Voriconazole resistance genes in Aspergillus flavus clinical isolates.

OBJECTIVE: The present study was designed to discover novel biomarkers involved in voriconazole resistance in clinical isolates of Aspergillus flavus. MATERIALS AND METHODS: Two voriconazole non-wild-type and two voriconazole-wild-type A. flavus clinical isolates were selected to evaluate possible molecular mechanism involved in A. flavus resistance to voriconazole using the mutation assessment, Quantitative real- time PCR of cyp51A and cyp51C genes and complementary DNA- amplified fragment length polymorphism technique. RESULTS: No mutations were seen in the cyp51A and cyp51C genes in voriconazole non-wild-type isolates compared to wild- type and reference strains. Regarding to mRNA expression results, no changes were observed in expression fold of cyp51A and cyp51C mRNA expression level in first non- wild- type isolate compared to wild-type isolate. For second isolate cyp51C mRNA expression level was down regulated (5.6 fold). The set of genes including ABC fatty acid transporter XM- 002375835 and aldehydereductase XM- 002376518 and three unknown functional genes were identified. Based on results, the over-expression of AKR1 and ABC fatty acid transporter in the voriconazole non- wild- type isolates suggests these genes could represent a novel molecular marker linked to the voriconazole resistance in A. flavus. CONCLUSION: The results obtained in this study showed a novel finding as the authors identified AKR1 and ABC fatty acid transporter genes as possible voriconazole target genes in Iranian clinical isolates of A. flavus.

Exploring the molecular mechanism of azole resistance in Aspergillus fumigatus.

Aspergillus infections are increasingly recognized as a global health problem because of limited antifungal drugs and occurrence of azole resistance worldwide. More cyp51-mediated and non-cyp51-mediated mechanisms of azole resistance have been identified in clinical and laboratory studies in recent years with applications of molecular biotechnology including next-generation sequencing, reverse genetics and so on. In this review, current research on the molecular mechanisms of azole resistance in A. fumigatus were presented and summarized and meanwhile the putative clinical relevance of these findings from bench work were discussed. Important aims are to gain more insight to mechanism of azole resistance and provide some efficient lead for prevention strategy.

Bacterial extract OM-85 BV protects mice against experimental chronic rhinosinusitis.

OBJECTIVES: To investigate the therapeutic effects of OM-85 BV as an adjunctive treatment on experimental chronic rhinosinusitis (CRS) in mice. METHODOLOGY: Female BALB/c mice aged 8-12 weeks were sensitized and administrated by intranasal Aspergillus fumigatis (AF) three times per week for 1 week, 3 weeks, 2 months and 3 months (n = 10 each time point). The mice were randomly and equally assigned to four groups: normal control group, model group, OM-85-BV plus amoxicillin group, and isolated amoxicillin group. Inflammatory changes were determined by hematoxylin-eosin (HE) staining. The expression levels of suppressor of cytokine signaling (SOCS) 1, SOCS3, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in samples were assessed by using real-time PCR (RT-PCR) and Western blotting. RESULTS: There were significantly inflammatory and structural changes between the model and other groups. Compared to the model group, the mRNA expression levels of SOCS1, SOCS3, TNF-α, and IFN-γ were significantly decreased in OM-85-BV plus amoxicillin group and isolated amoxicillin group, along with the protein levels. CONCLUSION: The bacterial extract OM-85 BV is a low-cost alternatively adjunctive drug to treat CRS with simple oral administration, good safety, and few side effects.

Breakthrough Aspergillus fumigatus and Candida albicans double infection during caspofungin treatment: laboratory characteristics and implication for susceptibility testing.

Caspofungin is used for the treatment of acute invasive candidiasis and as salvage treatment for invasive aspergillosis. We report characteristics of isolates of Candida albicans and Aspergillus fumigatus detected in a patient with breakthrough infection complicating severe gastrointestinal surgery and evaluate the capability of susceptibility methods to identify candin resistance. The susceptibility of C. albicans to caspofungin and anidulafungin was investigated by Etest, microdilution (European Committee on Antibiotic Susceptibility Testing [EUCAST] and CLSI), disk diffusion, agar dilution, and FKS1 sequencing and in a mouse model. Tissue was examined by immunohistochemistry, PCR, and sequencing for the presence of A. fumigatus and resistance mutations. The MICs for the C. albicans isolate were as follows: >32 microg/ml caspofungin and 0.5 microg/ml anidulafungin by Etest, 2 microg/ml caspofungin and 0.125 microg/ml anidulafungin by EUCAST methods, and 1 microg/ml caspofungin and 0.5 microg/ml anidulafungin by CLSI methods. Sequencing of the FKS1 gene revealed a mutation leading to an S645P substitution. Caspofungin and anidulafungin failed to reduce kidney CFU counts in animals inoculated with this isolate (P > 0.05 compared to untreated control animals), while both candins completely sterilized the kidneys in animals infected with a control isolate. Disk diffusion and agar dilution methods clearly separated the two isolates. Immunohistochemistry and sequencing confirmed the presence of A. fumigatus without FSK1 resistance mutations in liver and lung tissues. Breakthrough disseminated aspergillosis and candidiasis developed despite an absence of characteristic FKS1 resistance mutations in the Aspergillus isolates. EUCAST and CLSI methodology did not separate the candin-resistant clinical isolate from the sensitive control isolate as well as did the Etest and agar methods.

Detection of Aspergillus ribosomal RNA using biotinylated oligonucleotide probes.

Aspergillosis continues to be a devastating disease entity that results in significant mortality in immunosuppressed patients. Rapid diagnosis is often required to initiate appropriate therapy. Although the histopathologist may be able to visualize fungal organisms in tissue specimens, the histology of Aspergillus species may overlap with a variety of fungi, so diagnosis often relies on fungal cultures that can take weeks to complete. Recently, an in situ hybridization assay targeting Aspergillus 5S ribosomal RNA (rRNA) was reported. This assay proved to be useful when fungal cultures were negative or not performed but when fungi compatible with Aspergillus species were identified in tissue sections. That study was performed to compare the probe described in the previous study (5S-1 probe) with two other probes specific for Aspergillus. Two customly designed 21- and 23-base oligonucleotide probes complementary to 5S (5S-2 probe) and 18S (18S-1 probe) rRNA of Aspergillus were synthesized and labeled with multiple biotin moieties at the 3' termini. By GenBank analysis, the sequence of the 18S-1 probe was shown to have 90% to 100% homology to Aspergillus fumigatus group, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus parasiticus, Aspergillus tamarii, and Aspergillus glaucus group; the 5S-2 probe was homologous to Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus awamori, and Aspergillus wentii. In situ hybridization was performed on 43 cases of Aspergillus infection including 41 localized aspergillomas in the lung, brain, sinonasal tract, and ear, and 2 cases of invasive aspergillosis involving pleura and soft tissue of the scapular region. The results were compared with those obtained using a previously reported 5S-1 probe. In situ hybridization was positive in 38, 38, and 40 cases with the 5S-1, 5S-2, and 18S-1 probes, respectively. The 18S-1 probe was most useful because of a wider detection spectrum. In situ hybridization for Aspergillus rRNA provides a useful means for rapidly and accurately identifying Aspergillus in tissues and may be useful if fungal organisms suggestive of Aspergillus species are present but if cultures are negative or have not been performed.