Clinical Fungal Isolates Research Articles

Identification of dermatophyte species causing onychomycosis and tinea pedis by MALDI‐TOF mass spectrometry

Identification of dermatophytes is currently performed based on morphological criteria and is increasingly supported by genomic sequence comparison. The present study evaluates an alternative based on the analysis of clinical fungal isolates by mass spectrometry. Samples originating from skin and nail were characterized morphologically and by sequencing the internal transcribed spacer 1 (ITS1), ITS2 and the 5.8S rDNA regions of the rDNA clusters. In a blind comparative study, samples were analyzed by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF MS). The mass spectra were compared to a database comprising of the spectral data of reference strains by applying the saramis software package. All fungal isolates belonging to the taxa Trichophyton rubrum, T. interdigitale, T. tonsurans, Arthroderma benhamiae and Microsporum canis were correctly identified, irrespective of host origin and pathology. To test the robustness of the approach, four isolates were grown on five different media and analyzed. Although the resulting mass spectra varied in detail, a sufficient number of signals were conserved resulting in data sets exploitable for unequivocal species identification. Taken together, the usually widespread dermatophytes can be identified rapidly and reliably by mass spectrometry. Starting from pure cultures, MALDI-TOF MS analysis uses very simple sample preparation procedures, and a single analysis is performed within minutes. Costs for consumables as well as preparation time are considerably lower than for PCR analysis.

High-Throughput Identification of Clinical Pathogenic Fungi by Hybridization to an Oligonucleotide Microarray

Here we report the development of an oligonucleotide microarray method that can identify fungal pathogens in a single reaction. Specific oligonucleotide probes targeted to internal transcribed spacer 2 were designed and synthesized. Fungal DNA was amplified by universal primers, and the PCR product was hybridized with the oligonucleotide microarray. A series of specific hybridization profiles corresponding to species were obtained. The 122 strains of fungal pathogens, including standard and clinically isolated strains, used to test the specificity, stability, and sensitivity of the microarray system belonged to 20 species representing 8 genera. We found that the microarray system can successfully discriminate among the fungal pathogens to the species level, with high specificity and stability. The sensitivity was 15 pg/ml of DNA. This oligonucleotide microarray system represents a rapid, simple, and reliable alternative to conventional methods of identifying common clinical fungal isolates.

Intrinsic in vitro susceptibility of primary clinical isolates of Aspergillus fumigatus, Aspergillus terreus, Aspergillus nidulans, Candida albicans and Candida lusitaniae against amphotericin B

A total of 60 clinical fungal isolates from patients without prior amphotericin B treatment and three control strains were evaluated for their intrinsic susceptibility to amphotericin B (AmB) using microdilution, Etest and disc diffusion assays, on three media each, Roswell Park Memorial Institute (RPMI) 1640, Antibiotic Medium 3 (AM3) and High Resolution Medium. The fungal strains included isolates of Aspergillus fumigatus (n = 10), Aspergillus terreus (n = 12), Aspergillus nidulans (n = 9), Candida albicans (n = 6) and Candida lusitaniae (n = 23). The A. terreus strains were significantly less susceptible to AmB than the A. fumigatus strains in all nine experimental settings (P-values ranging from 0.009 to <0.00001). The A. nidulans strains were equally susceptible to AmB as the A. fumigatus strains in seven of nine experimental settings and less susceptible in two (microdilution performed on RPMI and AM3, P = 0.01 and 0.007). The C. lusitaniae strains were equally susceptible to AmB as the C. albicans strains in seven of nine experimental settings and more susceptible in two (microdilution and Etest, both performed on AM3, P = 0.01 and 0.0002). Thus, we confirmed that A. terreus is intrinsically less susceptible to AmB than A. fumigatus. In contrast, nine German clinical isolates of Aspergillus nidulans were found equally susceptible to AmB as 10 isolates of A. fumigatus. Furthermore, we found 23 German clinical isolates of C. lusitaniae from patients without prior treatment with AmB equally susceptible to AmB as C. albicans.

P6.16 Efficacy of Glucoprotamin Containing Disinfectant Against Different Bacterial and Fungal Clinical Isolates

P6.16 Efficacy of Glucoprotamin Containing Disinfectant Against Different Bacterial and Fungal Clinical Isolates

Differences in beta-glucan levels in culture supernatants of a variety of fungi

(1-->3)-beta-d-glucan is a well known cell wall constituent of fungal isolates that can be detected by assays in vivo and in vitro. Previous studies have shown that different fungal isolates may show different levels of reactivity with an assay for beta glucan. In this study we evaluated the in vitro reactivity of 127 clinical fungal isolates belonging to 40 different genera, with the Glucatell assay. The majority of the fungal isolates released high levels of beta glucan. Beta glucan test reactivity appears to be species-specific and this may reflect the beta glucan content of the organism.

Relationship between MIC and Minimum Sterol 14α-Demethylation-Inhibitory Concentration as a Factor in Evaluating Activities of Azoles against Various Fungal Species

The minimum growth-inhibitory concentrations (MICs) of azole antifungals were compared to their minimum sterol 14alpha-demethylation-inhibitory concentrations (MDICs) for clinical fungal isolates. The ascomycetous Candida yeasts tested were clearly divided into two groups: group I, consisting of C. albicans, C. tropicalis, and C. lusitaniae, had MICs that were much higher than the MDICs, whereas group II, comprising C. glabrata, C. parapsilosis, C. guilliermondii, and C. krusei, had MICs that were approximately equal to the MDICs. In the ascomycetous fungi Aspergillus fumigatus and Sporothrix schenckii, the MICs were indistinguishable from the MDICs. In the basidiomycetous fungi Cryptococcus (Filobasidiella) neoformans, C. curvatus, and Trichosporon asahii, the MICs and the MDICs were practically identical. These results support the notion that there are two distinct classes of fungi differing in their degree of tolerance to sterol 14alpha-demethylation deficiency. These findings have significant implications for both fungal physiology and antifungal chemotherapy.

ANTIFUNGAL ACTIVITY OF ETHANOLIC LEAF EXTRACT OF &lt;i&gt;Eucalyptus camaldulensis&lt;/i&gt; Dehn. AGAINST RINGWORM PATHOGENS

Ethanolic leaf extract of Eucalyptus camaldulensis, dispersed in a concentrated sugar solution had marked fungicidal effect against clinical dermatophytic fungal isolates; Microsporium gypseum and Trichophyton mentagrophytes. Microsporium gypseum at an inoculum level of 4.8 x 103 cfu/ml and T. mentagrophytes at 6.3x103 cfu/ml were killed to 7 cfu/ml and 10 cfu/ml respectively within 4 hours in Sabouraud broth supplemented with 63% (w/w) of dextrose and containing 100% (w/w) of crude eucalypt leaf extract. Added organic matter (bovine serum) did not affect its antifungal activity. Although distilled H2O extract conferred an equally significant (P = 0.05) antifungal activity, concentrated sugar solution provided a better vehicle for clinical application of the extracts. However the antifungal activity of the extract was dose-dependent. Minimum inhibitory concentrations (MIC) of 0.2mg/ml and 0.4mg/ml were recorded for M. gypseum and T. mentagrophytes respectively. KEY WORDS: Antifungal, Eucalypt leaf extract, ringworm. Global Jnl Pure & Applied Sciences Vol.10(1) 2004: 37-41

Cunninghamella bertholletiae Endocarditis

Cunninghamella bertholletiae Endocarditis

In vitro susceptibility of clinical isolates of Zygomycota to amphotericin B, flucytosine, itraconazole and voriconazole.

The in vitro susceptibility of 29 clinical isolates of fungi belonging to the division Zygomycota was reviewed. A broth microdilution test was performed by following the NCCLS reference method, with minor modifications. The species of Zygomycota tested were resistant in vitro to flucytosine, itraconazole and voriconazole. Amphotericin B showed the best activity against most of the strains evaluated.

Prospective study of Candida species in patients at a comprehensive cancer center.

Since most nosocomial systemic yeast infections arise from the endogenous flora of the patient, we prospectively evaluated the species stratification and antifungal susceptibility profile of Candida spp. associated with heavy colonization and systemic infection in patients at Memorial Sloan-Kettering Cancer Center in New York. A total of 349 Candida isolates were obtained from 223 patients during the later half of 1998. Cancer was the most common underlying disease, occurring in 91% of the patients, including 61.8% with organ and 23.7% with hematological malignancies; 4.4% of the patients had AIDS. Candida albicans was the predominant species (67.3%); among 114 non-albicans Candida spp., C. glabrata (45.6%) was the most frequent, followed by C. tropicalis (18.4%), C. parapsilosis (16.6%), and C. krusei (9.6%). The overall resistance to triazole-based agents among all yeast isolates was 9.4 and 10.8% for fluconazole and itraconazole, respectively. A total of 5% of C. albicans strains were resistant to triazole antifungals, whereas 30.8 and 46.2% of C. glabrata strains were resistant to fluconazole (MIC > or = 64 microg/ml) and itraconazole (MIC > or = 1 microg/ml), respectively. A significant association was observed between prior treatment with triazole and isolation of fluconazole-resistant C. albicans (P = 0.005, OR 36), although this relationship was not seen in C. glabrata isolates (P = 0.4). This study reinforces the importance of periodic, prospective surveillance of clinical fungal isolates to determine appropriate prophylactic, empiric, and preemptive antifungal therapy for the highly susceptible patient population.

Development of a PCR-Based Line Probe Assay for Identification of Fungal Pathogens

We report on a reverse-hybridization line probe assay (LiPA) which when combined with PCR amplification detects and identifies clinically significant fungal pathogens including Candida, Aspergillus, and Cryptococcus species. DNA probes have been designed from the internal transcribed-spacer (ITS) regions of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Candida dubliniensis, Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus versicolor, Aspergillus nidulans and Aspergillus flavus. The probes were incorporated into a LiPA for detection of biotinylated ITS PCR products, and the specificity of the probes was evaluated. We established LiPA detection limits for ITS 1 and for full ITS amplicons for genomic DNA from C. albicans, A. fumigatus, and C. neoformans. Further evaluation of the LiPA was carried out on clinical fungal isolates. One hundred twenty-seven isolates consisting of dimorphic yeasts and dermatophytic and filamentous fungi were tested by the LiPA, which correctly identified 77 dimorphic yeasts and 23 of the filamentous isolates; the remaining 27 isolates represented species of fungi for which probes were not included in the LiPA. The fungal-PCR-LiPA technology was applied to blood samples inoculated with Candida cells which were pretreated by minibead beating to mechanically disrupt the cells, with the DNA extracted by either a previously described guanidium thiocyanate-silica method or the commercially available QIAmp tissue kit. PCR amplification of the extracted DNA and subsequent DNA probe hybridization in the LiPA assay yielded detection limits of 2 to 10 cells/ml. An internal standard control was included in the PCR amplification to monitor for PCR inhibition. This fungal PCR-LiPA assay is robust and sensitive and can easily be integrated into a clinical-testing laboratory with the potential for same-day diagnosis of fungal infection.

Characterization of a fish antimicrobial peptide: gene expression, subcellular localization, and spectrum of activity.

Antimicrobial peptides are proposed to act as the first line of mucosal host defense by exerting broad-spectrum microbicidal activity against pathogenic microbes. Pleurocidin, a new 25-residue linear antimicrobial peptide, was recently isolated from the skin secretions of winter flounder (Pleuronectes americanus). The present study identifies the cDNA and gene encoding pleurocidin. The pleurocidin gene comprises four exons. Its upstream region demonstrates consensus binding sequences for transcription factors found in host defense genes in mammals, including sequences identical to the NF-IL6 and alpha and gamma interferon response elements. Pleurocidin is predicted to exist as a 68-residue prepropeptide that undergoes proteolytic cleavage of its amino-terminal signal and carboxy-terminal anionic propiece to form the active, mature peptide. Transmission electron microscopy localized pleurocidin to the mucin granules of skin and intestinal goblet cells. Significant synergy was shown to occur between pleurocidin and D-cycloserine targeting Mycobacterium smegmatis. Pleurocidin was functionally active at physiologic concentrations of magnesium and calcium; however, high concentrations of these divalent cations ablated pleurocidin's activity against a standard test strain, Escherichia coli D31. Pleurocidin was tested against bacterial and fungal clinical isolates and showed broad-spectrum antimicrobial activity. Together, these data support the hypothesis that pleurocidin participates in innate mucosal immunity, and it may prove to be a beneficial therapeutic agent.

Rapid identification of fungi by using the ITS2 genetic region and an automated fluorescent capillary electrophoresis system.

Invasive fungal disease often plays an important role in the morbidity and mortality of immunocompromised patients. The poor sensitivity of current fungal blood culture and histological practices has led to the development of highly sensitive and specific molecular techniques, such as the PCR. Sequence variability of the internal transcribed spacer 2 (ITS2) region of fungi is potentially useful in rapid and accurate diagnosis of clinical fungal isolates. PCR with fungus-specific primers targeted toward conserved sequences of the 5.8S and 28S ribosomal DNA (rDNA) results in amplification of the species-specific ITS2 regions, which are variable in amplicon length. We have made use of the ABI PRISM 310 genetic analyzer and the ABI PRISM 310 GeneScan analysis software for the determination of variable size differences of the ITS2 region of clinically important fungi, including Candida and non-Candida yeasts, Aspergillus species, and a variety of dermatophytes. No cross-reaction occurred when samples were tested against human and bacterial genomic DNA. We have found that most clinically significant fungal isolates can be differentiated by this method, and it therefore serves to be a promising tool for the rapid (<7 h) diagnosis of fungemia and other invasive fungal infections.

Stable phenotypic resistance of Candida species to amphotericin B conferred by preexposure to subinhibitory levels of azoles.

The fungicidal activity of amphotericin B (AmB) was quantitated for several Candida species. Candida albicans and C. tropicalis were consistently susceptible to AmB, with less than 1% survivors after 6 h of exposure to AmB. C. parapsilosis and variants of C. lusitaniae and C. guilliermondii were the most resistant, demonstrating 50 to 90% survivors in this time period and as high as 1% survival after a 24-h exposure time. All Candida species were killed (<1% survivors) after 24 h of exposure to AmB. In contrast, overnight exposure to either fluconazole or itraconazole resulted in pronounced increases in resistance to subsequent exposures to AmB. Most dramatically, C. albicans was able to grow in AmB cultures after azole preexposure. Several other Candida species did not grow in AmB but showed little or no reduction in viability after up to 24 h in AmB. Depending on the growth conditions, Candida cells preexposed to azoles may retain AmB resistance for days after the azoles have been removed. If this in vitro antagonism applies to the clinical setting, treatment of patients with certain antifungal combinations may not be beneficial. The ability of some Candida isolates to survive transient exposures to AmB was not reflected in the in vitro susceptibility changes as measured by standard MIC assays. This finding should be considered in studies attempting to correlate patient outcome with in vitro susceptibilities of clinical fungal isolates. Patients who fail to respond to AmB may be infected with isolates that are classified as susceptible by standard in vitro assays but that may be resistant to transient antifungal exposures which may be more relevant in the clinical setting.

Antifungal activity of the essential oil of Melaleuca alternifolia (tea tree oil) against pathogenic fungi in vitro.

The in vitro antifungal activity of tea oil, the essential oil of Melaleuca alternifolia, has been evaluated against 26 strains of various dermatophyte species, 54 yeasts, among them 32 strains of Candida albicans and other Candida sp. as well as 22 different Malassezia furfur strains. Minimum inhibitory concentrations (MIC) of tea tree oil were measured by agar dilution technique. Tea tree oil was found to be able to inhibit growth of all clinical fungal isolates. For the investigated dermatophytes MIC values from 1,112.5 to 4,450.0 micrograms/ml with a geometric mean of 1,431.5 micrograms/ml were demonstrated. Both C. albicans strains and the other strains belonging to the genus Candida and Trichosporon appeared to be slightly less susceptible to tea tree oil in vitro. However, their MIC values, which varied from 2,225.0 to 4,450.0 micrograms/ml (geometric mean 4,080 micrograms/ml), indicated moderate susceptibility to the essential oil of M. alternifolia. The lipophilic yeast M. furfur seemed to be most susceptible to tea tree oil. MIC values between 556.2 and 4,450.0 micrograms/ml (geometric mean 1,261.5 micrograms/ml) were found against the tested M. furfur strains. However, when calculated as percentage tea tree oil of the agar, the above-mentioned concentrations correspond to 0.5-0.44% tea tree oil content. These values are far below the usual relatively high therapeutic concentrations of the agent; approximately 5-10% solution or even the concentrated essential oil are used for external treatment. In comparison with tea tree oil, in vitro susceptibility against miconazole, an established topical antifungal, was tested. As expected, very low MIC values for miconazole were found for dermatophytes (geometric mean 0.2 microgram/ml), yeasts (geometric mean 1.0 microgram/ml), and M. furfur (geometric mean 2.34 micrograms/ml). It is suggested that the in vivo effect of tea tree oil ointment in the therapy of fungal infections of the skin and mucous membranes as well as in the treatment of dandruff, a mild form of seborrheic dermatitis, may be at least partly due to an antifungal activity of tea tree oil.

Susceptibility of clinical isolates of fungi to saperconazole.

The in vitro activity of saperconazole against 228 strains of mycotic agents belonging to 48 species was investigated. Susceptibility testing was performed using a microtiter broth dilution method. Isolates of Candida albicans, C. tropicalis and Torulopsis glabrata showed distinct intra-species variation of susceptibility with MIC values ranging from 0.045 to 100 mg l-1. The drug was inhibitory for the dermatophytes at a relatively narrow range of concentrations, most isolates being inhibited at MIC 0.78 mg l-1. The strongest antifungal potency of saperconazole was exerted against clinical isolates of the genus Aspergillus (MIC 90% = 0.19 mg l-1). Concentrations up to 100 mg l-1 had no macroscopically recognizable effect on the growth of zygomycetous fungi (Mucor, Rhizopus, Syncephalastrum). Species of the genus Absidia with their good sensitivity are an exception. Justification of in vitro susceptibility testing of triazoles is discussed. In the author's opinion, MIC values can serve as an informative parameter showing the range of indications of these antifungals for treatment. It is concluded that saperconazole exhibits a very good activity against a broad spectrum of medically important fungi in vitro and can be considered a promising antifungal drug.

Impregnated Disk Method for Antifungal Antibiotic Testing

Sixteen clinical fungal isolates were tested for antibiotic susceptibility by using disks impregnated with growth medium and antibiotic. The procedure was easier to perform, less time consuming, and as reliable as other currently available methods.