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http://dx.doi.org/10.1016/j.pt.2020.06.005DOI Listing
September 2020

Rapid diagnostic tests failing to detect infections by Plasmodium falciparum encoding pfhrp2 and pfhrp3 genes in a non-endemic setting.

Malar J 2020 May 11;19(1):179. Epub 2020 May 11.

University of Montpellier, CNRS, IRD, UMR MiVEGEC, Montpellier, France.

Background: Rapid diagnostic tests (RDTs) detecting the histidine-rich protein 2 (PfHRP2) have a central position for the management of Plasmodium falciparum infections. Yet, variable detection of certain targeted motifs, low parasitaemia, but also deletion of pfhrp2 gene or its homologue pfhrp3, may result in false-negative RDT leading to misdiagnosis and delayed treatment. This study aimed at investigating the prevalence, and understanding the possible causes, of P. falciparum RDT-negative infections at Montpellier Academic Hospital, France.

Methods: The prevalence of falsely-negative RDT results reported before and after the introduction of a loop-mediated isothermal amplification (LAMP) assay, as part as the malaria screening strategy in January 2017, was analysed. Negative P. falciparum RDT infections were screened for pfhrp2 or pfhrp3 deletion; and exons 2 were sequenced to show a putative genetic diversity impairing PfHRP2 detection.

Results: The overall prevalence of P. falciparum negative RDTs from January 2006 to December 2018 was low (3/446). Whereas no cases were reported from 2006 to 2016 (0/373), period during which the malaria diagnostic screen was based on microscopy and RDT, prevalence increased up to 4.1% (3/73) between 2017 and 2018, when molecular detection was implemented for primary screening. Neither pfhrp2/3 deletion nor major variation in the frequency of repetitive epitopes could explain these false-negative RDT results.

Conclusion: This paper demonstrates the presence of pfhrp2 and pfhrp3 genes in three P. falciparum RDT-negative infections and reviews the possible reasons for non-detection of HRP2/3 antigens in a non-endemic setting. It highlights the emergence of falsely negative rapid diagnostic tests in a non-endemic setting and draws attention on the risk of missing malaria cases with low parasitaemia infections using the RDT plus microscopy-based strategy currently recommended by French authorities. The relevance of a novel diagnostic scheme based upon a LAMP assay is discussed.
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http://dx.doi.org/10.1186/s12936-020-03251-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216663PMC
May 2020

Evaluation of six commercial kits for the serological diagnosis of Mediterranean visceral leishmaniasis.

PLoS Negl Trop Dis 2020 03 25;14(3):e0008139. Epub 2020 Mar 25.

Département de Parasitologie-Mycologie, Université de Montpellier, Centre Hospitalier Universitaire de Montpellier, Centre National de Référence des Leishmanioses, MIVEGEC, Montpellier, France.

Background: Zoonotic visceral leishmaniasis (VL) is endemic in the Mediterranean basin. However, large-scale comparative analyses of the commercial kits for the serological diagnosis of this neglected disease are lacking. This study compared the performances of four enzyme-linked immunosorbent assays (ELISA) and two immunochromatographic tests (ICT) as screening tests for the serodiagnosis of human VL in the Mediterranean region.

Methodology/principal Findings: Serum samples from 319 patients living in France, Tunisia or Morocco were tested using two ICT (IT LEISH and TruQuick LEISH IgG/IgM Meridian) and four ELISA reagents (NovaLisa Leishmania infantum IgG, Bordier Leishmania infantum, Ridascreen Leishmania IgG, and Vircell Leishmania). The population with proven VL (n = 181) included 65 immunocompromised patients. Significantly higher percentages of false-negative results were obtained with all assays in immunocompromised patients, compared with the immunocompetent population. In the whole population, sensitivity and specificity ranged from 80.7% to 93.9% and from 95.7% to 100%, respectively. The maximum accuracy was observed with the Bordier and Vircell ELISA kits (96.2%), and the lowest accuracy with Ridascreen reagent (88.7%). New thresholds of positivity are proposed for the Bordier, Vircell and NovaLisa ELISA kits to achieve 95% sensitivity with the highest possible specificity. Western blot (WB), used as a confirmation method, showed 100% sensitivity and identified 10.1% of asymptomatic carriers among the control population from the South of France.

Conclusions/significance: This is the first study that compared commercially available kits for VL serodiagnosis in the endemic region of the Mediterranean basin. It provides specific information about the tests' performance to help clinicians and biologists to select the right assay for VL screening.
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http://dx.doi.org/10.1371/journal.pntd.0008139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7135331PMC
March 2020

Universal highly efficient conditional knockout system in Leishmania, with a focus on untranscribed region preservation.

Cell Microbiol 2020 05 27;22(5):e13159. Epub 2020 Jan 27.

MiVEGEC, University of Montpellier, CNRS, IRD, CHU, Montpellier, France.

Trypanosomatids are divergent eukaryotes of high medical and economical relevance. Their biology exhibits original features that remain poorly understood; particularly, Leishmania is known for its high degree of genomic plasticity that makes genomic manipulation challenging. CRISPR-Cas9 has been applied successfully to these parasites providing a robust tool to study non-essential gene functions. Here, we have developed a versatile inducible system combining Di-Cre recombinase and CRISPR-Cas9 advantages. Cas9 is used to integrate the LoxP sequences, and the Cre-recombinase catalyses the recombination between LoxP sites, thereby excising the target gene. We used a Leishmania mexicana cell line expressing Di-Cre, Cas9, and T7 polymerase and then transfected donor DNAs and single guide RNAs as polymerase chain reaction (PCR) products. Because the location of LoxP sequences in the genomic DNA can interfere with the function and localisation of certain proteins of interest, we proposed to target the least transcribed regions upstream and/or downstream the gene of interest. To do so, we developed "universal" template plasmids for donor DNA cassettes with or without a tag, where LoxP sequences may be located either immediately upstream the ATG and downstream the stop codon of the gene of interest, or in the least transcribed areas of intergenic regions. Our methodology is fast, PCR-based (molecular cloning-free), highly efficient, versatile, and able to overcome the problems posed by genomic plasticity in Leishmania.
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http://dx.doi.org/10.1111/cmi.13159DOI Listing
May 2020

Evolutionary Divergence of Enzymatic Mechanisms for Tubulin Detyrosination.

Cell Rep 2019 12;29(12):4159-4171.e6

Tubulin Code Team, Institute of Human Genetics (IGH), CNRS-Université Montpellier, 141 rue de la Cardonille, 34293 Montpellier Cedex 5, France. Electronic address:

The two related members of the vasohibin family, VASH1 and VASH2, encode human tubulin detyrosinases. Here we demonstrate that, in contrast to VASH1, which requires binding of small vasohibin binding protein (SVBP), VASH2 has autonomous tubulin detyrosinating activity. Moreover, we demonstrate that SVBP acts as a bona fide activator of both enzymes. Phylogenetic analysis of the vasohibin family revealed that regulatory diversification of VASH-mediated tubulin detyrosination coincided with early vertebrate evolution. Thus, as a model organism for functional analysis, we used Trypanosoma brucei (Tb), an evolutionarily early-branched eukaryote that possesses a single VASH and encodes a terminal tyrosine on both α- and β-tubulin tails, both subject to removal. Remarkably, although detyrosination levels are high in the flagellum, TbVASH knockout parasites did not present any noticeable flagellar abnormalities. In contrast, we observed reduced proliferation associated with profound morphological and mitotic defects, underscoring the importance of tubulin detyrosination in cell division.
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http://dx.doi.org/10.1016/j.celrep.2019.11.074DOI Listing
December 2019

Inhibition of polymerase chain reaction: Pathogen-specific controls are better than human gene amplification.

PLoS One 2019 27;14(9):e0219276. Epub 2019 Sep 27.

Univ. Montpellier, Centre Hospitalier Universitaire (CHU) of Montpellier, Dept. of Parasitology-Mycology, Research Unit MiVEGEC, CNRS, IRD, Montpellier, France.

PCR inhibition is frequent in medical microbiology routine practice and may lead to false-negative results; however there is no consensus on how to detect it. Pathogen-specific and human gene amplifications are widely used to detect PCR inhibition. We aimed at comparing the value of PCR inhibitor detection using these two methods. We analysed Cp shifts (ΔCp) obtained from qPCRs targeting either the albumin gene or the pathogen-specific sequence used in two laboratory-developed microbiological qPCR assays. 3152 samples including various matrixes were included. Pathogen-specific amplification and albumin qPCR identified 62/3152 samples (2.0%), and 409/3152 (13.0%) samples, respectively, as inhibited. Only 16 samples were detected using both methods. In addition, the use of the Youden's index failed to determine adequate Cp thresholds for albumin qPCR, even when we distinguished among the different sample matrixes. qPCR targeting the albumin gene therefore appears not adequate to identify the presence of PCR inhibitors in microbiological PCR assays. Our data may be extrapolated to other heterologous targets and should discourage their use to assess the presence of PCR inhibition in microbiological PCR assays.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0219276PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6764677PMC
March 2020

The HslV Protease from and Its Activation by C-terminal HslU Peptides.

Int J Mol Sci 2019 Feb 26;20(5). Epub 2019 Feb 26.

Centre de Recherche en Biologie cellulaire de Montpellier (CRBM), CNRS, Université de Montpellier, 34090 Montpellier, France.

HslVU is an ATP-dependent proteolytic complex present in certain bacteria and in the mitochondrion of some primordial eukaryotes, including deadly parasites such as . It is formed by the dodecameric protease HslV and the hexameric ATPase HslU, which binds via the C-terminal end of its subunits to HslV and activates it by a yet unclear allosteric mechanism. We undertook the characterization of HslV from (LmHslV), a trypanosomatid that expresses two isoforms for HslU, LmHslU1 and LmHslU2. Using a novel and sensitive peptide substrate, we found that LmHslV can be activated by peptides derived from the C-termini of both LmHslU1 and LmHslU2. Truncations, Ala- and D-scans of the C-terminal dodecapeptide of LmHslU2 (LmC12-U2) showed that five out of the six C-terminal residues of LmHslU2 are essential for binding to and activating HslV. Peptide cyclisation with a lactam bridge allowed shortening of the peptide without loss of potency. Finally, we found that dodecapeptides derived from HslU of other parasites and bacteria are able to activate LmHslV with similar or even higher efficiency. Importantly, using electron microscopy approaches, we observed that the activation of LmHslV was accompanied by a large conformational remodeling, which represents a yet unidentified layer of control of HslV activation.
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http://dx.doi.org/10.3390/ijms20051021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6429459PMC
February 2019

EAPB0503: An Imiquimod analog with potent in vitro activity against cutaneous leishmaniasis caused by Leishmania major and Leishmania tropica.

PLoS Negl Trop Dis 2018 11 21;12(11):e0006854. Epub 2018 Nov 21.

Department of Pathology and Laboratory Medicine, American University of Beirut, Beirut, Lebanon.

Cutaneous Leishmaniasis (CL) is a parasitic infection classified by the WHO as one of the most uncontrolled spreading neglected diseases. Syria is endemic for Leishmania tropica and Leishmania major, causing CL in the Eastern Mediterranean. The large-scale displacement of Syrian refugees exacerbated the spread of CL into neighboring countries. Therapeutic interventions against CL include local, systemic and physical treatments. The high risk for drug-resistance to current treatments stresses the need for new therapies. Imiquimod is an immunomodulatory drug with a tested efficacy against L. major species. Yet, Imiquimod efficacy against L. tropica and the molecular mechanisms dictating its potency are still underexplored. In this study, we characterized the effect of Imiquimod against L. tropica and L. major, and characterized the molecular mechanisms dictating its anti-leishmanial efficacy against both strains. We also investigated the potency and molecular mechanisms of an Imiquimod analog, EAPB0503, against these two strains. We have tested the effect of Imiquimod and EAPB0503 on macrophages infected with either L. major, L. tropica strains, or patient-derived freshly isolated L. tropica parasites. The anti-amastigote activity of either drugs was assessed by quantitative real time PCR (RT-PCR) using kinetoplast specific primers, confocal microscopy using the Glycoprotein 63 (Gp63) Leishmania amastigote antibody or by histology staining. The mechanism of action of either drugs on the canonical nuclear factor kappa- B (NF-κB) pathway was determined by western blot, and confocal microscopy. The immune production of cytokines upon treatment of infected macrophages with either drugs was assessed by ELISA. Both drugs reduced amastigote replication. EAPB0503 proved more potent, particularly on the wild type L. tropica amastigotes. Toll-Like Receptor-7 was upregulated, mainly by Imiquimod, and to a lesser extent by EAPB0503. Both drugs activated the NF-κB canonical pathway triggering an immune response and i-NOS upregulation in infected macrophages. Our findings establish Imiquimod as a strong candidate for treating L. tropica and show the higher potency of its analog EAPB0503 against CL.
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http://dx.doi.org/10.1371/journal.pntd.0006854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6248897PMC
November 2018

Analysis of genetic polymorphisms and tropism in East African Leishmania donovani by Amplified Fragment Length Polymorphism and kDNA minicircle sequencing.

Infect Genet Evol 2018 11 19;65:80-90. Epub 2018 Jul 19.

Department of Microbiology and Molecular Genetics, Kuvin Centre for the Study of Infectious and Tropical Diseases, IMRIC, Hebrew University - Hadassah Medical School, Jerusalem, Israel. Electronic address:

Visceral leishmaniasis (VL), the most severe form of leishmaniasis, is caused by Leishmania donovani. In addition to fatal VL, these parasites also cause skin diseases in immune-competent and -suppressed people, post-kala azar dermal leishmaniasis (PKDL) and HIV/VL co-infections, respectively. Genetic polymorphism in 36 Ethiopian and Sudanese L. donovani strains from VL, PKDL and HIV/VL patients was examined using Amplified Fragment Length Polymorphism (AFLP), kDNA minicircle sequencing and Southern blotting. Strains were isolated from different patient tissues: in VL from lymph node, spleen or bone marrow; and in HIV/VL from skin, spleen or bone marrow. When VL and PKDL strains from the same region in Sudan were examined by Southern blotting using a DNA probe to the L. donovani 28S rRNA gene only minor differences were observed. kDNA sequence analysis distributed the strains in no particular order among four clusters (A - D), while AFLP analysis grouped the strains according to geographical origin into two major clades, Southern Ethiopia (SE) and Sudan/Northern Ethiopia (SD/NE). Strains in the latter clade were further divided into subpopulations by zymodeme, geography and year of isolation, but not by clinical symptoms. However, skin isolates showed significantly (p < 0.0001) fewer polymorphic AFLP fragments (average 10 strains = 348.6 ± 8.1) than VL strains (average 26 strains = 383.5 ± 3.8).
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http://dx.doi.org/10.1016/j.meegid.2018.07.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218636PMC
November 2018

Evolution of Toxoplasma-PCR methods and practices: a French national survey and proposal for technical guidelines.

Int J Parasitol 2018 08 8;48(9-10):701-707. Epub 2018 Jun 8.

Centre Hospitalier Universitaire (CHU) of Montpellier, University of Montpellier, Department of Parasitology-Mycology, Research Unit "MiVEGEC" (CNRS 5290/IRD 224/UM), Montpellier, France; Molecular Biology 'Pole' of the National Reference Center for Toxoplasmosis, Montpellier, France. Electronic address:

The molecular diagnosis of toxoplasmosis lacks standardisation due to the use of numerous methods with variable performance. This diversity of methods also impairs robust performance comparisons between laboratories. The harmonisation of practices by diffusion of technical guidelines is a useful way to improve these performances. The knowledge of methods and practices used for this molecular diagnosis is an essential step to provide guidelines for Toxoplasma-PCR. In the present study, we aimed (i) to describe the methods and practices of Toxoplasma-PCR used by clinical microbiology laboratories in France and (ii) to propose technical guidelines to improve molecular diagnosis of toxoplasmosis. To do so, a yearly self-administered questionnaire-based survey was undertaken in proficient French laboratories from 2008 to 2015, and guidelines were proposed based on the results of those as well as previously published work. This period saw the progressive abandonment of conventional PCR methods, of Toxoplasma-PCR targeting the B1 gene and of the use of two concomitant molecular methods for this diagnosis. The diversity of practices persisted during the study, in spite of the increasing use of commercial kits such as PCR kits, DNA extraction controls and PCR inhibition controls. We also observed a tendency towards the automation of DNA extraction. The evolution of practices did not always go together with an improvement in those, as reported notably by the declining use of Uracil-DNA Glycosylase to avoid carry-over contamination. We here propose technical recommendations which correspond to items explored during the survey, with respect to DNA extraction, Toxoplasma-PCR and good PCR practices.
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http://dx.doi.org/10.1016/j.ijpara.2018.03.011DOI Listing
August 2018

A new LAMP-based assay for the molecular diagnosis of toxoplasmosis: comparison with a proficient PCR assay.

Int J Parasitol 2018 05 22;48(6):457-462. Epub 2018 Feb 22.

CHU (University Hospital Centre) of Montpellier/University of Montpellier, Research Unit "MIVEGEC", CNRS, IRD, Faculty of Medicine, Laboratoire de Parasitologie-Mycologie, Montpellier, France; Pôle "Biologie Moléculaire" du Centre National de Référence de la Toxoplasmose, France. Electronic address:

Toxoplasmosis is generally a benign infection caused by the protozoan parasite Toxoplasma gondii but can have severe consequences in fetuses of mothers infected during pregnancy (congenital toxoplasmosis) and immunocompromised individuals. PCR-based diagnostic tests have become crucial for its diagnosis. However, this molecular diagnosis essentially relies upon laboratory-developed methods and suffers from a lack of standardization, leading to great variation in methods and performance among laboratories. With the need for accreditation of clinical microbiological laboratories, the use of commercial PCR kits has become an attractive alternative; but thorough evaluation of newly commercialized kits by proficient groups is necessary before any recommendation can be made to parasitology laboratories by health authorities or learned societies. Here, we compared the performance of an original commercial method, the Iam TOXO Q-LAMP (DiaSorin®), using Loop-mediated isothermal amplification (LAMP) technology, with our reference laboratory-developed method using real-time PCR. The kit was first tested using amniotic fluid (AF) and plasma samples (either negative or spiked with live T. gondii tachyzoites at different concentrations (from 7 to 10 tachyzoites/mL)). It was then assessed using a cohort of 11 AF, five placental and 32 blood clinical samples preserved at -20 °C. For the processing of placental/blood samples, a pretreatment step was used, which did not strictly follow the manufacturer's recommendations. The practical ease of use and compliance with good laboratory practices were also evaluated. Although the LAMP assay was less sensitive than the laboratory-developed method at very low parasite concentrations (0.1 T. gondii genome equivalents/mL), the two methods yielded identical results qualitatively and, in some instances, quantitatively, particularly for AF samples.
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http://dx.doi.org/10.1016/j.ijpara.2017.11.005DOI Listing
May 2018

Identification of the centromeres of : revealing the hidden pieces.

EMBO Rep 2017 11 21;18(11):1968-1977. Epub 2017 Sep 21.

Department of Parasitology-Mycology, Faculty of Medicine, University of Montpellier, Montpellier, France

affects millions of people worldwide. Its genome undergoes constitutive mosaic aneuploidy, a type of genomic plasticity that may serve as an adaptive strategy to survive distinct host environments. We previously found high rates of asymmetric chromosome allotments during mitosis that lead to the generation of such ploidy. However, the underlying molecular events remain elusive. Centromeres and kinetochores most likely play a key role in this process, yet their identification has failed using classical methods. Our analysis of the unconventional kinetochore complex recently discovered in (KKTs) leads to the identification of a KKT gene candidate (LmKKT1). The GFP-tagged LmKKT1 displays "kinetochore-like" dynamics of intranuclear localization throughout the cell cycle. By ChIP-Seq assay, one major peak per chromosome is revealed, covering a region of 4 ±2 kb. We find two largely conserved motifs mapping to 14 of 36 chromosomes while a higher density of retroposons are observed in 27 of 36 centromeres. The identification of centromeres and of a kinetochore component of chromosomes opens avenues to explore their role in mosaic aneuploidy.
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http://dx.doi.org/10.15252/embr.201744216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666652PMC
November 2017

Identification of Leishmania by Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry Using a Free Web-Based Application and a Dedicated Mass-Spectral Library.

J Clin Microbiol 2017 10 19;55(10):2924-2933. Epub 2017 Jul 19.

Laboratoire de Parasitologie-Mycologie, CHU Timone, Université d'Aix-Marseille, Marseille, France

Human leishmaniases are widespread diseases with different clinical forms caused by about 20 species within the genus. species identification is relevant for therapeutic management and prognosis, especially for cutaneous and mucocutaneous forms. Several methods are available to identify species from culture, but they have not been standardized for the majority of the currently described species, with the exception of multilocus enzyme electrophoresis. Moreover, these techniques are expensive, time-consuming, and not available in all laboratories. Within the last decade, mass spectrometry (MS) has been adapted for the identification of microorganisms, including However, no commercial reference mass-spectral database is available. In this study, a reference mass-spectral library (MSL) for isolates, accessible through a free Web-based application (mass-spectral identification [MSI]), was constructed and tested. It includes mass-spectral data for 33 different species, including species that infect humans, animals, and phlebotomine vectors. Four laboratories on two continents evaluated the performance of MSI using 268 samples, 231 of which were strains. All strains, but one, were correctly identified at least to the complex level. A risk of species misidentification within the , , and complexes was observed, as previously reported for other techniques. The tested application was reliable, with identification results being comparable to those obtained with reference methods but with a more favorable cost-efficiency ratio. This free online identification system relies on a scalable database and can be implemented directly in users' computers.
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http://dx.doi.org/10.1128/JCM.00845-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5625378PMC
October 2017

Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis.

J Clin Microbiol 2017 05 15;55(5):1369-1376. Epub 2017 Feb 15.

Département de Parasitologie-Mycologie, Centre Hospitalier Universitaire de Montpellier, and UMR CNRS 5290-IRD 224, Université de Montpellier, Montpellier, France.

Molecular diagnosis of congenital toxoplasmosis or disseminated toxoplasmosis is based mainly on PCR. The repeated DNA element has become the main DNA target used in most PCR methods, whether laboratory developed or commercial. In this multicenter study, we evaluated the ELITe MGB (Elitech) commercial kit by comparison with three reference quantitative PCR assays (RAs) used routinely in three proficient laboratories of the French National Reference Center for Toxoplasmosis network, using calibrated suspensions diluted to obtain a range of concentrations from 0.1 to 10,000 parasites/ml. These suspensions were extracted with either the DNA extraction kit (EXTRAblood; Elitech) recommended by the manufacturer or the QIAamp DNA minikit (Qiagen). The ELITe MGB assay was also evaluated on a panel of 128 clinical samples, including 56 amniotic fluid samples, 55 placenta samples, and various other samples, of which 95 originated from patients with proven toxoplasmosis. The ELITe MGB assay amplified low-concentration replicates (<10 parasites/ml) of calibrated suspensions less frequently than the RAs of 2/3 laboratories. Additionally, the combination of EXTRAblood and ELITe MGB yielded poorer sensitivity than the combination of QIAamp DNA minikit and ELITe MGB for low parasite concentrations ( < 0.001 for 1 parasite/ml). On clinical samples, the sensitivity and the specificity of the commercial assay were 89% and 100%, respectively. The sensitivity ranged from 79% (placenta samples) to 100% (amniotic fluid samples). Overall, this study shows that the ELITe MGB assay is suitable for the diagnosis of toxoplasmosis from non-cell-rich or non-hemoglobin-rich samples and that the EXTRAblood kit is not optimal.
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http://dx.doi.org/10.1128/JCM.02379-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5405254PMC
May 2017

Prevalence, risk factors for infection and subtype distribution of the intestinal parasite Blastocystis sp. from a large-scale multi-center study in France.

BMC Infect Dis 2016 Aug 26;16(1):451. Epub 2016 Aug 26.

Université de Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Centre d'Infection et d'Immunité de Lille, 1 rue du Professeur Calmette, BP 245, 59019, Lille cedex, France.

Background: Blastocystis sp. is the most common intestinal parasite of humans. Despite its potential public health impact, epidemiological data regarding the prevalence and molecular subtype distribution of Blastocystis sp. in Europe are rarely reported. Therefore, the first multi-center epidemiological survey performed in Europe was conducted in France to diagnose and subtype Blastocystis sp. and to identify risk factors for infection.

Methods: Stool samples from 788 patients were collected either in summer or winter in 11 hospitals throughout France together with patient data. All stool samples were tested for the presence of Blastocystis sp. by quantitative PCR targeting the SSU rDNA gene. Positive samples were sequenced to determine the distribution of the subtypes in our cohort. Statistical analyses were performed to identify potential risk factors for infection.

Results: Using quantitative PCR, the overall prevalence of Blastocystis sp. was shown to reach 18.1 %. The prevalence was significantly higher in summer (23.2 %) than in winter (13.7 %). Travellers or subjects infected with other enteric parasites were significantly more infected by Blastocystis sp. than non-travellers or subjects free of other enteric parasites, respectively. Different age-related epidemiological patterns were also highlighted from our data. The prevalence of Blastocystis sp. was not significantly higher in patients with digestive symptoms or diagnosed with chronic bowel diseases. Among symptomatic patients, Blastocystis sp. infection was significantly associated with abdominal pain. Gender, socioeconomic status, and immune status were not identified as potential risk factors associated with infection. Among a total of 141 subtyped isolates, subtype 3 was predominant (43.3 %), followed by subtype 1 and subtype 4 (20 %), subtype 2 (12.8 %), subtype 6 and subtype 7 (2.1 %). No association between ST and clinical symptoms was statistically evidenced.

Conclusions: A high prevalence of Blastocystis sp. infection was found in our French patient population. Seasonal impact on the prevalence of Blastocystis sp. was highlighted and recent travels and age were identified as main risk factors for infection. Most cases were caused by subtypes 1 to 4, with a predominance of subtype 3. Large variations in both prevalence and ST distribution between hospitals were also observed, suggesting distinct reservoirs and transmission sources of the parasite.
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http://dx.doi.org/10.1186/s12879-016-1776-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5002209PMC
August 2016

The Montpellier Leishmania Collection, from a Laboratory Collection to a Biological Resource Center: A 39-Year-Long Story.

Biopreserv Biobank 2016 Dec 5;14(6):470-479. Epub 2016 Jul 5.

Laboratory of Parasitology-Mycology, Faculty of Medicine, University of Montpellier-National Reference Centre for Leishmaniases-Unit MIVEGEC (CNRS 5290/IRD 224/University of Montpellier)-Academic Hospital Center (C.H.U.) of Montpellier , Montpellier, France .

We report the development of a laboratory collection of Leishmania that was initiated in 1975 and, after 39 years, has become an international Biological Resource Center (BRC-Leish, Montpellier, France, BioBank No. BB-0033-00052), which includes 6353 strains belonging to 36 Leishmania taxa. This is a retrospective analysis of the technical and organizational changes that have been adopted over time to take into account the technological advances and related modifications in the collection management and quality system. The technical improvements concerned the culture and cryopreservation techniques, strain identification by isoenzymatic and molecular techniques, data computerization and quality management to meet the changes in international standards, and in the cryogenic and microbiological safety procedures. The BRC is working toward obtaining the NF-S 96-900 certification in the coming years. Our long-term expertise in Leishmania storage and typing and collection maintenance should encourage field epidemiologists and clinical practitioners in endemic countries to secure their own strain collection with the help of the French BRC-Leish.
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http://dx.doi.org/10.1089/bio.2015.0101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5180084PMC
December 2016

Real-time PCR using FRET technology for Old World cutaneous leishmaniasis species differentiation.

Parasit Vectors 2016 May 3;9:255. Epub 2016 May 3.

National Reference Centre for Parasitology, Research Institute of the McGill University Health Centre, Montreal, QC, Canada.

Background: Recently, there has been a re-emergence of cutaneous leishmaniasis in endemic countries and an increase in imported cases in non-endemic countries by travelers, workers, expatriates, immigrants, and military force personnel. Old World cutaneous leishmaniasis is caused primarily by Leishmania major, L. tropica and L. aethiopica. Despite their low sensitivity, diagnosis traditionally includes microscopic and histopathological examinations, and in vitro cultivation. Several conventional PCR techniques have been developed for species identification, which are time-consuming and labour-intensive. Real-time PCR using SYBR green dye, although provides rapid detection, may generate false positive signals. Therefore, a rapid and easy method such as a FRET-based real-time PCR would improve not only the turn-around time of diagnosing Old World cutaneous Leishmania species but will also increase its specificity and sensitivity.

Methods: A FRET-based real-time PCR assay which amplifies the cathepsin L-like cysteine protease B gene encoding a major Leishmania antigen was developed to differentiate L. major, L. tropica, and L. aethiopica in one single step using one set of primers and probes. Assay performance was tested on cutaneous and visceral strains of Leishmania parasite cultures and isolates of other protozoan parasites as well as human biopsy specimen.

Results: The assay readily differentiates between the three Old World cutaneous leishmaniasis species based on their melting curve characteristics. A single Tm at 55.2 ± 0.5 °C for L. aethiopica strains was distinguished from a single Tm at 57.4 ± 0.2 °C for L. major strains. A double curve with melting peaks at 66.6 ± 0.1 °C and 48.1 ± 0.5 °C or 55.8 ± 0.6 °C was observed for all L. tropica strains. The assay was further tested on biopsy specimens, which showed 100% agreement with results obtained from isoenzyme electrophoresis and Sanger sequencing.

Conclusion: Currently, there are no published data on real-time PCR using FRET technology to differentiate between Old World cutaneous Leishmania species. In summary, our assay based on specific hybridization addresses the limitations of previous PCR technology and provides a single step, reliable method of species identification and rapid diagnostic applications.
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http://dx.doi.org/10.1186/s13071-016-1531-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4855858PMC
May 2016

TbFlabarin, a flagellar protein of Trypanosoma brucei, highlights differences between Leishmania and Trypanosoma flagellar-targeting signals.

Exp Parasitol 2016 Jul 6;166:97-107. Epub 2016 Apr 6.

Centre National de la Recherche Scientifique, Laboratoire de Parasitologie-Mycologie, UMR 5290, 39 Avenue Charles Flahault, F-34295 Montpellier Cedex 5, France; Université de Montpellier, 163 rue Auguste Broussonnet, F-34090 Montpellier, France; Centre Hospitalier Universitaire La Colombière, 39 Avenue Charles Flahault, F-34295 Montpellier Cedex 5, France; MIVEGEC, Unité 224, Institut de Recherches pour le Développement, 911 avenue Agropolis, BP64501, F-34394 Montpellier Cedex 5, Montpellier, France. Electronic address:

TbFlabarin is the Trypanosoma brucei orthologue of the Leishmania flagellar protein LdFlabarin but its sequence is 33% shorter than LdFlabarin, as it lacks a C-terminal domain that is indispensable for LdFlabarin to localize to the Leishmania flagellum. TbFlabarin is mainly expressed in the procyclic forms of the parasite and localized to the flagellum, but only when two palmitoylable cysteines at positions 3 and 4 are present. TbFlabarin is more strongly attached to the membrane fraction than its Leishmania counterpart, as it resists complete solubilization with as much as 0.5% NP-40. Expression ablation by RNA interference did not change parasite growth in culture, its morphology or apparent motility. Heterologous expression showed that neither TbFlabarin in L. amazonensis nor LdFlabarin in T. brucei localized to the flagellum, revealing non-cross-reacting targeting signals between the two species.
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http://dx.doi.org/10.1016/j.exppara.2016.04.004DOI Listing
July 2016

Leishmaniasis in Turkey: first clinical isolation of Leishmania major from 18 autochthonous cases of cutaneous leishmaniasis in four geographical regions.

Trop Med Int Health 2016 06 28;21(6):783-91. Epub 2016 Apr 28.

Department of Parasitology, Faculty of Medicine, Ege University, İzmir, Turkey.

Objective: To report isolation of Leishmania major strains obtained from 18 Turkish autochthonous cutaneous leishmaniasis (CL) patients infected with L. major between 2011 and 2014.

Methods: Initial diagnosis relied on microscopy and culture in enriched medium, prepared by adding specific amounts of liver extract, protein and lipid sources to NNN medium. Promastigotes were then transferred to RPMI medium including 10% of foetal calf serum for mass culture. Species-specific real-time PCR targeting ITS1 region of Leishmania spp. was performed using both lesion aspiration samples and cultured promastigotes. Two of 18 isolates were identified by isoenzyme analysis in the Leishmaniasis Reference Center in Montpellier, France. Each isolate was inoculated into the footpads of six mice to observe the pathogenicity of L. major. Developing lesions were observed, and the thickening of footpads was measured weekly.

Results: Melting curve analyses of 18 isolates showed a peak concordant with L. major, and two of them were confirmed by isoenzyme analyses as L. major zymodeme MON103. In the mouse model, acute lesions seen on day 21 were accepted as an indication of heavy infection. Severe impairments were observed on all mouse footpads over 3 weeks, which even progressed to extremity amputation.

Conclusion: Cutaneous leishmaniasis-causing L. major was recently identified in Adana province in southern Turkey, with PCR. Our study shows that such CL cases are not limited to Adana but currently present from western to Southeastern Anatolia, and along the Mediterranean coast. The role of small mammals, the main reservoirs of L. major in Anatolia, needs to be elucidated, as do the underlying factors that cause severe clinical manifestations in L. major infections in Turkey, contrary to the infections in neighbouring countries.
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http://dx.doi.org/10.1111/tmi.12698DOI Listing
June 2016

Single-molecule analysis of DNA replication reveals novel features in the divergent eukaryotes Leishmania and Trypanosoma brucei versus mammalian cells.

Sci Rep 2016 Mar 15;6:23142. Epub 2016 Mar 15.

University of Montpellier, Faculty of Medicine, Laboratory of Parasitology-Mycology, Montpellier, F34090, France.

Leishmania and Trypanosoma are unicellular parasites that possess markedly original biological features as compared to other eukaryotes. The Leishmania genome displays a constitutive 'mosaic aneuploidy', whereas in Trypanosoma brucei, the megabase-sized chromosomes are diploid. We accurately analysed DNA replication parameters in three Leishmania species and Trypanosoma brucei as well as mouse embryonic fibroblasts (MEF). Active replication origins were visualized at the single molecule level using DNA molecular combing. More than one active origin was found on most DNA fibres, showing that the chromosomes are replicated from multiple origins. Inter-origin distances (IODs) were measured and found very large in trypanosomatids: the mean IOD was 160 kb in T. brucei and 226 kb in L. mexicana. Moreover, the progression of replication forks was faster than in any other eukaryote analyzed so far (mean velocity 1.9 kb/min in T. brucei and 2.4-2.6 kb/min in Leishmania). The estimated total number of active DNA replication origins in trypanosomatids is ~170. Finally, 14.4% of unidirectional replication forks were observed in T. brucei, in contrast to 1.5-1.7% in Leishmania and 4% in MEF cells. The biological significance of these original features is discussed.
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http://dx.doi.org/10.1038/srep23142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4791591PMC
March 2016

Comparative genomic analysis of Leishmania (Viannia) peruviana and Leishmania (Viannia) braziliensis.

BMC Genomics 2015 Sep 18;16:715. Epub 2015 Sep 18.

Laboratório de Imunologia e Genômica de Parasitos, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

Background: The Leishmania (Viannia) braziliensis complex is responsible for most cases of New World tegumentary leishmaniasis. This complex includes two closely related species but with different geographic distribution and disease phenotypes, L. (V.) peruviana and L. (V.) braziliensis. However, the genetic basis of these differences is not well understood and the status of L. (V.) peruviana as distinct species has been questioned by some. Here we sequenced the genomes of two L. (V.) peruviana isolates (LEM1537 and PAB-4377) using Illumina high throughput sequencing and performed comparative analyses against the L. (V.) braziliensis M2904 reference genome. Comparisons were focused on the detection of Single Nucleotide Polymorphisms (SNPs), insertions and deletions (INDELs), aneuploidy and gene copy number variations.

Results: We found 94,070 variants shared by both L. (V.) peruviana isolates (144,079 in PAB-4377 and 136,946 in LEM1537) against the L. (V.) braziliensis M2904 reference genome while only 26,853 variants separated both L. (V.) peruviana genomes. Analysis in coding sequences detected 26,750 SNPs and 1,513 indels shared by both L. (V.) peruviana isolates against L. (V.) braziliensis M2904 and revealed two L. (V.) braziliensis pseudogenes that are likely to have coding potential in L. (V.) peruviana. Chromosomal read density and allele frequency profiling showed a heterogeneous pattern of aneuploidy with an overall disomic tendency in both L. (V.) peruviana isolates, in contrast with a trisomic pattern in the L. (V.) braziliensis M2904 reference. Read depth analysis allowed us to detect more than 368 gene expansions and 14 expanded gene arrays in L. (V.) peruviana, and the likely absence of expanded amastin gene arrays.

Conclusions: The greater numbers of interspecific SNP/indel differences between L. (V.) peruviana and L. (V.) braziliensis and the presence of different gene and chromosome copy number variations support the classification of both organisms as closely related but distinct species. The extensive nucleotide polymorphisms and differences in gene and chromosome copy numbers in L. (V.) peruviana suggests the possibility that these may contribute to some of the unique features of its biology, including a lower pathology and lack of mucosal development.
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http://dx.doi.org/10.1186/s12864-015-1928-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4575464PMC
September 2015

Molecular diagnosis of toxoplasmosis: value of the buffy coat for the detection of circulating Toxoplasma gondii.

Diagn Microbiol Infect Dis 2015 Aug 24;82(4):289-91. Epub 2015 Apr 24.

Laboratoire de Parasitologie et Mycologie, CHU de Grenoble et Université Grenoble Alpes, Grenoble 1 BP 217, 38043 Grenoble, France; Pôle "Biologie Moléculaire" du Centre National de Référence de la Toxoplasmose, Montpellier, France.

Early detection of Toxoplasma tachyzoites circulating in blood using PCR is recommended for immunosuppressed patients at high risk for disseminated toxoplasmosis. Using a toxoplasmosis mouse model, we show that the sensitivity of detection is higher using buffy coat isolated from a large blood volume than using whole blood for this molecular monitoring.
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http://dx.doi.org/10.1016/j.diagmicrobio.2015.04.004DOI Listing
August 2015

First efficient CRISPR-Cas9-mediated genome editing in Leishmania parasites.

Cell Microbiol 2015 Oct 19;17(10):1405-12. Epub 2015 Jun 19.

University of Montpellier, Faculty of Medicine, Laboratory of Parasitology-Mycology, Paris, France.

Protozoan pathogens that cause leishmaniasis in humans are relatively refractory to genetic manipulation. In this work, we implemented the CRISPR-Cas9 system in Leishmania parasites and demonstrated its efficient use for genome editing. The Cas9 endonuclease was expressed under the control of the Dihydrofolate Reductase-Thymidylate Synthase (DHFR-TS) promoter and the single guide RNA was produced under the control of the U6snRNA promoter and terminator. As a proof of concept, we chose to knockout a tandemly repeated gene family, the paraflagellar rod-2 locus. We were able to obtain null mutants in a single round of transfection. In addition, we confirmed the absence of off-target editions by whole genome sequencing of two independent clones. Our work demonstrates that CRISPR-Cas9-mediated gene knockout represents a major improvement in comparison with existing methods. Beyond gene knockout, this genome editing tool opens avenues for a multitude of functional studies to speed up research on leishmaniasis.
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http://dx.doi.org/10.1111/cmi.12456DOI Listing
October 2015

RNA interference screen reveals a high proportion of mitochondrial proteins essential for correct cell cycle progress in Trypanosoma brucei.

BMC Genomics 2015 Apr 15;16:297. Epub 2015 Apr 15.

Université Montpellier 1, UFR Médecine, Laboratoire de Parasitologie-Mycologie, CHRU de Montpellier, 39, Avenue Charles Flahault, 34295, Montpellier, Cedex 5, France.

Background: Trypanosomatid parasites possess a single mitochondrion which is classically involved in the energetic metabolism of the cell, but also, in a much more original way, through its single and complex DNA (termed kinetoplast), in the correct progress of cell division. In order to identify proteins potentially involved in the cell cycle, we performed RNAi knockdowns of 101 genes encoding mitochondrial proteins using procyclic cells of Trypanosoma brucei.

Results: A major cell growth reduction was observed in 10 cases and a moderate reduction in 29 other cases. These data are overall in agreement with those previously obtained by a case-by-case approach performed on chromosome 1 genes, and quantitatively with those obtained by "high-throughput phenotyping using parallel sequencing of RNA interference targets" (RIT-seq). Nevertheless, a detailed analysis revealed many qualitative discrepancies with the RIT-seq-based approach. Moreover, for 37 out of 39 mutants for which a moderate or severe growth defect was observed here, we noted abnormalities in the cell cycle progress, leading to increased proportions of abnormal cell cycle stages, such as cells containing more than 2 kinetoplasts (K) and/or more than 2 nuclei (N), and modified proportions of the normal phenotypes (1N1K, 1N2K and 2N2K).

Conclusions: These data, together with the observation of other abnormal phenotypes, show that all the corresponding mitochondrial proteins are involved, directly or indirectly, in the correct progress or, less likely, in the regulation, of the cell cycle in T. brucei. They also show how post-genomics analyses performed on a case-by-case basis may yield discrepancies with global approaches.
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http://dx.doi.org/10.1186/s12864-015-1505-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445814PMC
April 2015

Molecular diagnosis of toxoplasmosis in immunocompromised patients: a 3-year multicenter retrospective study.

J Clin Microbiol 2015 May 11;53(5):1677-84. Epub 2015 Mar 11.

Pôle "Biologie Moléculaire" du Centre National de Référence de la Toxoplasmose, Montpellier, France Centre Hospitalier Universitaire de Montpellier, Département de Parasitologie-Mycologie, UMR CNRS 5290, IRD 224, UM ("MiVEGEC"), Montpellier, France.

Toxoplasmosis is a life-threatening infection in immunocompromised patients (ICPs). The definitive diagnosis relies on parasite DNA detection, but little is known about the incidence and burden of disease in HIV-negative patients. A 3-year retrospective study was conducted in 15 reference laboratories from the network of the French National Reference Center for Toxoplasmosis, in order to record the frequency of Toxoplasma gondii DNA detection in ICPs and to review the molecular methods used for diagnosis and the prevention measures implemented in transplant patients. During the study period, of 31,640 PCRs performed on samples from ICPs, 610 were positive (323 patients). Blood (n = 337 samples), cerebrospinal fluid (n = 101 samples), and aqueous humor (n = 100 samples) were more frequently positive. Chemoprophylaxis schemes in transplant patients differed between centers. PCR follow-up of allogeneic hematopoietic stem cell transplant (allo-HSCT) patients was implemented in 8/15 centers. Data from 180 patients (13 centers) were further analyzed regarding clinical setting and outcome. Only 68/180 (38%) patients were HIV(+); the remaining 62% consisted of 72 HSCT, 14 solid organ transplant, and 26 miscellaneous immunodeficiency patients. Cerebral toxoplasmosis and disseminated toxoplasmosis were most frequently observed in HIV and transplant patients, respectively. Of 72 allo-HSCT patients with a positive PCR result, 23 were asymptomatic; all were diagnosed in centers performing systematic blood PCR follow-up, and they received specific treatment. Overall survival of allo-HSCT patients at 2 months was better in centers with PCR follow-up than in other centers (P < 0.01). This study provides updated data on the frequency of toxoplasmosis in HIV-negative ICPs and suggests that regular PCR follow-up of allo-HSCT patients could guide preemptive treatment and improve outcome.
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http://dx.doi.org/10.1128/JCM.03282-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4400795PMC
May 2015

Comments on Leishmania major in Gorilla Feces.

J Infect Dis 2015 Aug 3;212(3):505-6. Epub 2015 Mar 3.

National Reference Center for Leishmaniases, University Hospital Centre of Montpellier Laboratoire de Parasitologie-Mycologie, Faculty of Medicine.

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http://dx.doi.org/10.1093/infdis/jiv129DOI Listing
August 2015

Contribution of neonatal amniotic fluid testing to diagnosis of congenital toxoplasmosis.

J Clin Microbiol 2015 May 18;53(5):1719-21. Epub 2015 Feb 18.

Laboratoire de Parasitologie-Mycologie, CHU (University Hospital Centre) of Montpellier, UMR MIVEGEC CNRS 5290/IRD 224/University of Montpellier, Montpellier, France Pôle Biologie Moléculaire du Centre National de Référence de la Toxoplasmose, Centre Hospitalier Universitaire of Montpellier, Montpellier, France.

We evaluated the molecular diagnosis of congenital toxoplasmosis (CT) on neonatal amniotic fluid samples from 488 mother-child pairs. Maternal infection during pregnancy was diagnosed and dated or could not be ruled out. Forty-six cases of CT were defined according to the European Research Network on CT classification system and case definitions. Neonatal amniotic fluid testing had an overall sensitivity of 54% (95% confidence interval [95% CI], 39 to 69%) and a specificity of 100% (95% CI, 99 to 100%). Its sensitivity was 33% (95% CI, 13 to 59%) when antenatal diagnosis was positive and 68% (95% CI, 48 to 84%) when antenatal diagnosis was negative or lacking. This difference in sensitivity may have been due to treatment of antenatally diagnosed cases. Relative to postnatal serology, neonatal amniotic fluid testing allowed an earlier diagnosis to be made in 26% of the cases (95% CI, 9 to 51%).
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http://dx.doi.org/10.1128/JCM.02358-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4400781PMC
May 2015

The nucleoporin Mlp2 is involved in chromosomal distribution during mitosis in trypanosomatids.

Nucleic Acids Res 2015 Apr 17;43(8):4013-27. Epub 2015 Feb 17.

Laboratory of Parasitology-Mycology, Faculty of Medicine, University Montpellier 1, Montpellier F34090, France CNRS 5290-IRD 224-University Montpellier 1&2 (UMR 'MiVEGEC'), Montpellier F34090, France.

Nucleoporins are evolutionary conserved proteins mainly involved in the constitution of the nuclear pores and trafficking between the nucleus and cytoplasm, but are also increasingly viewed as main actors in chromatin dynamics and intra-nuclear mitotic events. Here, we determined the cellular localization of the nucleoporin Mlp2 in the 'divergent' eukaryotes Leishmania major and Trypanosoma brucei. In both protozoa, Mlp2 displayed an atypical localization for a nucleoporin, essentially intranuclear, and preferentially in the periphery of the nucleolus during interphase; moreover, it relocated at the mitotic spindle poles during mitosis. In T. brucei, where most centromeres have been identified, TbMlp2 was found adjacent to the centromeric sequences, as well as to a recently described unconventional kinetochore protein, in the periphery of the nucleolus, during interphase and from the end of anaphase onwards. TbMlp2 and the centromeres/kinetochores exhibited a differential migration towards the poles during mitosis. RNAi knockdown of TbMlp2 disrupted the mitotic distribution of chromosomes, leading to a surprisingly well-tolerated aneuploidy. In addition, diploidy was restored in a complementation assay where LmMlp2, the orthologue of TbMlp2 in Leishmania, was expressed in TbMlp2-RNAi-knockdown parasites. Taken together, our results demonstrate that Mlp2 is involved in the distribution of chromosomes during mitosis in trypanosomatids.
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http://dx.doi.org/10.1093/nar/gkv056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4417144PMC
April 2015

Characterisation of polyglutamylases in trypanosomatids.

Int J Parasitol 2015 Feb 13;45(2-3):121-32. Epub 2014 Nov 13.

Centre National de la Recherche Scientifique (CNRS), 5290-IRD 224-University Montpellier 1, Research Unit "MIVEGEC", Montpellier, France; CHU (Hospital University Centre) of Montpellier and University Montpellier 1 (Faculty of Medicine), Laboratoire de Parasitologie-Mycologie, Montpellier, France. Electronic address:

Microtubules are subject to post-translational modifications, which are thought to have crucial roles in the function of complex microtubule-based organelles. Among these, polyglutamylation was relatively recently discovered, and was related to centrosome stability, axonemal maintenance and mobility, and neurite outgrowth. In trypanosomatids, parasitic protozoa where microtubules constitute the essential component of the cytoskeleton, the function of polyglutamylated microtubules is unknown. Here, in order to better understand the role of this conserved but highly divergent post-translational modification, we characterised glutamylation and putative polyglutamylases in these parasites. We showed that microtubules are intensely glutamylated in all stages of the cell cycle, including interphase. Moreover, a cell cycle-dependent gradient of glutamylation was observed along the cell anteroposterior axis, which might be related to active growth of the microtubule 'corset' during the cell cycle. We also identified two putative polyglutamylase proteins (among seven analysed here) which appeared to be clearly and directly involved in microtubule polyglutamylation in in vitro activity assays. Paradoxically, in view of the importance of tubulins and of their extensive glutamylation in these organisms, RNA interference-based knockdown of all these proteins had no effect on cell growth, suggesting either functional redundancy or, more likely, subtle roles such as function modulation or interaction with protein partners.
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http://dx.doi.org/10.1016/j.ijpara.2014.09.005DOI Listing
February 2015