Laurence Lachaud

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.

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.

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.

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.

Novel insights into genome plasticity in Eukaryotes: mosaic aneuploidy in Leishmania.

Leishmania are unicellular eukaryotes that have many markedly original molecular features compared with other uni‐ or multicellular eukaryotes like yeasts or mammals. Genome plasticity in this parasite has been the subject of many publications, and has been associated with drug resistance or adaptability. Aneuploidy has been suspected by several authors and it is now confirmed using state‐of‐the‐art technologies such as high‐throughput DNA sequencing. The analysis of genome contents at the single cell level using fluorescence in situ hybridization (FISH) has brought a new light on the genome organization: within a cell population, every chromosome, in every cell, may be present in at least two ploidy states (being either monosomic, disomic or trisomic), and the chromosomal content varies greatly from cell to cell, thus generating a constitutive intra‐strain genomic heterogeneity, here termed ‘mosaic aneuploidy’. Mosaic aneuploidy deeply affects the genetics of these organisms, leading, for example, to an extreme degree of intra‐strain genomic diversity, as well as to a clearance of heterozygous cells in the population without however affecting genetic heterogeneity. Second, mosaic aneuploidy might be considered as a powerful strategy evolved by the parasite for adapting to modifications of environment conditions as well as for the emergence of drug resistance. On the whole, mosaic aneuploidy may be considered as a novel mechanism for generating phenotypic diversity driven by genomic plasticity.

Long-term monitoring of visceral leishmaniasis in patients with AIDS: Relapse risk factors value of polymerase chain reaction and potential impact on secondary prophylaxis.

Background:Molecular methods have become essential in the diagnosis of visceral leishmaniasis (VL) in patients who have AIDS. The present study aimed at (1) identifying relapse risk factors for VL and (2) assessing the value of long-range routine polymerase chain reaction (PCR) monitoring in such patients, (3) with a view to proposing decision-making elements for discontinuing specific secondary prophylaxis. Methods:A cohort of 27 HIV-positive patients was prospectively followed up during a period of 5 months to 9 years (median = 51 months) after a first episode of VL. The clinical and biologic follow-up protocol included routine Leishmania detection using peripheral blood and a previously validated PCR method. Quantitative and qualitative variables were statistically analyzed. Results:Sixteen patients relapsed, for a total of 38 relapses. CD4 counts <100 cells/μL and absence of highly active antiretroviral therapy at primary diagnosis and CD4 counts <100 cells/μL during follow-up were the major predictive factors for relapse. No relapse occurred when CD4 counts were >200 cells/μL. The Leishmania PCR assay was positive in all clinical relapses, and its negative predictive value was 100%. Conclusions:The PCR assay used here proved extremely useful for routine follow-up of VL in patients who had AIDS. Considering CD4 cell counts and Leishmania PCR assays, these results allow defining proposals for discontinuing secondary prophylaxis, and thus optimizing the clinical care of VL in these patients.

Colonization with the enteric protozoa Blastocystis is associated with increased diversity of human gut bacterial microbiota

Alterations in the composition of commensal bacterial populations, a phenomenon known as dysbiosis, are linked to multiple gastrointestinal disorders, such as inflammatory bowel disease and irritable bowel syndrome, or to infections by diverse enteric pathogens. Blastocystis is one of the most common single-celled eukaryotes detected in human faecal samples. However, the clinical significance of this widespread colonization remains unclear, and its pathogenic potential is controversial. To address the issue of Blastocystis pathogenicity, we investigated the impact of colonization by this protist on the composition of the human gut microbiota. For that purpose, we conducted a cross-sectional study including 48 Blastocystis-colonized patients and 48 Blastocystis-free subjects and performed an Ion Torrent 16S rDNA gene sequencing to decipher the Blastocystis-associated gut microbiota. Here, we report a higher bacterial diversity in faecal microbiota of Blastocystis colonized patients, a higher abundance of Clostridia as well as a lower abundance of Enterobacteriaceae. Our results contribute to suggesting that Blastocystis colonization is usually associated with a healthy gut microbiota, rather than with gut dysbiosis generally observed in metabolic or infectious inflammatory diseases of the lower gastrointestinal tract.

Traitement des leishmanioses en France : proposition d’un référentiel consensuel

Because it relies on potentially toxic, difficult-to-handle, or expensive compounds the therapy of leishmaniasis is still a complex issue in 2010, especially for visceral leishmaniasis in immuno-suppressed subjects, or in patients with cutaneous and mucosal involvement. This induces a wide diversity of observed therapeutic practices, some being sub-optimal. The Société de Pathologie Exotique organised a meeting dedicated to the therapy of leishmaniasis in France that led to the first consensus on therapeutic guidelines. Liposomal amphotericin B is the first-line option for visceral leishmaniasis both in immunocompetent, and immunosuppressed patients (cumulated doses of 20 mg/kg and 30-40 mg/kg, respectively). Secondary prophylaxis with either liposomal amphotericin B, pentamidine or meglumine antimoniate is proposed to patients with heavy immunosuppression until immunity has been restored for at least 6 months. While the efficacy of new topical formulations of paromomycin is being tested, patients with Old World cutaneous leishmaniasis may be left untreated, or be administered a combination of superficial cryotherapy plus intralesional antimony, or even--in complex situations--receive systemic therapy. The efficacy of a short course of pentamidine (L. guyanensis/L. panamensis) and a 20-day schedule of meglumine antimoniate (L. braziliensis) is solidly established. However, in well-defined situations, local therapy of New World cutaneous leishmaniasis is now considered acceptable.

Antifungal Susceptibility of 205 Candida spp. Isolated Primarily during Invasive Candidiasis and Comparison of the Vitek 2 System with the CLSI Broth Microdilution and Etest Methods

ABSTRACT Infections due to Candida spp. are frequent, particularly in immunocompromised and intensive care unit patients. Antifungal susceptibility tests are now required to optimize antifungal treatment given the emergence of acquired antifungal resistance in some Candida species. An antifungal susceptibility automated method, the Vitek 2 system (VK2), was evaluated. VK2 was compared to the CLSI broth microdilution reference method and the Etest procedure. For this purpose, 205 clinical isolates of Candida spp., including 11 different species, were tested for fluconazole, voriconazole, and amphotericin B susceptibility. For azoles, essential agreement ranged from 25% to 100%, depending on the method used and the Candida species tested. Categorical agreements for all of the species averaged 92.2% and ranged from 14.3 to 100%, depending on the 24-h or 48-h MIC reading by the Etest and CLSI methods and on the Candida species. Results obtained for Candida albicans showed excellent categorical and essential agreements with the two comparative methods. For Candida glabrata, the essential agreement was high with the CLSI method but low with the Etest method, and several very major errors in interpretation were observed between VK2 and the Etest method for both azoles. Low MICs of fluconazole were obtained for all of the Candida krusei isolates, but the VK2 expert software corrected all of the results obtained to resistant. Amphotericin B results showed MICs of ≤1 mg/liter for 201 (VK2), 190 (CLSI), and 202 (Etest) isolates. The AST-YS01 Vitek 2 card system (bioMérieux) is a reliable and practical standardized automated antifungal susceptibility test. Nevertheless, more assays are needed to better evaluate C. glabrata fluconazole sensitivity.

Traitement des leishmanioses en France : proposition d’un référentiel consensuel [Therapy of leishmaniasis in France: consensus on proposed guidelines].

Because it relies on potentially toxic, difficult-to-handle, or expensive compounds the therapy of leishmaniasis is still a complex issue in 2010, especially for visceral leishmaniasis in immuno-suppressed subjects, or in patients with cutaneous and mucosal involvement. This induces a wide diversity of observed therapeutic practices, some being sub-optimal. The Société de Pathologie Exotique organised a meeting dedicated to the therapy of leishmaniasis in France that led to the first consensus on therapeutic guidelines. Liposomal amphotericin B is the first-line option for visceral leishmaniasis both in immunocompetent, and immunosuppressed patients (cumulated doses of 20 mg/kg and 30-40 mg/kg, respectively). Secondary prophylaxis with either liposomal amphotericin B, pentamidine or meglumine antimoniate is proposed to patients with heavy immunosuppression until immunity has been restored for at least 6 months. While the efficacy of new topical formulations of paromomycin is being tested, patients with Old World cutaneous leishmaniasis may be left untreated, or be administered a combination of superficial cryotherapy plus intralesional antimony, or even--in complex situations--receive systemic therapy. The efficacy of a short course of pentamidine (L. guyanensis/L. panamensis) and a 20-day schedule of meglumine antimoniate (L. braziliensis) is solidly established. However, in well-defined situations, local therapy of New World cutaneous leishmaniasis is now considered acceptable.