Comparison of fungemia caused by Candida and non-Candida rare yeasts: a retrospective study from a tertiary care hospital.

Although Candida species are the most common cause of fungemia, non-Candida rare yeasts (NCY) have been increasingly reported worldwide. Although the importance of these yeast infections is recognized, current epidemiological information about these pathogens is limited, and they have variable antifungal susceptibility profiles. In this study, we aimed to evaluate the clinical characteristics for fungemia caused by NCY by comparing with candidemia. The episodes of NCY fungemia between January 2011 and August 2023 were retrospectively evaluated in terms of clinical characteristics, predisposing factor, and outcome. In addition, a candidemia group, including patients in the same period was conducted for comparison. Antifungal susceptibility tests were performed according to the reference method. A total of 85 patients with fungemia episodes were included: 25 with NCY fungemia and 60 with candidemia. Fluconazole had high minimal inhibitory concentration (MIC) values against almost all NCY isolates. The MIC values for voriconazole, posaconazole, and amphotericin B were ≤ 2 µg/ml, and for caspofungin and anidulafungin were ≥ 1 µg/ml against most of isolates. Hematological malignancies, immunosuppressive therapy, neutropenia and prolonged neutropenia, polymicrobial bacteremia/fungemia, preexposure to antifungal drugs, and breakthrough fungemia were associated with NCY fungemia, whereas intensive care unit admission, diabetes mellitus, urinary catheters, and total parenteral nutrition were associated with candidemia. In conclusion, the majority of fungemia due to NCY species was the problem, particularly in hematology units and patients with hematological malignancy. Preexposure to antifungal drugs likely causes a change in the epidemiology of fungemia in favor of non-albicans Candida and/or NCY.

Among all fungemia episodes, hematological malignancies, immunosuppressive therapy, neutropenia, and preexposure to antifungals were risk factors for non-Candida yeast fungemia; diabetes mellitus, urinary catheters, and total parenteral nutrition were risks for candidemia.

European candidaemia is characterised by notable differential epidemiology and susceptibility pattern: Results from the ECMM Candida III study.

The objectives of this study were to assess Candida spp. distribution and antifungal resistance of candidaemia across Europe. Isolates were collected as part of the third ECMM Candida European multicentre observational study, conducted from 01 to 07-07-2018 to 31-03-2022. Each centre (maximum number/country determined by population size) included ∼10 consecutive cases. Isolates were referred to central laboratories and identified by morphology and MALDI-TOF, supplemented by ITS-sequencing when needed. EUCAST MICs were determined for five antifungals. fks sequencing was performed for echinocandin resistant isolates. The 399 isolates from 41 centres in 17 countries included C. albicans (47.1%), C. glabrata (22.3%), C. parapsilosis (15.0%), C. tropicalis (6.3%), C. dubliniensis and C. krusei (2.3% each) and other species (4.8%). Austria had the highest C. albicans proportion (77%), Czech Republic, France and UK the highest C. glabrata proportions (25-33%) while Italy and Turkey had the highest C. parapsilosis proportions (24-26%). All isolates were amphotericin B susceptible. Fluconazole resistance was found in 4% C. tropicalis, 12% C. glabrata (from six countries across Europe), 17% C. parapsilosis (from Greece, Italy, and Turkey) and 20% other Candida spp. Four isolates were anidulafungin and micafungin resistant/non-wild-type and five resistant to micafungin only. Three/3 and 2/5 of these were sequenced and harboured fks-alterations including a novel L657W in C. parapsilosis. The epidemiology varied among centres and countries. Acquired echinocandin resistance was rare but included differential susceptibility to anidulafungin and micafungin, and resistant C. parapsilosis. Fluconazole and voriconazole cross-resistance was common in C. glabrata and C. parapsilosis but with different geographical prevalence.

Knockout of elF4E using CRISPR/Cas9 for large-scale production of resistant cucumber cultivar against WMV, ZYMV, and PRSV.

CRISPR/Cas9 is one of the most robust technologies for plant breeding enabling precise and efficient modifications in a genome. This technology is being used for the manipulation of target genes in a host to develop resistance against the plant pathogens. Cucumis sativus elF4E is one of the target genes playing a key role in viral infection during interaction with potyvirus viral proteins genome linked (VPg). Nevertheless, the allelic and positional effect of elF4E mutations in C. sativus is to be clarified in elF4E-VPg interaction. In addition, there are entanglements in the massive production of pathogen-resistant cultivars suitable for commercial production using CRISPR/Cas9 technology. Therefore, we targeted different positions of the elF4E in G27 and G247 inbred lines, using specific gRNA1 and gRNA2 for the first and third exons, respectively, and 1,221 transgene-free plants were selected in segregated T1 generation, where 192 G27 and 79 G247 plants had the least mutation at Cas9 cleavage site of gRNA1 or gRNA2. Crossing was performed to see allelic effects of elfF4E mutations in F1 populations, which were homozygous and heterozygous single (elF4E_1DEL or elF4E_3DEL) and double (elF4E_1-3DEL) mutants. Disease symptoms of watermelon mosaic virus (WMV), papaya ringspot virus (PRSV), and zucchini yellow mosaic virus (ZYMV) were evaluated in both non-edited and edited F1 plants, and we did not observe any symptom in homozygous elF4E_1-3DEL and elF4E_1DEL mutants. However, homozygous elF4E_3DEL was positive in reverse transcription polymerase chain reaction (RT-PCR), even if there were no significant symptoms on the inoculated leaves. ELISA and qRT-PCR indicated lower viral accumulation in homozygous elF4E_3DEL than heterozygous and non-edited plants. Regeneration and transformation protocols were also optimized comprehensively for both the genotypes. The average number of shoots/100 explants was determined for both G27 and G247 as 13.6 and 18.0, respectively. We could not detect any distinguishing difference between the non-edited and edited F1 plants for yield and morphology. Our results demonstrate an effective route for mass production of viral resistant cultivars of cucumber to WMV, ZYMV, and PRSV. In this way, the pathogen-resistant cultivars could be generated to reduce the losses caused by these pathogens in cucumber production.

Frequency of azole resistance in clinical and environmental strains of Aspergillus fumigatus in Turkey: a multicentre study.

OBJECTIVES: Aspergillus fumigatus causes several diseases in humans and azole resistance in A. fumigatus strains is an important issue. The aim of this multicentre epidemiological study was to investigate the prevalence of azole resistance in clinical and environmental A. fumigatus isolates in Turkey. METHODS: Twenty-one centres participated in this study from 1 May 2018 to 1 October 2019. One participant from each centre was asked to collect environmental and clinical A. fumigatus isolates. Azole resistance was screened for using EUCAST agar screening methodology (EUCAST E.DEF 10.1) and was confirmed by the EUCAST E.DEF 9.3 reference microdilution method. Isolates with a phenotypic resistance pattern were sequenced for the cyp51A gene and microsatellite genotyping was used to determine the genetic relationships between the resistant strains. RESULTS: In total, resistance was found in 1.3% of the strains that were isolated from environmental samples and 3.3% of the strains that were isolated from clinical samples. Mutations in the cyp51A gene were detected in 9 (47.4%) of the 19 azole-resistant isolates, all of which were found to be TR34/L98H mutations. Microsatellite genotyping clearly differentiated the strains with the TR34/L98H mutation in the cyp51A gene from the strains with no mutation in this gene. CONCLUSIONS: The rate of observed azole resistance of A. fumigatus isolates was low in this study, but the fact that more than half of the examined strains had the wild-type cyp51A gene supports the idea that other mechanisms of resistance are gradually increasing.

Correlation of Indeterminate Lesions of COVID-19 Pneumonia Detected on Computed Tomography with RT-PCR Results.

BACKGROUND: The typical findings of COVID-19 pneumonia include multilobar groundglass opacities and consolidation areas observed predominantly in the basal and peripheral parts of both lungs in computed tomography. OBJECTIVE: The current study aimed to correlate indeterminate lesions of COVID-19 pneumonia detected on computed tomography with the results of the reverse transcription-polymerase chain reaction (RT-PCR) test. METHODS: Patients with high-resolution computed tomography images that were reported to contain indeterminate lesions in terms of COVID-19 pneumonia were included retrospectively in the study. The lesions were categorized and the patterns were classified. The RT-PCR-positive and the RTPCR- negative patients were compared. P<0.05 was accepted as the statistical significance limit. RESULTS: The RT-PCR-positive patients exhibited a higher rate of peripheral lesions. Limited consolidation areas were not detected in the RT-PCR-positive patients. In the RT-PCR-negative patients, the rates of acinar nodules and the tree-in-bud pattern were significantly higher. The RTPCR- negative patients had higher nodular contour features and lesion coalescence. In the subgroup consisting of lesions with ground-glass opacities and/or ground-glass opacity around the nodule, the rate of nodular contour positivity was significantly higher in the RT-PCR- positive patients. CONCLUSION: COVID-19 pneumonia should be suspected when peripheral indeterminate lesions are detected. When indeterminate lesions, such as tree-in-bud pattern, acinar nodules and limited consolidation area are detected, alternative diagnoses should be considered first, even if there are ground glass opacities accompanying these lesions.

Effects of calcineurin inhibitors, cyclosporine A and tacrolimus (FK506), on the activity of antifungal drugs against Candida spp.

Introduction. The simultaneous use of antifungals with immunosuppressive agents has become a necessity for patients taking immunosuppressive therapy. However, antifungal drugs are problematic because of their limited target.Hypothesis. Scientists have been searching for new antifungals and some compounds with at least additive effects on antifungals. Calcineurin inhibitors used as immunosuppressive agents also attract attention due to their antifungal property.Aim. To evaluate the activity of two calcineurin inhibitors alone and in combination with amphotericin B (AMB), caspofungin (CAS), itraconazole (ITR), voriconazole (VOR) and fluconazole (FLU).Methodology. MICs of AMB, CAS, ITR, VOR, FLU and cyclosporine A (CsA) and tacrolimus (TAC) as calcineurin inhibitors were evaluated by the broth microdilution method against Candida albicans (n=13), C. krusei (n=7) and C. glabrata (n=10). Checkerboard and time-kill methods were performed to investigate the activity of combining calcineurin inhibitors with antifungal drugs.Results. The lowest MIC values were detected with VOR for all Candida isolates tested. Although we did not detect any inhibition for CsA or TAC alone at concentrations tested in this study, the combinations of CAS with CsA showed the highest synergistic activity (36.7%) by the checkerboard method, and CAS with CsA and ITR with TAC combinations exhibited apparent synergistic interaction by the time-kill method. However, the combinations of both CsA and TAC with AMB resulted in antagonistic interactions, especially against C. krusei isolate in time-kill testing.Conclusion. Synergistic interactions in the combinations of TAC or CsA with antifungal drugs, except for AMB, in many concentrations was found to be promising in terms of the treatment of patients with fungal infections.

In Vitro Effects of Farnesol Alone and in Combination with Antifungal Drugs Against Aspergillus Clinical Isolates.

Farnesol is an extracellular quorum-sensing molecule produced by Candida albicans. Farnesol is also a sesquiterpene alcohol existing in many herbal products and has various activity against fungal cells. We aimed to investigate the efficacy of farnesol alone and the contribution of farnesol on the activity of voriconazole and amphotericin B against Aspergillus clinical isolates in vitro. A total of 45 Aspergillus clinical isolates were used in this study. The MIC values of voriconazole, amphotericin B, and farnesol were determined using reference broth microdilution method. The interactions of farnesol with voriconazole and amphotericin B were investigated by the checkerboard method and evaluated based on the fractional inhibitor concentration index (FICI). The MIC ranges of farnesol, voriconazole, and amphotericin B were 1,500-6,000 µM, 0.125-1 µg/mL, and 0.125-0.5 µg/mL against Aspergillus fumigatus isolates, 3,000-12,000 µM, 0.125-0.5 µg/mL, and 0.25-2 µg/mL against Aspergillus flavus isolates, respectively. The most common interaction in combination tests was "no interaction," and synergistic interaction was not detected. The combinations of farnesol with voriconazole and amphotericin B had antagonistic activity against 38% and 27% of all isolates, respectively.We concluded that the responses of different fungal species against farnesol are variable, and different interactions may be observed when it is combined with different antifungals. Therefore, it should be noted that farnesol may have an adverse effect on some fungi or interact negatively with antifungals used in combination.

Does concomitant bacteraemia hide the fungi in blood cultures? An in vitro study.

Introduction. Polymicrobial infections including yeasts and bacteria are not rare and patients with polymicrobial bloodstream infection have higher early and overall case fatality rates. The diagnosis of invasive fungal and bacterial infections is mainly based on blood culture.Aim. The aim was to reveal the effect of concomitant bacteraemia on the detection of fungi from blood cultures in the presence of polymicrobial bloodstream infections involving Candida and non-Candida fungi and to show the superiority of blood culture bottles including selective fungal media in such situations.Methodology. Twenty-four polymicrobial bloodstream infection models - involving one fungus and one bacterium - were constituted by using clinical blood culture isolates (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Candida glabrata, Fusarium solani and Trichosporon asahii). The Plus Aerobic/F (PAF) and Mycosis IC/F (MICF) culture bottles were used with the BACTEC 9240 device. After a bottle signalled positive, direct microscopic examination and subcultures on agar plates were performed.Results. All of fungi that were inoculated alone and in combination were detected by both direct microscopic examination and subcultures on agar plates from MICF bottles, whereas direct microscopic examination only revealed the bacterial agents from PAF bottles including combinations. Furthermore, fungal growth was hidden by bacterial growth on blood agar subcultures from PAF bottles including combinations of F. solani, C. glabrata or T. asahii with bacteria.Conclusion. Blood culture bottles including selective fungal media that can allow selective growth of fungi and earlier detection of some species should be preferred in addition to non-selective blood culture bottles, especially in specific patient populations. Further, the use of selective agar plates such as inhibitory mould agar may contribute to the solution of this problem in clinical laboratories.

Mating genotypes and susceptibility profiles of clinical isolates of Candida glabrata from Turkey.

The sexual cycle of Candida glabrata is not known; however, genomic evidence is indicative of recombination among subpopulations and the genome harbours genes necessary for undergoing mating and meiosis, which may increase fitness. The relationship between specific mating type-like (MTL) loci and antifungal susceptibility is not well understood in C. glabrata. We investigated different combinations of clinical C. glabrata isolate mating types and their antifungal susceptibility profiles. Allele profiles of the mating genes of 103 clinical C. glabrata isolates were identified, and their antifungal susceptibility to azoles, echinocandins and amphotericin B were compared. The majority (88.3%) of screened isolates harboured the a allele in the locus. The MTL1, MTL2 and MTL3 loci harboured a (88.3%), a (95.1%), and α (71.8%) alleles, respectively. The C. glabrata isolates were susceptible to echinocandins but displayed high minimal inhibitory concentrations (MICs) for azoles. The MIC ranges and MIC90 values of all isolates were 1.0 to ≥64 and 8.0 µg mL-1 for fluconazole, 0.06 to ≥16.0 and 0.5 µg mL-1 for voriconazole, 0.06 to ≥16.0 and 1.0 µg mL-1 for posaconazole, ≤0.015 to 0.06, and 0.03 µg mL-1 for caspofungin, ≤0.015 to 0.06 and 0.015 µg mL-1 for anidulafungin and 0.5-2 and 2.0 µg mL-1 for amphotericin B, respectively. The mating gene alleles of the clinical C. glabrata isolates were not associated with differences in the MICs of the tested antifungals, except for the MTL3 α-allele and echinocandins. The mating genotypes of the clinical C. glabrata isolates had no recognisable common effect on antifungal susceptibility.

First multicentre report of in vitro resistance rates in candidaemia isolates in Turkey.

OBJECTIVES: This study investigated the antifungal resistance rates of isolates from candidaemia patients in 12 tertiary-care centres in Turkey. METHODS: A total of 1991 Candida spp. isolates from 12 centres isolated from 1997-2017 were included in the study. Species/species complex (SC) identification was performed using conventional methods in all centres, occasionally accompanied by MALDI-TOF/MS. Antifungal susceptibility testing was performed for amphotericin B, fluconazole, itraconazole, posaconazole, voriconazole and micafungin (as echinocandin class representative) using the CLSI microdilution method. Resistance rates were determined according to CLSI clinical breakpoints (CBPs). For drugs and species with undetermined CBPs, epidemiological cut-off values were used for wild-type (WT)/non-WT categorisation. RESULTS: No or low rates of resistance were detected in general for tested Candida spp. isolates. Specifically, overall resistance to fluconazole in isolates of Candida parapsilosis SC and Candida glabrata SC were 7.7% and 0.9%, respectively. Resistance rates for C. parapsilosis SC varied extensively from one center to other (0-47.1%). Importantly, no echinocandin resistance was detected. Rates of non-WT isolates were also generally low: fluconazole against Candida lusitaniae, 4.3%; posaconazole against C. parapsilosis SC, 3.5%; posaconazole against Candida krusei, 1.9%; and voriconazole against C. glabrata SC, 0.5%. CONCLUSION: This is the first multicentre report of antifungal resistance rates among candidaemia isolates in Turkey, suggesting low resistance rates in general. Due to varying rates of fluconazole resistance in C. parapsilosis SC isolates that was detected at remarkably high levels in some centres, further studies are warranted to explore the source, clonal relatedness and resistance mechanisms of the isolates.

Genetic Diversity and Antifungal Susceptibility of Candida parapsilosis Sensu Stricto Isolated from Bloodstream Infections in Turkish Patients.

Candida parapsilosis sensu stricto is an emerging cause of hospital-acquired Candida infections, predominantly in southern Europe, South America, and Asia. We investigated the genetic diversity and antifungal susceptibility profile of 170 independent C. parapsilosis sensu stricto strains obtained from patients with candidemia who were treated at the Ege University Hospital in Izmir, Turkey, between 2006 and 2014. The identity of each strain was confirmed via PCR amplification and digestion of the secondary alcohol dehydrogenase-encoding gene. The 24-h geometric mean minimum inhibitory concentrations of the antifungal agents, in increasing order, were as follows: posaconazole, 0.10 µg/mL; voriconazole, 0.21 µg/mL; caspofungin, 0.38 µg/mL; amphotericin B, 0.61 µg/mL; anidulafungin, 0.68 µg/mL; and fluconazole, 2.95 µg/mL. Microsatellite genotyping of the isolates (using fluorescently labeled primers and a panel of four different short-nucleotide repeat fragments) identified 25, 17, 17, and 8 different allelic genotypes at the CP6, B5, CP4, and CP1 locus, respectively. Posaconazole, caspofungin, and amphotericin B showed the greatest in vitro activity of the tested systemic azole, echinocandin, and polyene agents, respectively, and the observed antifungal susceptibility of the isolates was shown to be independent of their isolation source. We obtained a combined discriminatory power of 0.99 with a total of 130 genotypes for 170 isolates tested. Finally, microsatellite profiling analysis confirmed the presence of identical genotype between separate isolates, supporting that effective surveillance and infection-prevention programs are essential to limit the impact of C. parapsilosis sensu stricto on hospitalized patients' health.

Biofilm Formation and Resistance to Fungicides in Clinically Relevant Members of the Fungal Genus Fusarium.

Clinically relevant members of the fungal genus, Fusarium, exhibit an extraordinary genetic diversity and cause a wide spectrum of infections in both healthy individuals and immunocompromised patients. Generally, Fusarium species are intrinsically resistant to all systemic antifungals. We investigated whether the presence or absence of the ability to produce biofilms across and within Fusarium species complexes is linked to higher resistance against antifungals. A collection of 41 Fusarium strains, obtained from 38 patients with superficial and systemic infections, and three infected crops, were tested, including 25 species within the Fusarium fujikuroi species complex, 14 from the Fusarium solani species complex (FSSC), one Fusarium dimerum species complex, and one Fusarium oxysporum species complex isolate. Of all isolates tested, only seven strains from two species of FSSC, five F. petroliphilum and two F. keratoplasticum strains, recovered from blood, nail scrapings, and nasal biopsy samples, could produce biofilms under the tested conditions. In the liquid culture tested, sessile biofilm-forming Fusarium strains exhibited elevated minimum inhibitory concentrations (MICs) for amphotericin B, voriconazole, and posaconazole, compared to their planktonic counterparts, indicating that the ability to form biofilm may significantly increase resistance. Collectively, this suggests that once a surface adherent biofilm has been established, therapies designed to kill planktonic cells of Fusarium are ineffective.

Evaluation of direct antifungal susceptibility testing methods of Candida spp. from positive blood culture bottles.

BACKGROUND: Blood culture is the gold standard diagnostic method in candidemia in spite of long result time and low sensitivity rate. Early diagnosis is crucial for management of candidemia because a delay in treatment is related with increased mortality. We aimed to evaluate the direct applicability of antifungal susceptibility testing methods from positive blood culture bottles to save at least 24 hours. METHODS: Blood culture bottles were inoculated with 62 Candida isolates. Etest and broth microdilution (BMD) methods for six antifungals, disk diffusion (DD) method for two antifungals were performed, both directly from bottles and standardly. RESULTS: Essential agreements between direct and standard Etest methods were 87.1% for caspofungin and >90% for other antifungals, but the agreements of them with reference BMD were relatively low. Essential agreement between direct and standard BMD was >93%. Correlation between direct and standard DD methods was very high, negative correlations were observed between reference BMD and DD methods. CONCLUSION: BMD is a reference method to evaluate the antifungal susceptibility, direct application of BMD might provide reliable results at least 24 hours earlier. Direct DD method may be a qualitative alternative. Direct susceptibility testing methods may be very useful to initiating the appropriate treatment on time.

Evaluation of three different bottles in BACTEC 9240 automated blood culture system and direct identification of Candida species to shorten the turnaround time of blood culture.

PURPOSE: Candida spp. are the most common causes of fungemia. Rapid and accurate diagnostic methods are very important for appropriate management of candidemia. At present, blood culture is the essential diagnostic test despite having a long detection time and low sensitivity rate. We aimed to investigate the ways to shorten the turnaround time from blood culture collection to final identification in candidemia. METHODOLOGY: Sixty clinical bloodstream isolates of Candida were included, and Plus Aerobic/F, Peds Plus/F and Mycosis IC/Fbottles were used with a BACTEC 9240 blood culture instrument. Germ tube production, carbohydrate assimilation (API 20C AUX) and peptide nucleic acid fluorescent in situ hybridization yeast traffic light tests were performed directly from positive-signalled bottles. RESULTS: Time to positivity of blood cultures was affected by species of Candida, fungal load and bottle type. Candidatropicalis had the shortest and Candidaglabrata had the longest time to positivity. Mycosis IC/F culture bottle had a significant superiority in the isolation of yeasts, especially for C. glabrata and if there was a low fungal load in the bottle. Direct germ tube test had 90 % sensitivity and 97.6 % specificity for Candidaalbicans in two hours after signalling. The compliance between direct and classical assimilation tests was 98.3 %. Sensitivity and specificity of peptide nucleic acid fluorescent in situ hybridization were 100 %. CONCLUSION: We think that it is possible to shorten the turnaround time for the identification of Candida in blood culture even with currently available methods.

Prevalence and epidemiology of tinea pedis and toenail onychomycosis and antifungal susceptibility of the causative agents in patients with type 2 diabetes in Turkey.

BACKGROUND: Diabetes patients are particularly susceptible to fungal infections because their vascular and immunological systems are compromised. OBJECTIVES: The present study aimed to determine prevalences of tinea pedis and onychomycosis, factors predisposing to their development, and antifungal susceptibilities of causative fungal species against fluconazole, itraconazole, and terbinafine in patients with type 2 diabetes mellitus (DM). METHODS: Study groups were defined according to hemoglobin A1C rates of ≥6.5% for the diabetes group and ≤5.7% for control subjects. A total of 600 diabetes subjects and 152 control subjects were evaluated. Rates of onychomycosis and tinea pedis in diabetes patients, and associations with age, gender, blood glucose level, duration of diabetes and serum lipid profile were investigated, as were the distribution and antifungal susceptibility of agents isolated. RESULTS: Patients with onychomycosis and/or tinea pedis numbered 85 in the diabetes group and nine in the control group (P = 0.006). The development of onychomycosis or tinea pedis was significantly related to increasing age and male gender. Although the most common agents were dermatophytes, non-dermatophyte fungal isolates were not uncommon. Terbinafine was the most effective drug against dermatophytes but was invalid for non-dermatophyte isolates by in vitro antifungal susceptibility testing. CONCLUSIONS: The development of onychomycosis or tinea pedis was significantly related to type 2 DM, increasing age, and male gender. The most common isolate was Trichophyton rubrum. The isolation and identification of the fungus is important to the effective management of tinea pedis and onychomycosis in diabetes patients because non-dermatophyte fungi can cause these infections.

Methicillin-resistant S. aureus colonization in intensive care unit patients: Early identification and molecular typing.

INTRODUCTION: Early detection of methicillin-resistant Staphylococcus aureus (MRSA) in colonized patients is very important for infection control procedures to prevent MRSA spread. We aimed to monitor MRSA carriage in intensive care unit (ICU) patients and to evaluate the speed and efficiency of conventional culture, immunological, chromogenic, and molecular methods together with genotyping. METHODOLOGY: Nasal and axillar swab specimens were obtained from patients in the ICUs of the general surgery and neurosurgery wards in a tertiary hospital once a week over four weeks between December 2009 and July 2010. Oxacillin and cefoxitin disk diffusion tests, oxacillin agar screening test, latex agglutination test, chromogenic agar, and real-time polymerase chain reaction (PCR) tests were used for isolation and identification of MRSA. MRSA isolates were typed using ribotyping and pulsed-field gel electrophoresis (PFGE) typing. RESULTS: MRSA colonization was detected in 48 of 306 patients by real-time PCR. The MRSA colonization rate was 6.2%, 15.5%, and 38.5% at admission and in the first and second weeks, respectively. Sensitivity, specificity, positive and negative predictive values for all phenotypic tests were 98%, 99.6%, 98%, and 99.6%, respectively. The shortest handle time was observed in PCR. A total of three banding patterns were obtained from MRSA isolates by ribotyping, and PFGE analyses revealed 17 different pulsotypes varying from 11 to 18 distinct bands, showing high genetic diversity among the samples. CONCLUSION: Phenotypic MRSA screening tests in our study exhibited similar performances. The superiority of real-time PCR is its short turnaround time.

Time-Kill Kinetics and In Vitro Antifungal Susceptibility of Non-fumigatus Aspergillus Species Isolated from Patients with Ocular Mycoses.

Aspergillus species can cause ocular morbidity and blindness, and thus, appropriate antifungal therapy is needed. We investigated the in vitro activity of itraconazole, voriconazole, posaconazole, caspofungin, anidulafungin, and amphotericin B against 14 Aspergillus isolates obtained from patients with ocular mycoses, using the CLSI reference broth microdilution methodology. In addition, time-kill assays were performed, exposing each isolate separately to 1-, 4-, and 16-fold concentrations above the minimum inhibitory concentration (MIC) of each antifungal agent. A sigmoid maximum-effect (E max) model was used to fit the time-kill curve data. The drug effect was further evaluated by measuring an increase/decrease in the killing rate of the tested isolates. The MICs of amphotericin B, itraconazole, voriconazole, and posaconazole were 0.5-1.0, 1.0, 0.5-1.0, and 0.25 µg/ml for A. brasiliensis, A. niger, and A. tubingensis isolates, respectively, and 2.0-4.0, 0.5, 1.0 for A. flavus, and 0.12-0.25 µg/ml for A. nomius isolates, respectively. A. calidoustus had the highest MIC range for the azoles (4.0-16.0 µg/ml) among all isolates tested. The minimum effective concentrations of caspofungin and anidulafungin were ≤0.03-0.5 µg/ml and ≤0.03 µg/ml for all isolates, respectively. Posaconazole demonstrated maximal killing rates (E(max) = 0.63 h(-1), r(2) = 0.71) against 14 ocular Aspergillus isolates, followed by amphotericin B (E(max) = 0.39 h(-1), r(2) = 0.87), voriconazole (E(max) = 0.35 h(-1), r(2) = 0.098), and itraconazole (E(max) = 0.01 h(-1), r(2) = 0.98). Overall, the antifungal susceptibility of the non-fumigatus Aspergillus isolates tested was species and antifungal agent dependent. Analysis of the kinetic growth assays, along with consideration of the killing rates, revealed that posaconazole was the most effective antifungal against all of the isolates.

The effect of clinical characteristics on the performance of galactomannan and PCR for the diagnosis of invasive aspergillosis in febrile neutropenic patients.

Rapid diagnosis and early treatment of invasive aspergillosis is crucial for the management of the patients with haematological malignancy. We evaluated 358 sera from 78 febrile neutropenic episodes in patient with invasive aspergillosis (IA) (one proven, 17 probable, and 60 possible) and 83 episodes in patients with no IA according to the EORTC/MSG criteria. Patient's specimens were tested by Mycassay Aspergillus PCR (first commercial real-time PCR test) and in house real-time PCR to investigate the presence of Aspergillus DNA, and by ELISA for detect the galactomannan (GM) antigen. We systematically investigated the medical background that can be effective on the test results. The hospitalisation period was longer in proven/probable episodes when compared with no IA (P = 0.001) and possible episodes. With regard to duration of neutropenia, the differences between both proven/probable with no IA (P = 0.023) and possible with no IA (P = 0.002) were highly significant. Similarly, the rates of T cell suppressant therapy in group proven/probable and possible episodes were significantly higher than in no IA (P = 0.005). There are significant differences in the performance of GM and PCR-based tests among studies, and standardisation is required. Therefore, it can be useful to determine the effective factors on these tests. The use of larger volume of sera improved the performance of real-time PCR for detection of Aspergillus DNA in high-risk adult patients in the present study. Some host factors such as duration of neutropenia and administration of T cell suppressants related to the development of IA.

Is the extraction by Whatman FTA filter matrix technology and sequencing of large ribosomal subunit D1-D2 region sufficient for identification of clinical fungi?

Although conventional identification of pathogenic fungi is based on the combination of tests evaluating their morphological and biochemical characteristics, they can fail to identify the less common species or the differentiation of closely related species. In addition these tests are time consuming, labour-intensive and require experienced personnel. We evaluated the feasibility and sufficiency of DNA extraction by Whatman FTA filter matrix technology and DNA sequencing of D1-D2 region of the large ribosomal subunit gene for identification of clinical isolates of 21 yeast and 160 moulds in our clinical mycology laboratory. While the yeast isolates were identified at species level with 100% homology, 102 (63.75%) clinically important mould isolates were identified at species level, 56 (35%) isolates at genus level against fungal sequences existing in DNA databases and two (1.25%) isolates could not be identified. Consequently, Whatman FTA filter matrix technology was a useful method for extraction of fungal DNA; extremely rapid, practical and successful. Sequence analysis strategy of D1-D2 region of the large ribosomal subunit gene was found considerably sufficient in identification to genus level for the most clinical fungi. However, the identification to species level and especially discrimination of closely related species may require additional analysis.

Potential of polymerase chain reaction and galactomannan for the diagnosis of invasive aspergillosis in patients with febrile neutropenia.

The incidence of invasive aspergillosis (IA) has increased over the last years, especially in immuncompromised patients with high mortality rates. Because of difficulties about the diagnosis; serological methods [galactomannan (GM) antigen test] and polymerase chain reaction (PCR) developed in recent years. MycAssay Aspergillus PCR performance in the diagnosis of IA was evaluated and compared with the GM and in-house PCR. This study was conducted with 358 serum samples obtained from 99 patient with febrile neutropenic episodes who were followed in haematology and bone marrow transplantation units. Patients were classified by the European Organization for the Research and Treatment of Cancer/Mycoses Study Group criteria, 18 of them is proven and probable IA. GM antigen test and two different real-time PCR; one of them is fist commercial PCR for IA; Mycassay Aspergillus and the other one is in-house real-time PCR performed. Sensitivity values were Mycassay Aspergillus PCR, in-house PCR, and GM 65.38%, 11.53% and 23.07%, respectively. The high sensitivity obtained from Mycassay Aspergillus PCR and sensitivity is increased by using a combination of diagnostic methods. GM antigen test and real-time PCR could be beneficial for early diagnosis and treatment of IA. For routine usage of PCR as diagnostic assay more studies needed in future.