Genotype distribution of clinical isolates of Trichosporon asahii based on sequencing of intergenic spacer 1

The sequence polymorphisms of intergenic transcribed spacer and the antifungal susceptibility profile of 18 Trichosporon asahii isolates from Spain, Argentina, and Brazil together with 43 intergenic transcribed spacer 1 sequences deposited in the GenBank were analyzed. Six genotypes were detected instead of 5 genotypes described previously. Genotype 1 was the most common found comprising 57.3% of all strains, followed by genotype 3 (14.7%) and genotype 5 (13.1%). Spanish strains had members in all genotypes except 2, whereas South American isolates were grouped with genotypes 1, 3, and 6. Our results indicate that all genotypes are present in at least 2 countries suggesting a worldwide distribution. On the other hand, genotype 6 was not previously described but was only composed of 2 South American strains isolated from a subcutaneous abscess and skin. All isolates showed amphotericin B MICs>or=2 mg/L supporting the in vitro resistance of this species to this antifungal. Three isolates from South America showed high MICs to all antifungals analyzed. The true epidemiologic usefulness of classifying T. asahii in genotypes should be ascertaining analyzing a high number of isolates from many countries.     

In vitro activity of terbinafine against medically important non-dermatophyte species of filamentous fungi

OBJECTIVES: The activity in vitro of terbinafine against 442 clinical isolates of several species of filamentous fungi was analysed. METHODS: A broth microdilution test was carried out following the National Committee for Clinical Laboratory Standards reference method, with modifications described previously. RESULTS: The geometric mean (GM) of MICs of terbinafine for non-Aspergillus fumigatus species was 0.24 mg/L whereas the GM for A. fumigatus rose as far as 2.92 mg/L. Terbinafine showed a very strong activity in vitro against Penicillium spp., Paecilomyces spp., Trichoderma spp., Acremonium spp. and Arthrographis spp. with GMs <1 mg/L. However, some species such as Scedosporium spp., Fusarium spp., Scopulariopsis brevicaulis, and most of Mucorales exhibited high MICs of the allylamine with GMs >/= 4 mg/L. CONCLUSIONS: Overall, the GM of MICs of terbinafine was 1.57 mg/L, but significant differences in susceptibilities were seen between genera and species.     

Value of Serial Quantification of Fungal DNA by a Real-Time PCR-Based Technique for Early Diagnosis of Invasive Aspergillosis in Patients with Febrile Neutropenia

A study was designed to assess the reliability of the serial detection of Aspergillus sp. DNA to diagnose invasive aspergillosis (IA) in patients with febrile neutropenia. Two blood and two serum samples were taken weekly from 83 patients. A total of 2,244 samples were analyzed by real-time quantitative PCR. Twelve (14.4%) patients were diagnosed with IA. Taking two consecutive positive results as the diagnostic criterion, PCR detected 11 cases, with 4 false positives, giving sensitivity, specificity, positive, and negative predictive values of 91.6%, 94.4%, 73.3%, and 98.5%, respectively. On analyzing in conjunction with high-resolution chest tomography (HRCT) and galactomannan (GM) testing, the combination of serial PCR and GM detected 100% of aspergillosis cases, with a positive predictive value of 75.1%. This diagnostic strategy presented, according to CART analysis, a receiver-operator curve with an area under the curve of 0.97 (95% confidence interval, 0.895 to 1.032; P < 0.01), with a relative risk of IA 6.92 times higher than the control population and with predictive success of 95.2%. As regards early diagnosis, the serial detection of Aspergillus DNA took on average 21 days less than HRCT and 68 days less than GM. The serial detection of Aspergillus DNA using real-time quantitative PCR has great diagnostic applicability, which increases when combined with GM quantification.     

In vitro activities of 10 combinations of antifungal agents against the multiresistant pathogen Scopulariopsis brevicaulis

The activities of 10 combinations of antifungal agents against 25 clinical isolates of Scopulariopsis brevicaulis were tested by the checkerboard technique. An average indifferent effect was detected for all combinations. Synergy was observed for some isolates and combinations, particularly with posaconazole-terbinafine (68% of strains), amphotericin B-caspofungin (60%), and posaconazole-caspofungin (48%).     

Correlation between the procedure for antifungal susceptibility testing for Candida spp. of the European Committee on Antibiotic Susceptibility Testing (EUCAST) and four commercial techniques

The correlation between results obtained with the European Committee on Antibiotic Susceptibility Testing (EUCAST) antifungal susceptibility testing procedure (document 7.1) and four commercial systems was evaluated for a collection of 93 clinical isolates of Candida spp. Overall, agreement between the EUCAST procedure and the Sensititre YeastOne and Etest methods was 75% and 90.4%, respectively. The correlation indices (p < 0.01) between the EUCAST and commercial methods were 0.92 for Sensititre YeastOne, 0.89 for Etest, - 0.90 for Neo-Sensitabs, and 0.95 for Fungitest. Amphotericin B MICs obtained by Sensititre YeastOne were consistently higher than with the EUCAST method and, although very major errors were not observed, 91% of MICs were misclassified. Amphotericin B- and fluconazole-resistant isolates were identified correctly with Sensititre YeastOne, Etest and Fungitest. Neo-Sensitabs identified amphotericin B-resistant isolates, but misclassified > 5% of fluconazole-resistant isolates as susceptible. The commercial methods, particularly Etest and Fungitest, appeared to be suitable alternatives to the EUCAST procedure for antifungal susceptibility testing of clinical isolates of Candida.     

Interaction between Pathogenic Bacteria and Intrauterine Leukocytes Triggers Alternative Molecular Signaling Cascades Leading to Labor in Women

Increased risk of preterm labor has been linked to cervicovaginal infection with Ureaplasma urealyticum and group B streptococci. Although various experimental models have been developed to study the role of amniochorion infection in preterm labor, they typically exclude the initial interaction between intrauterine leukocytes (recruited from decidual vessels into the avascular fetal membranes) and infecting bacteria. In this work, we ascertained whether inflammatory molecules secreted by bacterium-activated intrauterine leukocytes stimulate the amniochorion production of mediators involved in human labor. Using a two-step process beginning with placental circulating leukocytes as a proxy for intrauterine leukocytes, we found that coincubation of amniochorion explants with plasma from placental whole blood preincubated with group B streptococci resulted in a significant increase in tumor necrosis factor alpha (TNF-α) and matrix metalloproteinase 9 (MMP-9) levels in tissue. Extensive changes in the connective tissue arrangement and a decrease in collagen content demonstrated the degradation of the extracellular matrix following this treatment. In contrast, plasma from blood preconditioned with U. urealyticum induced a highly significant secretion of interleukin-1β (IL-1β) and prostaglandin E₂ (PGE₂) by the amniochorion without changes in the extracellular matrix organization or content. These data demonstrate that group B streptococci induce degradation of the amniochorion as a result of MMP-9 production, probably via TNF-α, whereas U. urealyticum stimulates the secretion of PGE₂, probably via IL-1β, potentially stimulating myometrial contraction. Our study provides novel evidence that the immunological cells circulating within the uterine microenvironment respond differentially to an infectious agent, triggering alternative molecular signaling pathways leading to human labor.Colonization of the cervicovaginal tract with pathogenic microorganisms, such as group B streptococci (GBS) and Ureaplasma urealyticum, during pregnancy has been associated with premature rupture of the fetal membranes (14, 32, 41). Bacteria colonizing the lower genital tract may ascend to the gestational tissues, triggering an inflammatory response mediated primarily by interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and IL-6 (14, 25, 41). These cytokines stimulate the secretion of additional cytokines and other mediators, including uterotonic compounds, such as prostaglandin E₂ (PGE₂) (9, 27, 42) and matrix metalloproteinases (MMPs) (3, 33, 37), which participate in the weakening of the fetal membranes and their consequent rupture (41, 47). However, the interactions between these microorganisms and the fetal membranes that lead to the secretion of these biochemical mediators, and their cellular origins, are poorly understood.Most in vitro experiments that have explored the effects of microorganisms on the fetal membranes involve the exposure of ex vivo explants of these tissues to whole bacteria or their products (5, 21, 36). However, none of these experimental models considered the possible contribution of other local cells that are components of the choriodecidual microenvironment. This perspective may be critical to an understanding of the rupture of the membranes in the presence of infection, because several leukocyte populations are accumulated selectively in the gestational tissues (6, 31, 46). There, they interact directly with the avascular fetal membranes, leading to further leukocyte recruitment, especially under infectious conditions (18, 44, 52). Additionally, we recently demonstrated that the soluble products of leukocytes circulating in the placental blood induce collagenolysis in the fetal membranes (13). Further characterization indicated that these leukocytes are phenotypically and functionally different than those in the peripheral circulation (49), and because they are sources of activated matrix metalloproteinase 9 (MMP-9), PGE₂ and the proinflammatory cytokines IL-1β and TNF-α (49) may play prominent roles in the process that leads to the rupture of the fetal membranes.To define the role of local leukocytes in the infection-mediated rupture of the fetal membranes, in this study, using an in vitro model, we first explored whether direct contact between two distinct pathogenic bacteria and leukocytes circulating in the placental blood induced the differential secretion of mediators responsible for the initiation of an inflammatory response. Second, we examined the subsequent effects of the products derived from this initial interaction on the amniochorion. Our results showed that GBS induce the production of TNF-α and MMP-9, promoting degradation of the amniochorion, whereas U. urealyticum induces secretion of IL-1β and PGE₂ potentially stimulating myometrial contraction. These findings suggest that local leukocytes are able to respond specifically to the infectious agents triggering alternative molecular signaling cascades leading to human labor.