Patrick Bastien

Comparison of Six PCR Methods Using Peripheral Blood for Detection of Canine Visceral Leishmaniasis

ABSTRACT The objectives of this study were to compare the sensitivities and reliabilities of different PCR methods for the diagnosis and epidemiological study of canine visceral leishmaniasis (CVL) using dog blood. We chose to work with peripheral blood, as this type of sampling is noninvasive, straightforward, and easy to repeat. Six PCR methods were compared: three primer pairs target genomic DNA, and the other three target kinetoplast (mitochondrial) DNA. Sensitivity, specificity, reproducibility, and ease of interpretation without hybridization were evaluated for each method. The assessment was first performed using artificial samples. All methods could detect less than one parasite per reaction tube. However, the sensitivities varied among the different methods by a factor of 500 on purified cultivated parasites and by a factor of 10,000 on seeded dog blood samples (i.e., from 10 to 10−3 parasite per ml of blood for the latter). Only four methods were found sufficiently reliable for the diagnosis of CVL. They were tested on 37 dogs living in an area of endemicity and grouped according to clinical status and specific serology. Only the two methods targeting kinetoplast DNA (K13A-K13B and RV1-RV2) could detect the parasite in 100% of symptomatic infected dogs. Similarly, all seropositive dogs were found PCR positive by these methods versus 62% by the genomic-DNA-based methods. Finally, these kinetoplast-based methods proved clearly superior to the others in the detection of Leishmania in asymptomatic dogs. Our data allow the discussion of the advantages and drawbacks of highly sensitive versus moderately sensitive PCR methods in diagnosis and prevalence studies of CVL.

Quantitative real-time PCR is not more sensitive than "conventional" PCR.

Molecular methods, essentially based upon PCR, have become an indispensable tool in the diagnosis of infectious diseases. Real-time quantitative PCR (qrtPCR), as a novel technology, has revolutionized molecular diagnostics by adding reliability and speed ([15][1], [28][2]). However, Apfalter et al

Comparison of Various Sample Preparation Methods for PCR Diagnosis of Visceral Leishmaniasis Using Peripheral Blood

ABSTRACT We have compared various sample preparation methods for the PCR diagnosis of visceral leishmaniasis (VL) using peripheral blood samples and tested the influence of these protocols upon sensitivity. Four methods of lysis-DNA extraction were used with two types of blood samples: whole blood (WB) and buffy coat (BC). Comparisons were first carried out with seeded samples at various parasite concentrations. At high concentrations (≥1,000 parasites/ml), there were no significant differences in PCR sensitivity among the methods tested. At concentrations of ≤100 parasites/ml, proteinase K (PK)-based methods proved clearly superior to guanidine-EDTA-based methods. Moreover, a 10-fold increase in sensitivity was observed for BC over that for WB. Thus, the best sensitivity was obtained with the BC prepared with PK-based methods. With this combination, the PCR reliably detected 10 parasites/ml but was inconsistent when the sample contained 1 parasite/ml of blood. The methods that yielded the highest sensitivities were evaluated with seven dogs and four human VL patients. Again, the utilization of the BC prepared with PK-based methods gave the best results. The optimization of each step of the assay (sample preparation, DNA extraction, and PCR conditions) yielded a highly sensitive tool for the diagnosis of VL using patient blood, thus avoiding more invasive diagnostic procedures and allowing the detection of low parasitemia during posttherapeutic follow-up.

Genetic nomenclature for Trypanosoma and Leishmania

Christine Clayton *, Mark Adams , Renata Almeida , Theo Baltz , Mike Barrett , Patrick Bastien , Sabina Belli , Stephen Beverley , Nicolas Biteau , Jenefer Blackwell , Christine Blaineau , Michael Boshart , Frederic Bringaud , George Cross , Angela Cruz , Wim Degrave , John Donelson , Najib El-Sayed , Gioliang Fu , Klaus Ersfeld , Wendy Gibson , Keith Gull , Alasdair Ivens , John Kelly , Daniel Lawson , John Lebowitz , Phelix Majiwa , Keith Matthews , Sara Melville , Gilles Merlin , Paul Michels , Peter Myler , Alan Norrish , Fred Opperdoes , Barbara Papadopoulou , Marilyn Parsons , Thomas Seebeck , Deborah Smith , Kenneth Stuart , Michael Turner , Elisabetta Ullu , Luc Vanhamme aa

Conserved linkage groups associated with large-scale chromosomal rearrangements between Old World and New World Leishmania genomes

The genus Leishmania can be taxonomically separated into three main groups: the Old World subgenus L. (Leishmania), the New World subgenus L. (Leishmania) and the New World subgenus L. (Viannia). The haploid genome of Old World Leishmania species has been shown to contain 36 chromosomes defined as physical linkage groups; the latter were found entirely conserved across species. In the present study, we tried to verify whether this conservation of the genome structure extends to the New World species of Leishmania. 300 loci were explored by hybridization on optimized pulsed field gel electrophoresis separations of the chromosomes of polymorphic strains of the six main pathogenic Leishmania species of the New World. When comparing these New World karyotypes with their Old World counterparts, 32 out of 36 linkage groups were found conserved among all species. Four chromosomal rearrangements were found. All species belonging to the L. (Viannia) subgenus were characterized by the presence (i) of a short sequence exchange between chromosomes 26 and 35, and (ii) more importantly, of a fused version of chromosomes 20 and 34 which are separated in all Old World species. 69 additional markers were isolated from a plasmid library specifically constructed from the rearranged chromosomes 20+34 in an attempt to detect mechanisms other than a fusion or breakage: only two markers out of 40 did not belong to the linkage groups 20 and 34. On the other hand, all strains belonging to the New World subgenus L. (Leishmania) were characterized by two different chromosomal rearrangements of the same type (fusion/breakage) as above as compared with Old World species: chromosomes 8+29 and 20+36. Consequently, these two groups of species have 35 and 34 heterologous chromosomes, respectively. Overall, these results show that large-scale chromosomal rearrangements occurred during the evolution of the genus Leishmania, and that the three main groups of pathogenic species are characterized by different chromosome numbers. Nevertheless, translocations seem particularly rare, and the conservation of the major linkage groups should be an essential feature for the compared genetics between species of this parasite.

Parasite Susceptibility to Amphotericin B in Failures of Treatment for Visceral Leishmaniasis in Patients Coinfected with HIV Type 1 and Leishmania infantum

BACKGROUND Visceral leishmaniasis (VL) is an opportunistic infection that can occur among patients infected with human immunodeficiency virus type 1 (HIV-1) in areas where both infections are endemic. Highly active antiretroviral therapy has decreased the incidence of VL in southern Europe among HIV-1-infected patients, but VL is still observed among patients with low CD4 cell counts, and most coinfected patients receiving highly active antiretroviral therapy experienced relapse, despite initial treatment with liposomal amphotericin B. METHODS Through long-term monitoring of VL in 10 patients with HIV-1 infection and/or AIDS, we compared parasite strains derived from primary and secondary episodes of VL. All the patients have received many courses of amphotericin B treatment and/or prophylaxis. RESULTS Through molecular techniques, we have shown that secondary episodes of VL can be attributable to relapse (7 of 10 episodes) or reinfection (3 of 10). We developed an assay to measure amphotericin B susceptibility and found no evidence of decreased susceptibility among strains isolated from patients, some of whom were infected with the same isolate for up to 10 years. CONCLUSIONS This apparent absence of resistance, as determined by in vitro susceptibility testing, has important consequences and suggests that amphotericin B will remain a useful drug of choice against VL, even after repetitive treatments or prophylactic use.

Global Distribution Maps of the Leishmaniases

The leishmaniases are vector-borne diseases that have a broad global distribution throughout much of the Americas, Africa, and Asia. Despite representing a significant public health burden, our understanding of the global distribution of the leishmaniases remains vague, reliant upon expert opinion and limited to poor spatial resolution. A global assessment of the consensus of evidence for leishmaniasis was performed at a sub-national level by aggregating information from a variety of sources. A database of records of cutaneous and visceral leishmaniasis occurrence was compiled from published literature, online reports, strain archives, and GenBank accessions. These, with a suite of biologically relevant environmental covariates, were used in a boosted regression tree modelling framework to generate global environmental risk maps for the leishmaniases. These high-resolution evidence-based maps can help direct future surveillance activities, identify areas to target for disease control and inform future burden estimation efforts. DOI: http://dx.doi.org/10.7554/eLife.02851.001

Comparison of Two Widely Used PCR Primer Systems for Detection of Toxoplasma in Amniotic Fluid, Blood, and Tissues

ABSTRACT The PCR diagnosis of toxoplasmosis suffers from lack of standardization. Interlaboratory comparative studies of PCR methods have been performed, but intralaboratory comparisons are scarce. Here, we optimized and compared the technical performances of two PCR primer systems widely used for Toxoplasma detection. The differences between the two methods were visible only at low parasite concentrations (≤1 Toxoplasma genome per reaction tube). Nevertheless, when clinical samples were tested, both methods significantly differed in their technical sensitivities and specificities. Only one method appeared adequate for samples containing blood or tissue.

Leishmania: Sex, lies and karyotype

The exploration of the genome of the tryponosomotid protozoan Leishmania has been difficult until recently owing to a number of obstacles, not least our ignorance of the ploidy and of the number of chromosomes (as in many other protozoa, the latter do not condense during mitosis), the uncertainty of the species concept in these allegedly asexual protozoa and the absence of classical genetic studies. Here, Patrick Bastien, Christine Bloineou and Michel Pages discuss the advances in this field brought about by the advent of molecular biology and its techniques, with on emphasis on ploidy and genetic exchange. In particular, they discuss the data from pulsed field gel electrophoresis (PFGE). When coupled with DNA restriction analysis, PFGE constitutes a powerful tool for the direct examination o f chromosomes of protozoa.

FISH analysis reveals aneuploidy and continual generation of chromosomal mosaicism in Leishmania major

The protozoan parasite Leishmania is generally considered to be diploid, although a few chromosomes have been described as aneuploid. Using fluorescence in situ hybridization (FISH), we determined the number of homologous chromosomes per individual cell in L. major (i) during interphase and (ii) during mitosis. We show that, in Leishmania, aneuploidy appears to be the rule, as it affects all the chromosomes that we studied. Moreover, every chromosome was observed in at least two ploidy states, among monosomic, disomic or trisomic, in the cell population. This variable chromosomal ploidy among individual cells generates intra‐strain heterogeneity, here precisely chromosomal mosaicism. We also show that this mosaicism, hence chromosome ploidy distribution, is variable among clones and strains. Finally, when we examined dividing nuclei, we found a surprisingly high rate of asymmetric chromosome allotments, showing that the transmission of genetic material during mitosis is highly unstable in this ‘divergent’ eukaryote: this leads to continual generation of chromosomal mosaicism. Using these results, we propose a model for the occurrence and persistence of this mosaicism. We discuss the implications of this additional unique feature of Leishmania for its biology and genetics, in particular as a novel genetic mechanism to generate phenotypic variability from genomic plasticity.