Nadine François

Multilocus Sequence Typing Reveals Intrafamilial Transmission and Microevolutions of Candida albicans Isolates from the Human Digestive Tract

ABSTRACT Candida albicans is a human commensal that is also responsible for superficial and systemic infections. Little is known about the carriage of C. albicans in the digestive tract and the genome dynamics that occur during commensalisms of this diploid species. The aim of this study was to evaluate the prevalence, diversity, and genetic relationships among C. albicans isolates recovered during natural colonization of the digestive tract of humans, with emphasis on Crohns disease patients who produce anti-yeast antibodies and may have altered Candida sp. carriage. Candida sp. isolates were recovered from 234 subjects within 25 families with multiple cases of Crohns disease and 10 control families, sampled at the oral and fecal sites. Prevalences of Candida sp. and C. albicans carriage were 53.4% and 46.5%, respectively, indicating frequent commensal carriage. No differences in prevalence of carriage could be observed between Crohns disease patients and healthy subjects. Multilocus sequence typing (MLST) of C. albicans isolates revealed frequent colonization of a subject or several members of the same family by genetically indistinguishable or genetically close isolates. These latter isolates differed by loss-of-heterozygosity events at one or several of the MLST loci. These loss-of-heterozygosity events could be due to either chromosome loss followed by duplication or large mitotic recombination events between complementary chromosomes. This study was the first to jointly assess commensal carriage of C. albicans, intrafamilial transmission, and microevolution. The high frequency of each of these events suggests that the digestive tract provides an important and natural niche for microevolutions of diploid C. albicans through the loss of heterozygosity.

Candida albicans colonization and ASCA in familial Crohn's disease

OBJECTIVES:Anti-Saccharomyces cerevisiae antibodies (ASCAs) are present in 50–60% of patients with Crohns disease (CD) and in 20–25% of their healthy relatives (HRs). The yeast, Candida albicans, has been shown to generate ASCAs, but the presence of C. albicans in the digestive tract of CD patients and their HRs has never been investigated. Therefore, we studied C. albicans carriage in familial CD and its correlation with ASCAs.METHODS:Study groups consisted of 41 CD families composed of 129 patients and 113 HRs, and 14 control families composed of 76 individuals. Mouth swabs and stool specimens were collected for isolation, identification, and quantification of yeasts. Serum samples were collected for detection of ASCAs and anti-C. albicans mannan antibodies (ACMAs).RESULTS:C. albicans was isolated significantly more frequently from stool samples from CD patients (44%) and their HRs (38%) than from controls (22%) (P<0.05). The prevalence of ACMAs was similar between CD patients, their HRs, and controls (22, 19, and 21%, respectively, P=0.845), whereas the prevalence of ASCAs was significantly increased in CD families (72 and 34% in CD and HRs, respectively, in contrast to 4% in controls, P<0.0001). AMCA levels correlated with C. albicans colonization in all populations. ASCA levels correlated with C. albicans colonization in HRs but not in CD patients.CONCLUSIONS:CD patients and their first-degree HRs are more frequently and more heavily colonized by C. albicans than are controls. ASCAs correlate with C. albicans colonization in HRs but not in CD. In HRs, ASCAs could result from an altered immune response to C. albicans. In CD, a subsequent alteration in sensing C. albicans colonization could occur with disease onset.

Synthesis and antifungal activity of a ferrocene-fluconazole analogue.

A novel ferrocene fluconazole analogue was synthesized and its antifungal properties investigated against yeast strains of medical importance, including those intrinsically resistant to fluconazole. In vitro tests revealed a slight increase in fungal growth and a reversal of the effect of fluconazole at minimal inhibitory concentrations.

Pneumocystis carinii f. sp. hominis Is Not Infectious for SCID mice

ABSTRACT The infectious power of Pneumocystis carinii f. sp. hominis was explored by inoculating SCID mice intranasally with either P. carinii f. sp. hominis or P. carinii f. sp. muris isolates. Only mice inoculated with mouse parasites developed Pneumocystis pneumonia, as assessed by microscopy and PCR. These results suggest that humans do not contract pneumocystosis from animals.

Evaluation of MALDI-TOF mass spectrometry for the identification of medically-important yeasts in the clinical laboratories of Dijon and Lille hospitals

Conventional identification (CI) of yeasts is based on morphological, biochemical and/or immunological methods. Matrix-assisted laser desorption/ionization - time of flight (MALDI-TOF or MT-MS) mass spectrometry has been proposed as a new method for the identification of microorganisms. This prospective study compared the performance of MT-MS and CI for the identification of yeasts isolated from clinical samples. Sequencing of the internal transcribed spacer (ITS) regions of ribosomal DNA was used as the reference method in the analysis of a total of 1207 yeast isolates. Concordance between MT-MS and CI was observed for 1105 isolates (91.5%), while 74 isolates (6.1%) were misidentified. Molecular identification revealed that 73 of these 74 isolates were identified correctly by MT-MS and CI correctly identified the last one. Concordance between the two techniques was excellent for the medically-important species (98-100%), including the identification of closely-related species (Candida albicans/C. dubliniensis; C. inconspicua/C. norvegensis; C. parapsilosis/C. metapsilosis/C. orthopsilosis). Only 2.3% of isolates belonging to C. famata, C. lambica and C. magnoliae or to Geotrichum spp. and Trichosporon spp. were not identified by MT-MS. This investigation highlights the potential of MT-MS-based yeast identification as a reliable, time and cost-efficient alternative to CI.

Members 5 and 6 of the Candida albicans BMT family encode enzymes acting specifically on β-mannosylation of the phospholipomannan cell-wall glycosphingolipid

A family of nine genes encoding proteins involved in the synthesis of β-1,2 mannose adhesins of Candida albicans has been identified. Four of these genes, BMT1-4, encode enzymes acting stepwise to add β-mannoses on to cell-wall phosphopeptidomannan (PPM). None of these acts on phospholipomannan (PLM), a glycosphingolipid member of the mannose-inositol-phosphoceramide family, which contributes with PPM to β-mannose surface expression. We show that deletion of BMT5 and BMT6 led to a dramatic reduction of PLM glycosylation and accumulation of PLM with a truncated β-oligomannoside chain, respectively. Disruptions had no effect on sphingolipid biosynthesis and on PPM β-mannosylation. β-Mannose surface expression was not affected, confirming that β-mannosylation is a process based on specificity of acceptor molecules, but liable to global regulation.

Application of Mass Spectrometry Technology to Early Diagnosis of Invasive Fungal Infections

ABSTRACT We recently developed a mass spectrometry (MS) procedure based on the detection of a serum disaccharide (MS-DS) in patients with invasive candidiasis (IC). Here, we compare the performance of MS-DS for the diagnosis of IC, invasive aspergillosis (IA), and mucormycosis (MM) with those of commercially available antigen detection tests. This retrospective study included 48 patients (23 IC patients [74 serum samples], 15 IA patients [40 serum samples], and 10 MM patients [15 serum samples]) and 49 appropriate controls (102 serum samples). MS-DS, mannan (Mnn), galactomannan (GM), and (1,3)-β-d-glucan (BDG) were detected by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS, Platelia, and Fungitell assays, respectively. For IC, the sensitivity and specificity of the MS-DS index, BDG detection, and Mnn detection were 62% and 84%, 82% and 60%, and 33% and 94% per serum sample and 83% and 69%, 96% and 31%, and 39% and 86% per patient, respectively. For IA, the corresponding values in comparison to BDG and GM detection were 83% and 81%, 62% and 95%, and 62% and 100% per serum sample and 93% and 76%, 87% and 90%, and 93% and 100% per patient, respectively. Nine of the 10 MM patients had a positive MS-DS result. MS-DS gave an early diagnosis in IC (73% positivity before blood culture), IA (positive before GM detection in six patients), and MM (positivity mainly preceded the date of diagnosis) patients. For IC, persisting MS-DS was associated with a poor prognosis. The different biomarkers were rarely detected simultaneously, suggesting different kinetics of release and clearance. For IA, MS-DS provided better complementation to GM monitoring than BDG monitoring. MS-DS detects panfungal molecules circulating during invasive fungal infections. The performance of MS-DS compared favorably with those of biological tests currently recommended for monitoring at-risk patients. Further validation of this test in multicenter studies is required.

Variants of NOD1 and NOD2 genes display opposite associations with familial risk of crohn's disease and anti‐saccharomyces cerevisiae antibody levels

Background: NOD2 is involved in Crohns disease (CD), but the role of NOD1 remains unclear. Anti‐Saccharomyces cerevisiae antibodies (ASCA) are higher in CD patients and some of their relatives. Using family‐based analyses we investigated the relationships between NOD2 mutations, NOD1 +32656 variant, and both the risk of CD and ASCA levels. We compared allelic frequencies between families with multiple CD cases (multiplex), those with one case of CD (simplex), and control families, searching for a gradient of at risk alleles according to the prevalence of the disease among families. Methods: In all, 93 CD patients, 160 healthy relatives from 22 multiplex families, 22 CD patients and 81 healthy relatives from 22 simplex families, and 169 subjects from 27 control families were included in the study. ASCA levels were determined by enzyme‐linked immunosorbent assay. NOD1 +32656, NOD2 R702W, G908R, and 1007fs were genotyped by polymerase chain reaction / restriction fragment length polymorphism. Results: In family‐based analyses NOD2 mutations and the NOD1 wildtype allele were associated with CD in multiplex families, with a synergetic effect when risk alleles of both genes were transmitted. Lower ASCA levels were strongly associated with the NOD1 variant allele. Simplex families had a lower frequency of the “at risk” +32656 allele than multiplex families. Conclusions: The +32656 variant was associated with low ASCA level and low risk of CD in multiplex families. NOD2 and NOD1 variants displayed antagonist effects on the risk of CD and ASCA level. A gradient of NOD1, NOD2 at‐risk alleles was associated with the variable prevalence of CD in families. (Inflamm Bowel Dis 2012;)

Strategy for Overcoming Serum Interferences in Detection of Serum (1,3)-β-d-Glucans

The (1,3)-β-d-glucan (BG) is a cell-wall polysaccharide of most fungi. Regular measurement of BG levels in serum is recognized as a useful diagnostic marker for invasive fungal diseases in immunocompromised and intensive care unit (ICU) patients ([1][1]). Previous studies have identified several

Short fungal fractions of β-1,3 glucans affect platelet activation

Platelets are capable of binding, aggregating, and internalizing microorganisms, which enhances the elimination of pathogens from the blood. The yeast Candida albicans is a pathobiont causing life-threatening invasive infections. Its cell wall contains β-1,3 glucans that are known to trigger a wide range of host cell activities and to circulate during infection. We studied the effect of β-1,3 glucan fractions (BGFs) consisting of diglucosides (Glc2), tetraglucosides (Glc4), and pentaglucosides (Glc5) on human platelets, their mechanisms of action, and their possible impact on host defenses. The effect of BGFs on the coagulation process was determined by measuring thrombin generation. Platelets pretreated with BGFs were analyzed in terms of activation, receptor expression, aggregation, and adhesion to neutrophils and to C. albicans The results show that BGFs affected the endogenous thrombin potential in a concentration-dependent manner. For platelet activation, BGFs at a low concentration (2 μmol/l) reduced ATP release and prevented the phosphorylation of protein kinase C. BGFs diminished the expression of P-selectin and the activation of αIIbβ3 BGFs decreased platelet aggregation and the interaction between thrombin-stimulated platelets and neutrophils, fibrinogen, and C. albicans GLc5 decreased ATP release and TGF-β1 production in response to TLR4 upregulation in thrombin-stimulated platelets, but TLR4 blockage abolished the effect of BGFs on platelets. This study provides evidence that fungal pentaglucosides modulate platelet activity mediated via TLR4 stimulation and reduce platelet-neutrophil interaction.