Search

The synthetic peptide T11F (TCRVDHRGLTF), with sequence identical to a fragment of the constant region of human IgM, and most of its alanine-substituted derivatives proved to possess a significant candidacidal activity in vitro. In this study, the therapeutic efficacy of T11F, D5A, the derivative most active in vitro, and F11A, characterized by a different conformation, was investigated in Galleria mellonella larvae infected with Candida albicans. A single injection of F11A and D5A derivatives, in contrast with T11F, led to a significant increase in survival of larvae injected with a lethal inoculum of C. albicans cells, in comparison with infected animals treated with saline. Peptide modulation of host immunity upon C. albicans infection was determined by hemocyte analysis and larval histology, highlighting a different immune stimulation by the studied peptides. F11A, particularly, was the most active in eliciting nodule formation, melanization and fat body activation, leading to a better control of yeast infection. Overall, the obtained data suggest a double role for F11A, able to simultaneously target the fungus and the host immune system, resulting in a more efficient pathogen clearance.

Galleria mellonella has been described as a cheap and an easy-to-reproduce model for the study of fungal infections. We hypothesized that yeasts with higher virulence potential decrease survival and significantly trigger an immune response in G. mellonella through the regulation of innate immunity-related genes encoding antimicrobial peptides (AMPs) such as gallerimycin and galiomicin. Candida albicans SC5314 and Candida dubliniensis CBS 7987, selected because of their different virulence potential, were used for a killing assay followed by the determination of gene expression using qPCR. In vivo results confirmed a significantly (p = 0.0321) lower pathogenicity for C. dubliniensis than for C. albicans. Accordingly, the induction of C. dubliniensis AMPs was lower at all the selected time points post-infection (1 h, 24 h, 48 h). Moreover, we observed an extremely high regulation of the galiomicin gene compared to the gallerimycin one, suggesting a different role of the tested AMPs in protecting G. mellonella from candidiasis.

The widely accepted dogma of intrauterine sterility and initial colonization of the newborn during birth has been blurred by recent observations of microbial presence in meconium, placenta, and amniotic fluid. Given the importance of a maternal-derived in utero infant seeding, it is crucial to exclude potential environmental or procedural contaminations and to assess fetal colonization before parturition. To this end, we analyzed sterilely collected intestinal tissues, placenta, and amniotic fluid from rodent fetuses and tissues from autoptic human fetuses. Total bacterial DNA was extracted from collected samples and analyzed by Next Generation Sequencing (NGS) techniques using hypervariable 16S ribosomal RNA (rRNA) regions (V3-V4). Colonizing microbes were visualized in situ, using labeled probes targeting 16S ribosomal DNA by fluorescent in situ hybridization. The NGS analysis showed the presence of pioneer microbes in both rat and human intestines as well as in rodent placentas and amniotic fluids. Microbial communities showed fetus- and dam-dependent clustering, confirming the high interindividual variability of commensal microbiota even in the antenatal period. Fluorescent in situ hybridization analysis confirmed the microbes’ presence in the lumen of the developing gut. These findings suggest a possible antenatal colonization of the developing mammalian gut.

Differences in relative proportions of gut microbial communities in adults have been correlated with intestinal diseases and obesity. In this study we evaluated the gut microbiota biodiversity, both bacterial and fungal, in obese and normal-weight school-aged children.We studied 28 obese (mean age 10.03 ± 0.68) and 33 age- and sex-matched normal-weight children. BMI z-scores were calculated, and the obesity condition was defined according to the WHO criteria. Fecal samples were analyzed by 16S rRNA amplification followed by denaturing gradient gel electrophoresis (DGGE) analysis and sequencing. Real-time polymerase chain reaction (PCR) was performed to quantify the most representative microbial species and genera.DGGE profiles showed high bacterial biodiversity without significant correlations with BMI z-score groups. Compared to bacterial profiles, we observed lower richness in yeast species. Sequence of the most representative bands gave back Eubacterium rectale, Saccharomyces cerevisiae, Candida albicans, and C. glabrata as present in all samples. Debaryomyces hansenii was present only in two obese children. Obese children revealed a significantly lower abundance in Akkermansia muciniphyla, Faecalibacterium prausnitzii, Bacteroides/Prevotella group, Candida spp., and Saccharomyces spp. (P = 0.031, P = 0.044, P = 0.003, P = 0.047, and P = 0.034, respectively).Taking into account the complexity of obesity, our data suggest that differences in relative abundance of some core microbial species, preexisting or diet driven, could actively be part of its etiology. This study improved our knowledge about the fungal population in the pediatric school-age population and highlighted the need to consider the influence of cross-kingdom relationships.

Candida albicans is the most common human fungal pathogen with an estimated crude mortality rate of 40%. The ability of the organism to switch from the yeast to hyphal form and produce biofilms are important virulence factors. C. albicans infections are combatted by the host immune system. However, Candida triggers a strong inflammatory response that, if not appropriately regulated, can damage host tissues. Therefore, it is important that the host immune response eliminates the fungus but limits tissue damage. This study provides evidence that targeting cholinergic receptors cannot only curb the virulence of C. albicans by inhibiting filamentous growth and biofilm formation but can also appropriately regulate the host immune response to induce rapid clearance with limited damage to vital tissues. This article provides evidence that repurposing licensed drugs that target cholinergic receptors may offer novel therapeutic solutions for the prevention or treatment of fungal infections., Acetylcholine modulates the virulence of Candida albicans and regulates an appropriate immune response to infection in a Galleria mellonella infection model. Indeed, the evidence suggests that C. albicans possesses a functional cholinergic receptor that can regulate filamentous growth and biofilm formation. Furthermore, G. mellonella immune cell subsets possess repertories of cholinergic receptors which regulate an effective and appropriate cellular immune response to C. albicans infection. This study aimed to investigate the cholinergic receptor subtype involved in regulation of filamentous growth and biofilm formation by C. albicans and determine the roles of cholinergic receptors in modulation of G. mellonella immune cell subsets. The general muscarinic receptor agonist, pilocarpine hydrochloride, inhibited C. albicans biofilm formation and pathogenicity, a phenomenon that could be reversed using the general muscarinic receptor antagonist, scopolamine. Pilocarpine hydrochloride protected G. mellonella larvae from C. albicans infection via inhibition of C. albicans filamentation and appropriate regulation of cellular immunity. However, scopolamine abrogated the capacity of pilocarpine hydrochloride to protect G. mellonella larvae from C. albicans infection. Furthermore, acetylcholine and pilocarpine hydrochloride exhibited differential modulatory capabilities on Galleria mellonella hemocyte responses to C. albicans. The data in this article demonstrate that a muscarinic receptor modulates C. albicans filamentation and biofilm formation. Furthermore, the results suggest that G. mellonella hemocyte subsets possess unique repertoires of cholinergic receptors that regulate their differentiation, activation, and function in contrasting manners. Therefore, targeting cholinergic receptors by repurposing currently licensed cholinergic drugs may offer novel therapeutic solutions for the prevention or treatment of fungal infections. IMPORTANCE Candida albicans is the most common human fungal pathogen with an estimated crude mortality rate of 40%. The ability of the organism to switch from the yeast to hyphal form and produce biofilms are important virulence factors. C. albicans infections are combatted by the host immune system. However, Candida triggers a strong inflammatory response that, if not appropriately regulated, can damage host tissues. Therefore, it is important that the host immune response eliminates the fungus but limits tissue damage. This study provides evidence that targeting cholinergic receptors cannot only curb the virulence of C. albicans by inhibiting filamentous growth and biofilm formation but can also appropriately regulate the host immune response to induce rapid clearance with limited damage to vital tissues. This article provides evidence that repurposing licensed drugs that target cholinergic receptors may offer novel therapeutic solutions for the prevention or treatment of fungal infections.

Candida species (spp.) are a part of the normal human microbiota. Candida dubliniensis mostly colonizes the oral cavity and/or respiratory tract (Mahelová and Růžička 2017), especially in HIV-infected individuals (Coleman et al.1997; Sullivan et al.2004; Wahab et al.2014), while Candida albicans is a common inhabitant of the gastrointestinal tract, urogenital tract and oral cavity (Sardi et al.2013; Höfs, Mogavero and Hube 2016). Candidiasis is the most common global fungal infection (Sardi et al.2013). Candida albicans has been isolated in more than 50% of candidiasis; however, the number of non-albicans spp. able to cause serious candidiasis has increased in recent years (Yapar 2014; Pu et al.2015; Sandhu et al.2017). Although C. dubliniensis is phylogenetically very similar to C. albicans, it differs in some genes, especially those coding for virulence-associated proteins. Candida dubliniensis lacks more than 168 genes characteristic of its ‘yeast-cousin’ C. albicans (Jackson et al.2009), the majority of them encoding proteins related to the yeast-to-hyphae transition, tissue invasion or biofilm development (Moran et al.2004; Jackson et al.2009; Moran, Coleman and Sullivan 2012). Moreover, C. dubliniensis manifests a higher predisposition to develop resistance to fluconazole (Sullivan et al.1995; Moran, Coleman and Sullivan 2012; Jordan et al.2014). On the other hand, both C. albincans and C. dubliniensis are able to form a biofilm (Sullivan et al.2004; Borghi et al.2014). Adherence is the first and most crucial step in biofilm development, and various surface antigens participate in this process (Chaffin 2008; Gow and Hube 2012; Hebecker et al.2014). CR3-RP (complement receptor 3-related protein) is one of the cell surface antigens of Candida spp. with functional and structural similarity to the human complement receptor 3 (CR3) expressed on neutrophils, macrophages and monocytes. CR3-RP has been demonstrated to bind human complement fragment iC3b and to mediate leukocyte diapedesis (Heidenreich and Dierich 1985; Bujdáková et al.1997). Additionally, CR3-RP seems to be an important immunogenic mannoprotein participating in adhesion and biofilm development (Bujdáková et al.2008, 2010). A fragment of CR3-RP was sequenced (DINGGGATLPQ), and according to this sequence, CR3-RP was categorized into the DING protein family (named after DINGGG N termini) (Bujdáková et al.2008; Bernier 2013). Some other surface proteins contributing to biofilm development have been described, such as Eap protein, the Als protein family, the Hwp1 or MP65 proteins (Gomez et al.1996; Nailis et al.2010; Finkel and Mitchell 2011; Araújo, Henriques and Silva 2017). Additionally, antibodies generated after the immunization of animals with some of the above proteins seems to be promising in tools focused on fighting yeast infections (Fujibayashi et al.2009; Mishra, Ali and Shukla 2015; Torosantucci et al.2017). Recent studies showed that antibodies targeting Als3 (Coleman et al.2009), MP65 (De Bernardis et al.2007) or another 42.7 kDa unnamed surface antigen in the Candida cell wall (Mishra, Ali and Shukla 2015) decreased adhesion and biofilm formation.

The widely accepted dogma of intrauterine sterility and initial colonisation of the newborn during birth has been blurred by recent observations of microbial presence in meconium, placenta and amniotic fluid. Given the importance of a maternal-derived in utero infant seeding, it is crucial to exclude potential environmental or procedural contaminations, and to assess foetal colonisation before parturition. To ascertain antenatal microbial colonisation in mammals, we analysed sterilely collected intestinal tissues from rodent foetuses in parallel with experimental controls, and tissues from autoptic human foetuses. Next generation sequencing (NGS) showed the presence of pioneer microbes in both rat and human intestines, as well as in rodent placentas and amniotic fluids. Live microbes were isolated from culture-dependent analyses from homogenized rat foetal intestines.Microbial communities showed foetus- and dam-dependent clustering, confirming the high interindividual variability of microbiota even in the antenatal period. Fluorescent in situ hybridisation analysis confirmed the microbes’ existence in the lumen of the developing gut.These findings have vast implications for an emerging field of enhancing the management of healthy pregnancies, and for understanding how the infant microbiome starts and it is thus shaped.

Gut microbiota is considered a separate organ with endocrine capabilities, actively contributing to tissue homeostasis. It consists of at least two separate microbial populations, the lumen-associated (LAM) and the mucosa-associated microbiota (MAM). In the present study, we compared LAM and MAM, by collecting stools and sigmoid brush samples of forty adults without large-bowel symptoms, and through a 16S rRNA gene next-generation sequencing (NGS) approach. MAM sample analysis revealed enrichment in aerotolerant Proteobacteria, probably selected by a gradient of oxygen that decreases from tissue to lumen, and in Streptococcus and Clostridium spp., highly fermenting bacteria. On the other hand, LAM microbiota showed an increased abundance in Bacteroides, Prevotella, and Oscillospira, genera able to digest and to degrade biopolymers in the large intestine. Predicted metagenomic analysis showed LAM to be enriched in genes encoding enzymes mostly involved in energy extraction from carbohydrates and lipids, whereas MAM in amino acid and vitamin metabolism. Moreover, LAM and MAM communities seemed to be influenced by different host factors, such as diet and sex. LAM is affected by body mass index (BMI) status. Indeed, BMI negatively correlates with Faecalibacterium prausnitzii and Flavonifractor plautii abundance, putative biomarkers of healthy status. In contrast, MAM microbial population showed a significant grouping according to sex. Female MAM was enriched in Actinobacteria (with an increased trend of the genus Bifidobacterium), and a significant depletion in Veillonellaceae. Interestingly, we found the species Gemmiger formicilis to be associated with male and Bifidobacterium adolescentis, with female MAM samples. In conclusion, our results suggest that gut harbors microbial niches that differ in both composition and host factor susceptibility, and their richness and diversity may be overlooked evaluating only fecal samples.

Staphylococcus aureus is a known major cause of foodborne illnesses, and raw milk and dairy products are often contaminated by enterotoxigenic and antimicrobial-resistant S. aureus strains. In the present study, 35 S. aureus strains were isolated from 383 raw milk samples collected from various dairy herds in the province of Milan (northern Italy). The isolates were characterized based on their antimicrobial susceptibility patterns and the presence of genes encoding staphylococcal enterotoxins (sea, seb, sec, sed, and see). About half (45.7%) of the strains were enterotoxigenic, and 37.1% were resistant to at least one of the antimicrobial drugs tested. Seven (20%) of 35 isolates were identified as methicillin-resistant S. aureus (MRSA), and SCCmec typing performed with a multiplex PCR assay revealed the presence of gene cassettes IV and V, typical of community-acquired MRSA, and I and II, characteristic of health care-associated MRSA. The MRSA strains were evaluated for the presence of the Panton-Valentine leukocidin gene, but this gene was not found. The results of the present study revealed the presence of toxin-producing S. aureus and MRSA strains in raw milk. MRSA and enterotoxigenic S. aureus in dairy farms are an important risk factor for the spread of staphylococcal infections; therefore, further studies are needed to find strategies for monitoring and controlling the presence of S. aureus, especially MRSA, in dairy products.

Invasive Candida infections are well established infectious entities of immunocompromised or critically ill patients and are characterised by high morbidity and mortality. Owing to the common eukaryotic structure of fungi and humans, a limited number of antifungal drugs is available for therapeutic purposes. In this unsatisfactory scenario, the emergence of drug resistance represents an important health problem. Failure of antifungal treatment can be related to host factors, to the pharmacokinetic and pharmacodynamic parameters of the drug, or to morphological, reproductive modalities and biofilm production of the fungus itself. Innate or acquired antifungal resistance derives from the presence or onset of molecular mechanisms related to the toxic activity of the drug itself. The resulting resistance can thus be extended to different molecules of the same class according to a greater or lesser affinity of the molecules for the target. In addition, non-specific cellular mechanisms of extrusion of toxic substances, such as overexpression of efflux pumps, can play a role involving different antifungal classes. Here we briefly review the current antifungal susceptibility testing methods and their usefulness as predictors of antifungal resistance in Candida spp., focusing on assessment of the involved molecular mechanisms.

Background and aims Phenylalanine (Phe) restricted diet, combined with Phe-free l -amino acid supplementation, is the mainstay of treatment for phenylketonuria (PKU). Being the diet a key factor modulating gut microbiota composition, the aim of the present paper was to compare dietary intakes, gut microbiota biodiversity and short chain fatty acids (SCFAs) production in children with PKU, on low-Phe diet, and in children with mild hyperphenylalaninemia (MHP), on unrestricted diet. Methods and Results We enrolled 21 PKU and 21 MHP children matched for gender, age and body mass index z-score. Dietary intakes, including glycemic index (GI) and glycemic load (GL), and fecal microbiota analyses, by means of denaturing gradient gel electrophoresis (DGGE) and Real-time PCR were assessed. Fecal SCFAs were quantified by gas chromatographic analysis. Results We observed an increased carbohydrate (% of total energy), fiber and vegetables intakes (g/day) in PKU compared with MHP children (p = 0.047), as well a higher daily GI and GL (maximum p Compared with MHP, PKU showed a lower degree of microbial diversity and a decrease in fecal butyrate content (p = 0.02). Accordingly, two of the most abundant butyrate-producing genera, Faecalibacterium spp. and Roseburia spp., were found significantly depleted in PKU children (p = 0.02 and p = 0.03, respectively). Conclusion The low-Phe diet, characterized by a higher carbohydrate intake, increases GI and GL, resulting in a different quality of substrates for microbial fermentation. Further analyses, thoroughly evaluating microbial species altered by PKU diet are needed to better investigate gut microbiota in PKU children and to eventually pave the way for pre/probiotic supplementations.

Anorexia nervosa (AN) is a psychiatric disease with devastating physical consequences, with a pathophysiological mechanism still to be elucidated. Metagenomic studies on anorexia nervosa have revealed profound gut microbiome perturbations as a possible environmental factor involved in the disease. In this study we performed a comprehensive analysis integrating data on gut microbiota with clinical, anthropometric and psychological traits to gain new insight in the pathophysiology of AN. Fifteen AN women were compared with fifteen age-, sex- and ethnicity-matched healthy controls. AN diet was characterized by a significant lower energy intake, but macronutrient analysis highlighted a restriction only in fats and carbohydrates consumption. Next generation sequencing showed that AN intestinal microbiota was significantly affected at every taxonomic level, showing a significant increase of Enterobacteriaceae, and of the archeon Methanobrevibacter smithii compared with healthy controls. On the contrary, the genera Roseburia, Ruminococcus and Clostridium, were depleted, in line with the observed reduction in AN of total short chain fatty acids, butyrate, and propionate. Butyrate concentrations inversely correlated with anxiety levels, whereas propionate directly correlated with insulin levels and with the relative abundance of Roseburia inulinivorans, a known propionate producer. BMI represented the best predictive value for gut dysbiosis and metabolic alterations, showing a negative correlation with Bacteroides uniformis (microbiota), with alanine aminotransferase (liver function), and with psychopathological scores (obsession-compulsion, anxiety, and depression), and a positive correlation with white blood cells count. In conclusion, our findings corroborate the hypothesis that the gut dysbiosis could take part in the AN neurobiology, in particular in sustaining the persistence of alterations that eventually result in relapses after renourishment and psychological therapy, but causality still needs to be proven.

Although there has been an overall good coverage of Candida glabrata infections by the echinocandins, emergence of antifungal resistance during therapy has been reported. We investigated, by using an invertebrate host model, the fitness of sequential C. glabrata isolates with different echinocandins susceptibility patterns. The studied strains were isolated from a case of recurrent fungemia with a fatal outcome due to C. glabrata that developed cross-resistance to echinocandins during caspofungin therapy. The sequential strains isolated post-therapy showed a S663P mutation in the Fks2p hot spot 1. In vivo study in the invertebrate host Galleria mellonella did not suggest a fitness cost related to the acquired antifungal resistance, the three isolates displayed a similar rate of killing (P = 0.54). We observed a clear correlation between emergence of antifungal resistance and persistence of the causal agent, probably aided by the unchanged fitness and unresponsiveness in vivo to the adopted therapy.

A phosphorylated peptide, named K40H, derived from the constant region of IgMs was detected in human serum by liquid chromatography coupled to high-resolution mass spectrometry. Synthetic K40H proved to exert a potent in vitro activity against fungal pathogens, and to inhibit HIV-1 replication in vitro and ex vivo. It also showed a therapeutic effect against an experimental infection by Candida albicans in the invertebrate model Galleria mellonella. K40H represents the proof of concept of the innate role that naturally occurring antibody fragments may exert against infectious agents, shedding a new light upon the posthumous role of antibodies and opening a new scenario on the multifaceted functionality of humoral immunity.

An altered gut microbiota has been linked to obesity in adulthood, although little is known about childhood obesity. The aim of this study was to characterize the composition of the gut microbiota in obese (n = 42) and normal-weight (n = 36) children aged 6 to 16. Using 16S rRNA gene-targeted sequencing, we evaluated taxa with differential abundance according to age- and sex-normalized body mass index (BMI z-score). Obesity was associated with an altered gut microbiota characterized by elevated levels of Firmicutes and depleted levels of Bacteroidetes. Correlation network analysis revealed that the gut microbiota of obese children also had increased correlation density and clustering of operational taxonomic units (OTUs). Members of the Bacteroidetes were generally better predictors of BMI z-score and obesity than Firmicutes, which was likely due to discordant responses of Firmicutes OTUs. In accordance with these observations, the main metabolites produced by gut bacteria, short chain fatty acids (SCFAs), were higher in obese children, suggesting elevated substrate utilisation. Multiple taxa were correlated with SCFA levels, reinforcing the tight link between the microbiota, SCFAs and obesity. Our results suggest that gut microbiota dysbiosis and elevated fermentation activity may be involved in the etiology of childhood obesity.

Over the past decade, the emergence of biofilm-related invasive fungal diseases has been the subject of numerous studies focused on antifungal resistance and its impact on antifungal therapy in severely ill patients. The majority of the studies investigated the molecular mechanisms involved in antifungal resistance and pathogenicity of biofilm production by Candida albicans and Aspergillus fumigatus, the most common etiologic agents of yeast and mold invasive infections. The main mechanism characterizing biofilm-related antifungal resistance is the production of extracellular matrix, a physical barrier preventing the drugs from entering and expressing their activity. However, over-expression of efflux pumps, genetic changes of drug targets, persister cells, biofilm-host immune system interaction, proteins leading to filamentation, all together contribute to the onset of biofilm antifungal resistance. Some of these mechanisms are shared with planktonic cells and are often related to developmental phases of biofilm formation. All physical and genetic factors leading to biofilm-related antifungal resistance have been briefly discussed.

Over the past decade, the emergence of biofilm-related invasive fungal diseases has been the subject of numerous studies focused on antifungal resistance and its impact on antifungal therapy in severely ill patients. The majority of the studies investigated the molecular mechanisms involved in antifungal resistance and pathogenicity of biofilm production by Candida albicans and Aspergillus fumigatus, the most common etiologic agents of yeast and mold invasive infections. The main mechanism characterizing biofilm-related antifungal resistance is the production of extracellular matrix, a physical barrier preventing the drugs from entering and expressing their activity. However, over-expression of efflux pumps, genetic changes of drug targets, persister cells, biofilm-host immune system interaction, proteins leading to filamentation, all together contribute to the onset of biofilm antifungal resistance. Some of these mechanisms are shared with planktonic cells and are often related to developmental phases of biofilm formation. All physical and genetic factors leading to biofilm-related antifungal resistance have been briefly discussed.

The Candida parapsilosis group encompasses three species: C. parapsilosis, C. orthopsilosis, and C. metapsilosis. Here, we describe the incidence and echinocandin susceptibility profile of bloodstream isolates of these three species collected from patients admitted to an Italian university hospital from 2007 to 2014. Molecular identification of cryptic species of the C. parapsilosis complex was performed using polymerase chain reaction amplification of the gene encoding secondary alcohol dehydrogenase, followed by digestion with the restriction enzyme BanI. Minimum inhibitory concentrations were determined using the broth microdilution method according to European Committee for Antimicrobial Susceptibility Testing (EUCAST EDef 7.2) and Clinical Laboratory Standards Institute (CLSI M27-A3) guidelines, and the results were compared with those obtained using the E-test and Sensititre methods. Of the 163 C. parapsilosis complex isolates, 136 (83.4%) were identified as C. parapsilosis, and 27 (16.6%) as C. orthopsilosis. The species-specific incidences were 2.9/10,000 admissions for C. parapsilosis and 0.6/10,000 admissions for C. orthopsilosis. No resistance to echinocandins was detected with any of the methods. The percent essential agreement (EA) between the EUCAST and E-test/Sensititre methods for anidulafungin, caspofungin, and micafungin susceptibility was, respectively, as follows: C. parapsilosis, 95.6/97.8, 98.5/88.2, and 93.4/96.3; C. orthopsilosis, 92.6/92.6, 96.3/77.8, and 63.0/66.7. The EA between the CLSI and E-test/Sensititre methods was, respectively, as follows: C. parapsilosis, 99.3/100, 98.5/89.0, and 96.3/98.5; C. orthopsilosis, 96.3/92.6, 100/81.5, and 92.6/88.9. Only minor discrepancies, ranging from 16.9% (C. parapsilosis) to 11.1% (C. orthopsilosis), were observed between the CLSI and E-test/Sensititre methods. In conclusion, this epidemiologic study shows a typical C. parapsilosis complex species distribution, no echinocandin resistance, and it reinforces the relevance of using commercially available microbiological methods to assess antifungal susceptibility. These data improve our knowledge of the national distribution of species of the psilosis group, as there are very few studies of these species in Italy.

In the present work, we studied the distribution of Candida parapsilosis complex species and the antifungal susceptibility of clinical isolates collected during an Italian surveillance study of yeast invasive fungal infections (IFIs) in intensive care units (ICUs). Minimum inhibitory concentrations (MICs) were determined using the Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution method. BanI digestion patterns of the secondary alcohol dehydrogenase polymerase chain reaction (PCR) products were used to identify C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis. A total of 138 C. parapsilosis isolates were stored (January 2007-December 2008). The overall frequency of C. parapsilosis complex in IFIs was 22%. Of the 138 tested isolates, 95% were C. parapsilosis sensu stricto, 3.6% were C. orthopsilosis, and 1.4% were C. metapsilosis. The MIC(50) values (expressed as μg/ml) for anidulafungin, caspofungin, and micafungin for C. parapsilosis complex were 2, 1, and 2, respectively, and the MIC(90) values were 4, 2, and 4, respectively. The MIC(50) and MIC(90) values for itraconazole and posaconazole were 0.12 and 0.25, respectively, and for fluconazole, they were 1 and 4, respectively. This study, the most comprehensive study conducted to date to evaluate the frequency and antifungal susceptibility profiles of C. parapsilosis complex isolates from critically ill patients in Italy, highlights the low prevalence of C. orthopsilosis and C. metapsilosis in IFIs.

Abnormally viscous bronchial secretions, a characteristic feature of cystic fibrosis (CF), may trap bacteria and fungi, allowing transient or chronic lung colonization. We report here a case of persistent Scedosporium apiospermum colonization in a patient with CF, who subsequently developed a lung mycetoma, and died with neurological symptoms suggestive of cerebral fungal involvement. Six isolates from consecutive sputum samples were molecularly typed by random amplification of polymorphic DNA (RAPD) using primers UBC701, UBC703, and GC70. Moreover, in vitro susceptibility of these isolates to current antifungals (amphotericin B, itraconazole, voriconazole, posaconazole, caspofungin and anidulafungin) was investigated by means of both E-test and CLSI methods. Antifungal susceptibility testing showed low minimum inhibitory concentration values only for triazole drugs. However, a unique genotype was isolated over a 12-month period, despite antifungal treatment with voriconazole for three months. This case report illustrates the therapy-refractory feature of this fungus, and provides new evidence that, as already reported, once a genotype of S. apiospermum has established colonization, it seems not to be replaced by others.

The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically changed over the past 10 y with the emergence of community-associated MRSA (CA-MRSA). Recent studies have reported a frequent association of these strains with hospital outbreaks, and an incidence varying over time and by region. In order to evaluate the MRSA lineages circulating in our area of Italy, we performed a molecular characterization of CA-MRSA isolates prospectively collected from April 2006 to July 2007 at the San Paolo Hospital of Milan. We investigated the protein A-encoding gene (spa-typing), the staphylococcal chromosomal cassette SCCmec, the presence of Panton-Valentine leukocidin (PVL), and 3 adhesin genes. Twenty-five CA-MRSA isolates cultured from 25 patients were collected; an equal number of healthcare-associated (HA)-MRSA strains, from 25 patients hospitalized in various wards, were collected for comparison purposes. SCCmec type IV emerged as the most frequent genotype in both CA- and HA-MRSA. Seventeen different spa types were identified: t515 was the most common (36%), followed by t008 (20%). We detected 3 PVL-positive strains, only among the CA-MRSA. On the whole, our local MRSA epidemiology appears to be heterogeneous, with a predominant t515 spa type, only recently considered to belong to clonal EMRSA-15.

Patients with inefficient CD4+ T-cell recovery on virogically suppressive highly active antiretroviral therapy constitute a major clinical hurdle given the threat of HIV/AIDS disease progression. We show heightened circulating lipopolysaccharide associated with plasma enterobacterial DNA and highly activated Ki67+CD4+CD8+ in 24 immunologic-nonresponders (CD4+ T-cellor = 200; HIV-RNAor = 50) compared with 11 full responders (CD4+ T-cellor= 400; HIV-RNAor = 50). These data provide novel insight into INRs pathogenesis, since they correlate augmented systemic translocation of microbial bioproducts with T-cell hyperactivation.

Identification of new molecular scaffolds structurally unrelated to known antimalarials may represent a valid strategy to overcome resistance of P. falciparum (Pf) to currently available drugs. We describe herein the investigation of a new polycyclic pharmacophore, related to clotrimazole, to develop innovative antimalarial agents. This study allowed us to discover compounds characterized by a high in vitro potency, particularly against Pf CQ-resistant strains selectively targeting free heme, which are easy to synthesize by low-cost synthetic strategies.

Fungal infections have dramatically increased in the last decades in parallel with an increase of populations with impaired immunity, resulting from medical conditions such as cancer, transplantation or other chronic diseases. Such opportunistic infections result from a complex relationship between fungi and host, and can range from self-limiting to chronic or life-threatening infections. Modern medicine, characterized by a wide use of biomedical devices, offers new niches for fungi to colonize and form biofilm communities. The capability of fungi to form biofilms is well documented and associated with increased drug tolerance and resistance. In addition, biofilm formation facilitates persistence in the host promoting a persistent inflammatory condition. \ud With a limited availability of antifungals within our arsenal, new therapeutic approaches able to address both host and pathogenic factors that promote fungal disease progression, i.e. chronic inflammation and biofilm-formation, could represent an advantage in the clinical setting. \ud In this paper we discuss the antifungal properties of Myriocin, Fulvic Acid and Acetylcholine in light of their already known anti-inflammatory activity and as candidate dual action therapeutics to treat opportunistic fungal infections.

Background: The human pathogenic mold Aspergillus fumigatus is able to form a complex biofilm embedded in extracellular matrix. Biofilms confer antimicrobial resistance and it is well known that aspergillosis is often refractory to the conventional antifungal therapy. The treatment of biofilm-related infections poses a significant clinical challenge on a daily basis, promoting the search for new therapeutic agents. Our aim was to exploit the modulation of sphingolipid mediators as new therapeutic target to overcome antifungal resistance in biofilm-related infections. Results: Antifungal susceptibility testing was performed on 20 clinical isolates of Aspergillus fumigatus and one reference strain (A. fumigatus Af293) according the EUCAST protocol. Sessile MICs were assessed on 24-h preformed-biofilm by means of XTT-reduction assay. Myriocin (0.25-64 mg/L), a commercial sphingolipid synthesis inhibitor, was used. The MEC50 value (mg/L) of Myriocin was 8 (range 4-16) for both planktonic and sessile cells. Drug-induced morphological alterations were analyzed by optical and electron microscopy (TEM) on 24h preformed A. fumigatus Af293 biofilms. An evident hyphal damage, resulting in short, stubby, and highly branched hyphae was observed by optical microscopy. At 24h, TEM studies showed important morphological alterations, such as invaginations of the cell membrane, modification in the vacuolar system and presence of multilamellar bodies, in some cases within vacuoles. Conclusions: The direct antifungal activity, observed on both planktonic and sessile fungi, suggests that inhibition of sphingolipid synthesis could represent a new target to fight biofilm-related A. fumigatus resistance.

Voriconazole, amphotericin B and itraconazole were tested in vitro against 18 strains of Aspergillus fumigatus isolated from cystic fibrosis patients. Susceptibility was tested with the broth microdilution method (M38-A protocol-NCCLS). Results of this reference method were compared with those of an experimental commercial microdilution broth method (Sensititre). Two different inocula, prepared from 2- and 7-day cultures, were used. Minimum inhibitory concentrations (MICs) of the reference method ranged from 0.25 to 2 microg/ml for voriconazole, 0.06 to 1 microg/ml for amphotericin B, 0.016 to16 microg/ml for itraconazole. There were no significant differences in the MIC ranges or MIC90 values obtained with the two testing methods or with the two types of inocula. These findings confirm the good in vitro activity of voriconazole, itraconazole and amphotericin B against A. fumigatus. They also indicate that reliable susceptibility data can be generated more rapidly by commercial systems and use of 2-day cultures for inoculum preparation.

Summary Resistance to fluconazole is a possible event during prolonged suppressive drug therapy for cryptococ- cal meningitis, the most frequently encountered life- threatening manifestation of cryptococcosis. The knowledge of this resistance at the molecular level is important for management of cryptococcosis. In order to identify genes involved in azole resistance in Cryptococcus neoformans , a cDNA subtraction library technique was chosen as a strategy. First, a fluconazole-resistant mutant BPY22.17 was obtained from a susceptible clinical isolate BPY22 by in vitro exposure to the drug. Then, a subtractive hybridiza- tion procedure was used to compare gene expression between the obtained strains. We identified a cDNA overexpressed in the fluconazole-resistant strain BPY22.17 that was used as a probe to isolate the entire gene in a C. neoformans genomic library. Sequence analysis of this gene identified an ATP Binding Cassette (ABC) transporter-encoding gene called C. neoformans AntiFungal Resistance 1 ( CnAFR1 ). Disruption of CnAFR1 gene in the resistant isolate (BPY22.17) resulted in an enhanced suscepti- bility of the knock-out mutant cnafr1 against flucona- zole, whereas reintroduction of the gene in cnafr1 resulted in restoration of the resistance phenotype, thus confirming that CnAFR1 is involved in flucona- zole resistance of C. neoformans . Our findings there- fore reveal that an active drug efflux mechanism can be involved in the development of azole resistance in this important human pathogen.

Fluconazole susceptibility among 800 clinical Candida isolates (60% C. albicans ) and two control strains ( C. krusei ATCC 6258 and C. parapsilosis ATCC 22019) was tested with the NCCLS M27-A method (gold standard) and six commercial products (Candifast, disk, Etest, Fungitest, Integral System Yeasts, and Sensititre YeastOne). Results were classified as susceptible, susceptible-dose dependent, or resistant using M27-A breakpoints or, for Fungitest, Integral System Yeasts, and Candifast, as susceptible, intermediate, or resistant, according to the manufacturers' instructions. Concordance with NCCLS M27-A results was analyzed with the χ 2 test. Intra- and interlaboratory reproducibility was also evaluated. NCCLS M27-A (90.1%), Etest (93.1%), Sensititre YeastOne (93.1%), disk (96.7%), Fungitest (92.6%), Integral System Yeasts (40.6%), and Candifast (6.0%) classified the indicated percentages of C. albicans isolates as susceptible. Among non- C. albicans strains, the percentages of susceptible isolates were as follows: NCCLS M27-A, 74.0%; Etest, 83.8%; Sensititre YeastOne, 64.1%; disk, 60.6%; Fungitest, 76.6%; Integral System Yeasts, 28.3%; and Candifast, 27.4%. All methods except Candifast and Integral System Yeasts showed good agreement with NCCLS M27-A results for both C albicans and non- C. albicans isolates. Intralaboratory reproducibility was excellent for NCCLS M27-A, Etest, Sensititre YeastOne, disk, and Fungitest (88 to 91%). Similar results emerged from the interlaboratory reproducibility evaluation. Our findings indicate that some commercial methods can be useful for fluconazole susceptibility testing of clinical Candida isolates. Those characterized by a lack of medium standardization and/or objective interpretative criteria should be avoided. Particular caution is necessary when testing is being done for clinical and epidemiological purposes.

Invasive aspergillosis has been mainly reported among immunocompromised patients during prolonged periods of neutropenia. Recently, however, non-neutropenic patients in the ICU population have shown an increasing risk profile for aspergillosis. Associations with chronic obstructive pulmonary disease and corticosteroid therapy have been frequently documented in this cohort. Difficulties in achieving a timely diagnosis of aspergillosis in non-neutropenic patients is related to the non-specificity of symptoms and to lower yields with microbiological tests compared to neutropenic patients. Since high mortality rates are typical of invasive aspergillosis in critically ill patients, a high level of suspicion and prompt initiation of adequate antifungal treatment are mandatory. Epidemiology, risk factors, diagnostic algorithms, and different approaches in antifungal therapy for invasive aspergillosis in non-neutropenic patients are reviewed.

ABSTRACT: Aim: The aim of our study was to investigate whether biofilm production by Candida albicans clinical isolates could be a hallmark of virulence in vivo. Materials & methods: Twenty clinical isolates of C. albicans were examined via histological studies on larvae infected with various fungal doses (from 103 to 105 CFU/larva) of biofilm producer and nonproducer strains. Results: The poor prognostic role of infection due to a biofilm-producing isolate was confirmed by the Wald test (hazard ratio: 2.63; 95% CI: 2.03–3.41). Histological examinations at 24 h showed a strong innate immune response, with evidence of melanization for both infection groups. However, at 48 h, we found huge differences in filamentation and tissue invasion capability between biofilm nonproducing and producing isolates, the latter being highly organized into biofilm and invading the larval intestinal tract. Invasion corroborated survival data. Conclusion: The histological results demonstrate that the production of biofilm could enhance the invasiveness of C. albicans.

The hyphal wall protein 1 (HWP1) gene of Candida albicans encodes for a fungal cell wall protein, required for hyphal development and yeast adhesion to epithelial cells; yet, its role in pathogenesis remains largely unknown. In the present study, we analyzed two C. albicans laboratory strains, the DAY286 (HWP1/HWP1) and the null mutant FJS24 (hwp1/hwp1) and six clinical isolates [3 harbouring the homozygous HWP1 gene (HWP1/HWP1) and 3 the heterologous gene (HWP1/hwp1)]. Biofilm production, fungal HWP1 mRNA levels and ultrastructural morphology were investigated; also, the susceptibility of these strains to microglial cells was evaluated, in terms of fungal damage and immune cell-mediated secretory response. When comparing the two laboratory strains, biofilm was produced to a similar extent independently on the genetic background, while the susceptibility to microglial cell-mediated damage was higher in the hwp1/hwp1 mutant than in the HWP1/HWP1 counterpart. Also, transmission electron microscopy revealed differences between the two in terms of abundance in surface adhesin-like structures, fungal cell wall shape and intracellular granules. When comparing the clinical isolates grouped according to their HWP1 genotype, reduced biofilm production and increased susceptibility to microglial cell-mediated damage occurred in the HWP1/hwp1 isolates with respect to the HWP1/HWP1 counterparts; furthermore, upon exposure to microglial cells, the HWP1/HWP1 isolates, but not the HWP1/hwp1 counterpart, showed enhanced HWP1 mRNA levels. Finally, both laboratory and clinical isolates exhibited reduced ability to stimulate TNFα and nitric oxide production by microglial cells in the case of heterozygous or null mutant HWP1 genotype. Overall, these data indicate that C. albicans HWP1 genotype influences pathogen morphological structure as well as its interaction with microglial cells, while fungal biofilm production results unaffected, thus arguing on its role as virulence factor that directly affects host mediated defences.

A PCR-based assay was developed to detect and identify medically important yeasts in clinical samples. Using a previously described set of primers (G. Morace et al., J. Clin. Microbiol. 35:667–672, 1997), we amplified a fragment of the ERG11 gene for cytochrome P-450 lanosterol 14α-demethylase, a crucial enzyme in the biosynthesis of ergosterol. The PCR product was analyzed in a reverse cross blot hybridization assay with species-specific probes directed to a target region of the ERG11 gene of Candida albicans (pCal), C. guilliermondii (pGui), C. ( Torulopsis ) glabrata (pGla), C. kefyr (pKef), C. krusei (pKru), C. parapsilosis (pPar), C. tropicalis (pTro), the newly described species C. dubliniensis (pDub), Saccharomyces cerevisiae (pSce), and Cryptococcus neoformans (pCry). The PCR-reverse cross blot hybridization assay correctly identified multiple isolates of each species tested. No cross-hybridization was detected with any other fungal, bacteria, or human DNAs tested. The method was tested against conventional identification on 140 different clinical samples, including blood and cerebrospinal fluid, from patients with suspected fungal infections. The results agreed with those of culture and phenotyping for all but six specimens (two of which grew yeasts not included in the PCR panel of probes and four in which PCR positivity-culture negativity was justified by clinical findings). Species identification time was reduced from a mean of 4 days with conventional identification to 7 h with the molecular method. The PCR-reverse cross blot hybridization assay is a rapid method for the direct detection and identification of yeasts in clinical samples.

Technical complications and nosocomial bloodstream infections associated with short-term central venous catheterization remain a heavy burden in terms of morbidity, mortality and cost in HIV-positive subjects. Between 1994 and 1997, 327 central venous catheters (CVCs) inserted in 212 patients for a total of 5005 catheter days were investigated. Forty-two technical complications (13%) occurred in 40 patients. Logistic regression analysis revealed that a high APACHE III score was associated with development of CVC-related complications (P = 0.01). One hundred and eight of 327 CVCs (33%) were suspected as being infected. However only 61 episodes (61/327, 19%) were finally diagnosed as CVC-related sepsis. Three variables affecting the rate of CVC-related sepsis were identified: 1) administration of TPN (P = 0.01); 2) low number of circulating CD4+ cells (P = 0.04); 3) high APACHE III score (P = 0. 04). Doctors responsible for AIDS patients should carefully consider the relative risks and benefits of CVC insertion in an individual patient.

Summary. We describe a patient with acute lymphoblastic leukaemia who developed rhinocerebral zygomycosis during the aplastic phase induced by antineoplastic chemotherapy. The patient was treated with fluconazole intravenously (400 mg daily) for 30 days and underwent surgical debridement. As a result of this treatment a complete remission of the zygomycosis-associated symptoms was observed. The possibility of treating zygomycosis with fluconazole is discussed. Zusammenfassung. Eine an akuter lymphoblastischer Leukamie erkrankte Patientin entwickelte wahrend der Aplasiephase nach antineoplastischer Chemotherapie eine rhinozerebrale Zygomykose. Die Patientin wurde mit 400 mg Fluconazol taglich intravenos uber einen Zeitraum von 30 Tagen behandelt, und es wurde ein chirurgisches Debridement durchgefuhrt. Unter dieser Behandlung kam es zu einem volli-gen Abklingen der Mykosesymptome. Die Mog-lichkeit, Fluconazol zur Zygomykose-Behandlung einzusetzen, wird diskutiert.

The epidemiological features of 37 episodes of candidemia in HIV-infected subjects were analysed in a retrospective matched case-control study conducted over an 8-year period (1990-1997). Univariate analysis identified eight risk factors that were significantly associated with candidemia (P0.05): i) use of central venous catheters; ii) administration of total parenteral nutrition; iii) previous antifungal therapy; iv) previous therapy with glycopeptides; v) presence of oral/ esophageal candidiasis; vi) concomitant bacterial infections; vii) neutropenia; and viii) concomitant AIDS dementia complex. Stepwise logistic regression analysis revealed that the only independent risk factor for developing candidemia was the use of central venous catheters (P = 0.0001). Candida albicans was the most frequently isolated pathogen, accounting for 18 (48%) episodes of candidemia, followed by Candida tropicalis (19%) and Candida glabrata (11%). The crude mortality rate was 62%. On univariate analysis concomitant opportunistic infections, presence of non-Candida albicans species of Candida and neutropenia were shown to be predictive of death. Multivariate analysis revealed that the presence of non-Candida albicans strains of Candida was the only significant factor associated with a worse prognosis (P = 0.001). In conclusion, candidemia appears to be more common in patients with advanced HIV disease. Of the factors which influenced the onset of candidemia, use of central venous catheters seemed to be the most important one.

This study was conducted to evaluate the efficacy of highly active anti-retroviral therapy (HAART) in preventing recurrence of oral candidosis (OC) associated with HIV. A prospective case-controlled observational study was performed in an inner-city university-hospital HIV/AIDS clinic. Ninety-three HIV-positive study subjects with a history of recurrent OC were divided into two groups: protease inhibitors (PI)-treated patients (group 1, n = 30) and non-PI-treated patients (group 2, n = 63). Study subjects were matched for sex, age, stage of HIV infection, and peripheral CD4+ T-cell counts. The non-PI-treated group was further subdivided into the following three subgroups: HIV-positive study subjects treated with reverse transcriptase inhibitors (RTI; groups 2a and 2c) and HIV-positive study subjects not treated with RTIs (group 2b). Group 2c met the same inclusion criteria as group 2a had but was matched 6 months after the beginning of the study. We also assessed in vitro peripheral blood mononuclear cells (PBMC) and their lymphoproliferative response, as well as cutaneous delayed-type hypersensitivity (DTH) response to Candida-associated antigens in a randomly selected sample of study subjects divided into those treated with PIs and those who were not. During a 1-year follow-up, OC was diagnosed in 2 (7%) PI-treated and 23 (36%) non-PI-treated patients (p

A Rhodococcus equi pulmonary infection in a 63-year-old man receiving chemotherapy and radiotherapy for spinocellular carcinoma is described. The patient, a knife-grinder, was promptly treated with levofloxacin plus amikacin followed by rifampicin for 2 months, and he is still in good clinical condition after an 8-month follow-up.

Aim: We investigated the pathogenic role of biofilm and the therapeutic efficacy of anidulafungin in experimental infections of the wax moth Galleria mellonella by Candida albicans clinical strains. Materials & methods: On the basis of the in vitro propensity to form biofilm, five biofilm-producer (BP) and four nonproducer (NP) C. albicans clinical strains were used in this study. For each strain, we assessed the virulence by infecting G. mellonella larvae and observing survival. Anidulafungin was administered 2 h after yeast inoculum at 0.6 µg, according to the therapeutic dose recommended for humans. Results: Biofilm-forming ability highly influenced the larva-killing rate. A significant (p < 0.0001) survival decrease was observed in the BP group, with 80% of the infected larvae dying within 72 h. NP isolates did not reach the same killing rate, even at the end of experiments (216 h). Larval survival was enhanced (p < 0.0001) by anidulafungin administration in both groups. Survival rate at 72 h was similar in both groups (BP 78.5% and NP 87.5%); whereas there were still differences at the end of the experiments, with a higher survival in the NP group (75 vs 48%). Conclusion: Our data confirm the pathogenic role of biofilm in C. albicans infections. Its importance was further enhanced by a lack of contribution from extracellular enzymes, detected in both NP and BP strains. In addition, we demonstrated anidulafungin efficacy in treating biofilm-related invasive candidiasis.

Candida albicans is the most prevalent human fungal pathogen associated with biofilm formation on indwelling medical devices. Under this form, Candida represents an infectious reservoir difficult to eradicate and possibly responsible for systemic, often lethal infections. Currently, no information is available on the occurrence and persistence of pathogenic viruses within C. albicans biofilm. Therefore, the aim of this study was to investigate whether Herpes Simplex Virus type 1 (HSV-1) and Coxsackievirus type B5 (CVB5) can be encompassed in Candida biofilm, retain their infectivity and then be released. Thus, cell-free virus inocula or HSV-1-infected cells were added to 24h-old fungal biofilm in tissue culture plates; 48 h later, the biofilm was detached by washing and energetic scratching and the presence of virus in the rescued material was end-point titrated on VERO cells. Planktonic Candida cultures and samples containing only medium were run in parallel as controls. We found that both HSV-1 and CVB5 free virus particles, as well as HSV-1 infected cells remain embedded in the biofilm retaining their infectivity. As a second step, the influence of biofilm on virus sensitivity to sodium hypochlorite and to specific neutralizing antibodies was investigated. The results showed that virus encompassment in fungal biofilm reduces virus sensitivity to chemical inactivation but does not affect antibody neutralization. Overall, these data provide the first in vitro evidence that viruses can be encompassed within Candida biofilm and then be released. Thus, it may be speculated that Candida biofilm can be a reservoir of viruses too, posing a further health risk.

The objective of this case-control study, conducted in a large Italian university hospital over a 12-month period, was to evaluate the risk factors associated with the emergence of azole resistant oral candidosis in 64 Human Immunodeficiency Virus (HIV) infected patients. A swab was obtained from each patient by brushing candidal lesions. Candida albicans was isolated in 41 patients (64%), Candida glabrata in ten (16%), Candida krusei in five (8%), Candida kefyr in two (3%), Candida tropicalis in two (3%), and Candida lipolytica and Candida guilliermondii in one case, respectively. Two patients suffered a double infection i.e. C. albicans + C. krusei and C. albicans + C. glabrata, respectively. Candida species were tested in vitro for their susceptibility to ketoconazole, fluconazole, itraconazole and amphotericin B. MICs of the four antifungal drugs were obtained for each yeast using a microdilution broth method developed in our laboratory. Twenty four (37%) of the isolated strains were resistant both to itraconazole and fluconazole, five (8%) to fluconazole alone, and two (3%) to ketoconazole alone, while none of the isolated strains was resistant to amphotericin B. Patients with oral candidosis caused by a strain resistant to one or more azole drug were compared to control patients with azole-susceptible oral candidosis. On univariate analysis, more than five episodes of oral candidosis in the last year (P = 0.01), previous use of azole therapy (P = 0.001), C2-3 category of HIV infection (P = 0.01) and low number of circulating CD4+ T-cells (P = 0.03) were significantly associated with an increased risk for the development of azole resistance. However, previous use of azole therapy was the only factor selected by a stepwise logistic regression analysis which was independently associated with the isolation of azole resistant strains (P = 0.003). Our findings indicate that, in view of the potential risk for the emergence and selection of azole resistant strains of Candida in patients with AIDS, it is important to carefully choose the antifungal drug for the therapy of mild fungal infections after evaluation of the in-vitro susceptibility of the isolated strains.