Nathalie Bourgeois

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.

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.

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.

Microtubule-severing proteins are involved in flagellar length control and mitosis in Trypanosomatids.

Microtubules are key players in the biology of Trypanosomatid parasites, not only as classical components of the mitotic spindle, microtubule‐organizing centres and flagellum but also as the essential constituent of the cytoskeleton. Their length dynamics are regulated by, among others, microtubule‐severing proteins. Four and six genes encoding microtubule‐severing proteins can be found bioinformatically in the Leishmania major and Trypanosoma brucei genome respectively. We investigated all these proteins in these organisms, which include the katanin, katanin‐like, spastin and fidgetin, and looked at their subcellular localization as well as their putative function by examining ‘loss‐of‐function’ phenotypes. The katanin‐like KAT60b was found implicated in flagellar length reduction, but not in its size increase, while the katanin p80 subunit appeared clearly involved in cytokinesis. Fidgetin and spastin homologues were both localized in the nucleus: the first as a discrete and variable number of dots during most of the cell cycle, redistributing to the spindle and midbody during mitosis; the second concentrated as ≤ 5 perinucleolar punctuations, similar to the electron‐dense plaques identified in T. brucei, which were assimilated to kinetochores. This first study of microtubule‐severing proteins in ‘divergent’ eukaryotes gives further insight into the multiple functions of these proteins identified in the hitherto studied models.

Antifungal susceptibility of 205 Candida sp. primarily isolated during invasive candidiasis. Comparison of the Vitek2 system with 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.

Constitutive mosaic aneuploidy is a unique genetic feature widespread in the Leishmania genus.

Using fluorescence in situ hybridization, we determined the ploidy of four species of Leishmania: Leishmania infantum, Leishmania donovani, Leishmania tropica and Leishmania amazonensis. We found that each cell in a strain possesses a combination of mono-, di- and trisomies for all chromosomes; ploidy patterns were different among all strains/species. These results extend those we previously described in Leishmania major, demonstrating that mosaic aneuploidy is a genetic feature widespread to the Leishmania genus. In addition to the genetic consequences induced by this mosaicism, the apparent absence of alternation between haploid/diploid stages questions the modality of genetic exchange in Leishmania sp.

Value of 2 IgG avidity commercial tests used alone or in association to date toxoplasmosis contamination

Toxoplasmosis acquired during pregnancy exposes the fetus to congenital toxoplasmosis. Avidity tests are commonly used to date time of infection to evaluate the fetal risk and to offer prenatal diagnosis. This study evaluated and compared 2 commercial avidity tests: Platelia Toxo IgG Avidity (Bio-Rad, Marnes la Coquette, France) and Liaison(R) Toxo IgG Avidity II (Diasorin, Saluggia, Italy) kits. In complement, a study of specific IgG and IgM in the 2 systems was carried out. Sensitivity and specificity of the avidity tests were 94.4% and 87.8% for the Liaison(R) and 91.3% and 98.5% for the Platelia methods, respectively. Percentages of complete discrepancies, partial discrepancies, and agreement between both tests were 1.1%, 23.6%, and 75.3%, respectively. Moreover, the combination of both avidity tests may be useful in some cases. Indeed, that strategy permitted to confirm without delay a chronic toxoplasmosis in 23 cases and avoid treatment in these pregnant women.

Comparison of three real-time PCR methods with blood smears and rapid diagnostic test in Plasmodium sp. infection

In cases of malaria, rapid and accurate diagnosis of Plasmodium sp. is essential. In this study three different quantitative, real-time PCR methods were compared with routine methods used for malaria diagnosis. A comparative study was conducted prospectively in the laboratories of Montpellier and Nîmes University Hospitals. The methods used for routine diagnostic malaria testing consisted of microscopic examination of Giemsa-stained blood smears and rapid diagnostic tests. Three quantitative real-time PCR methods (qRT-PCR) were tested: qRT-PCR1 amplified a specific sequence on the P. falciparum Cox1 gene, qRT-PCR2 amplified a species-specific region of the multicopy 18S rDNA, and qRT-PCR3 amplified a mitochondrial DNA sequence. Among the 196 blood samples collected, 73 samples were positive in at least one of the five tests. Compared with the routine method, there were no false negatives for P. falciparum diagnosis in either qRT-PCR1 or qRT-PCR3. In all P. ovale, P. vivax and P. malariae infections diagnosed from blood smears, qRT-PCR1 was negative, as expected, whereas qRT-PCR2 and qRT-PCR3 were positive and concordant (simple kappa coefficient = 1). One negative sample from microscopy was positive with both qRT-PCR2 and qRT-PCR3. Together, qRT-PCR3 and the combined qRT-PCR1 and qRT-PCR2 were concordant with routine methods for malaria diagnosis (99% and 99.5%, respectively). These three rapid, molecular qRT-PCR methods, used alone or in association, showed excellent results, with high concordance, accuracy and reliability in malaria diagnosis.

A case of Candida haemulonii osteitis: clinical features, biochemical characteristics, and antifungal resistance profile

We describe here the first case of osteitis caused by Candida haemulonii in a young immunocompetent patient. This patient presented a history of severe peripheral vascular disease associated with a lack of hygienic conditions as the only risk factors for such an uncommon infection. Clinical signs and histological examination allowed us to determine that it was a C. haemulonii infection and not colonization. The outcome was favourable with oral voriconazole therapy and surgical revascularization. An environmental cause of such infections is most probable, as C. haemulonii has previously been isolated from different non-human sources. Identification methods, results obtained with three in vitro antifungal susceptibility methods and clinical features are reported.