Fluconazole resistance is not a predictor of poor outcome in patients with cryptococcosis.

BACKGROUND: Cryptococcus isolates with high MICs to fluconazole are increasingly reported, and a potential clinical impact has been advocated. However, there are different methods to evaluate fluconazole MICs and comparative analysis among such techniques and their comprehensive correlation with clinical outcome are not available. METHODS: Over a 13-year period (2000-2013), fluconazole MICs were determined for 62 cryptococcal isolates recovered from 22 patients with cryptococcosis using CLSI M27-A3, EUCAST, E test and Sensititre YeastOne, simultaneously. The relationship between the fluconazole MICs and the clinical outcome at week 10 was assessed in patients who received fluconazole as induction or maintenance therapy (n = 16). RESULTS: The percentage of cryptococcal strains with MIC values ≥16 µg/mL according to different methods was CLSI 1.6%, EUCAST 16.1%, E test 31.6% and Sensititre YeastOne 53.2%. Among the 16 patients treated with fluconazole, no correlation between clinical outcome and any MIC value obtained with either method was observed. The only variable independently associated with a poor outcome was having a disseminated disease. CONCLUSIONS: There is a weak correlation between fluconazole MICs against Cryptococcus spp. as determined by CLSI, EUCAST, E test and Sensititre YeastOne. Neither procedure could predict the clinical outcome of patients with cryptococcosis receiving fluconazole-based therapy. With present methods, fluconazole resistance in Cryptococcus may be clinically misleading.

Insight into the Significance of Aspergillus fumigatus cyp51A Polymorphisms.

Triazole antifungal compounds are the first treatment choice for invasive aspergillosis. However, in the last decade the rate of azole resistance among Aspergillus fumigatus strains has increased notoriously. The main resistance mechanisms are well defined and mostly related to point mutations of the azole target, 14-α sterol demethylase (cyp51A), with or without tandem repeat integrations in the cyp51A promoter. Furthermore, different combinations of five Cyp51A mutations (F46Y, M172V, N248T, D255E, and E427K) have been reported worldwide in about 10% of all A. fumigatus isolates tested. The azole susceptibility profile of these strains shows elevated azole MICs, although on the basis of the azole susceptibility breakpoints, these strains are not considered azole resistant. The purpose of the study was to determine whether these cyp51A polymorphisms (single nucleotide polymorphisms [SNPs]) are responsible for the azole susceptibility profile and whether they are reflected in a poorer azole treatment response in vivo that could compromise patient treatment and outcome. A mutant with a cyp51A deletion was generated and became fully susceptible to all azoles tested. Also, three cyp51A gene constructions with different combinations of SNPs were generated and reintroduced into an azole-susceptible wild-type (WT) strain (the ΔakuBKU80 strain). The alternative model host Galleria mellonella was used to compare the virulence and voriconazole response of G. mellonella larvae infected with A. fumigatus strains with WT cyp51A or cyp51A with SNPs. All strains were pathogenic in G. mellonella larvae, although they did not respond similarly to voriconazole therapeutic doses. Finally, the full genomes of these strains were sequenced and analyzed in comparison with those of A. fumigatus WT strains, revealing that they belong to different strain clusters or lineages.

Molecular Identification and Susceptibility Testing of Molds Isolated in a Prospective Surveillance of Triazole Resistance in Spain (FILPOP2 Study).

Antifungal resistance is increasing by the emergence of intrinsically resistant species and by the development of secondary resistance in susceptible species. A previous study performed in Spain revealed levels of azole resistance in molds of between 10 and 12.7%, but secondary resistance in Aspergillus fumigatus was not detected. We used itraconazole (ITZ)-supplemented medium to select resistant strains. A total of 500 plates supplemented with 2 mg/liter of ITZ were sent to 10 Spanish tertiary hospitals, and molecular identification and antifungal susceptibility testing were performed. In addition, the cyp51A gene in those A. fumigatus strains showing azole resistance was sequenced. A total of 493 isolates were included in the study. Sixteen strains were isolated from patients with an infection classified as proven, 104 were isolated from patients with an infection classified as probable, and 373 were isolated from patients with an infection classified as colonization. Aspergillus was the most frequent genus isolated, at 80.3%, followed by Scedosporium-Lomentospora (7.9%), Penicillium-Talaromyces (4.5%), Fusarium (2.6%), and the order Mucorales (1%). Antifungal resistance was detected in Scedosporium-Lomentospora species, Fusarium, Talaromyces, and Mucorales Three strains of A. fumigatus sensu stricto were resistant to azoles; two of them harbored the TR34+L98H mechanism of resistance, and the other one had no mutations in cyp51A The level of azole resistance in A. fumigatus remains low, but cryptic species represent over 10% of the isolates and have a broader but overall higher range of antifungal resistance.

Could the determination of Aspergillus fumigatus mating type have prognostic value in invasive aspergillosis?

A clear link between mating type and virulence has been demonstrated for some fungal pathogens, but not for Aspergillus fumigatus as of yet. An association between mating type and invasiveness has recently been established. The mating type proportion (MAT1-1:MAT1-2) of 213 A. fumigatus strains was determined (48.5%:51.5%) and results were in agreement with previous studies. However, these percentages changed when the strain collection was divided into azole-susceptible and -resistant strains. The 163 susceptible strains kept these proportions, but among the 50 azole-resistant strains 60.0% MAT1-1 and 40% MAT1-2 were found. Moreover, looking at the clinical outcome associated to 27 azole-resistant strains, we found that MAT1-1 was linked to a high mortality rate (64%), whereas the rate associated to MAT1-2 genotype was markedly lower (15%). The pathogenicity linked to the Mat type was tested in a Galleria mellonella model of infection, showing that MAT1-1 strains were consistently more pathogenic than MAT1-2, independently of their susceptibility phenotype. This data would suggest that A. fumigatus mating type determination at the time of diagnosis could have a prognostic value in invasive aspergillosis.

Assessment of biofilm production in Candida isolates according to species and origin of infection.

The biofilm production (BP) of 200 clinical strains of Candida isolated during 2010-2013 were assessed using an in vitro model and a comparison of the results was made between species and between origins of the infections. The BP was assessed using the crystal violet assay, and the strains were classified as low, moderate, or high biofilm producers. Candida tropicalis had the highest values for BP, which varied depending on the origin of the infection. Assessment of BP is a key diagnostic tool that enables us to better understand Candida infections.

Antifungal Susceptibility Testing of Ascomycetous Yeasts Isolated from Animals.

Recent studies suggest that antifungal resistance in yeast isolates of veterinary origin may be an underdiagnosed threat. We tested a collection of 92 ascomycetous yeast isolates that were obtained in Spain from birds, mammals and insects for antifungal susceptibility. MICs to amphotericin B and azoles were low, and no resistant isolates were detected. Despite these results, and given the potential role of animals as reservoirs of resistant strains, continuous monitoring of antifungal susceptibility in the veterinary setting is recommended.

Genotyping and antifungal susceptibility testing of multiple Malassezia pachydermatis isolates from otitis and dermatitis cases in pets: is it really worth the effort?

A total of 216 colonies of Malassezia pachydermatis from 28 cases of fungal otitis or dermatitis in pets were genotyped by M13 fingerprinting and tested for antifungal susceptibility. A huge genetic diversity was found (157 M13 types in total), with all animals having a polyclonal pattern of infection (5.4 ± 1.5 genotypes/sample). Furthermore, analysis of molecular variance (AMOVA) revealed that most genetic diversity (44%) was found at the within sample level. In contrast, variability in antifungal susceptibility among isolates from the same sample was less important, with different M13 types displaying in most cases identical or very similar MIC results. Most isolates displayed high in vitro susceptibility to amphotericin B, terbinafine and all azoles tested except fluconazole, for which MIC values were always ≥4 µg/ml and a 26.9% of isolates displayed values ≥32 µg/ml. We conclude that although characterization of multiple yeast isolates results in a considerable increase in laboratory workload and expenses, it may help to get a better understanding of the epidemiology of M. pachydermatis in a given patient population.

Therapeutic drug monitoring of voriconazole helps to decrease the percentage of patients with off-target trough serum levels.

We monitored trough voriconazole serum concentrations from 107 patients (n = 258 samples) at 6 hospitals in Madrid. Most of the patients were male (67%) and had the following underlying conditions: hematological cancer (42%), solid organ transplantation (15%), chronic obstructive pulmonary disease (14%), human immunodeficiency virus infection (8.4%), solid cancer (5.6%), and other (29%). The indication for voriconazole administration was aspergillosis treatment (74.6%) and prophylaxis (14%). The main reasons for voriconazole trough drug monitoring were initiation of treatment/prophylaxis (33%), patient monitoring (47%), and suspected toxicity (3.5%). Levels (µg/ml) were subtherapeutic (<1; 18.2%), on-target (1-5.5; 71.3%), and high (>5.5; 10.5%). The samples percentage with on-target levels was significantly lower for the first sample than for subsequent samples (62.6% vs. 77.5%). "Subsequent samples," "admission in nonpediatric wards," "voriconazole used for treatment of invasive aspergillosis," and "use of proton pump inhibitors" were predictors of voriconazole therapeutic levels (≥1 µg/ml).

Water Sources in a Zoological Park Harbor Genetically Diverse Strains of Clostridium Perfringens Type A with Decreased Susceptibility to Metronidazole.

The presence of Clostridium perfringens in water is generally regarded as an indicator of fecal contamination, and exposure to waterborne spores is considered a possible source of infection for animals. We assessed the presence and genetic diversity of C. perfringens in water sources in a zoological park located in Madrid (Spain). A total of 48 water samples from 24 different sources were analyzed, and recovered isolates were toxinotyped, genotyped by fluorophore-enhanced repetitive polymerase chain reaction (rep-PCR) fingerprinting and tested for antimicrobial susceptibility. C. perfringens was recovered from 43.8 % of water samples and 50 % of water sources analyzed. All isolates (n = 70) were type A and 42.9 % were ß2-toxigenic (i.e., cpb2+), but none contained the enterotoxin-encoding gene (cpe). Isolates belonged to 15 rep-PCR genotypes and most genetic diversity (88 %) was distributed among isolates obtained from the same sample. Most isolates displayed intermediate susceptibility (57.1 %; MIC = 16 µg ml-1) or resistance (5.7 %; MIC ≥ 32 µg ml-1) to metronidazole. No resistance to other antimicrobials was detected, although some isolates showed elevated MICs to erythromycin and/or linezolid. Finally, a marginally significant association between absence of cpb2 and decreased susceptibility to metronidazole (MIC ≥ 16 µg ml-1) was detected. In conclusion, our results reveal a high prevalence of C. perfringens type A in the studied water reservoirs, which constitutes a health risk for zoo animals. The elevated MICs to metronidazole observed for genetically diverse isolates is a cause of additional concern, but more work is required to clarify the significance of reduced metronidazole susceptibility in environmental strains.

[Application of mass spectrometry in mycology]. / Aplicación de la espectrometría de masas en micología.

MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry (MS) is becoming an essential tool in most microbiology laboratories. At present, by using a characteristic fungal profile obtained from whole cells or through simple extraction protocols, MALDI-TOF MS allows the identification of pathogenic fungi with a high performance potential. This methodology decreases the laboratory turnaround time, optimizing the detection of mycoses. This article describes the state-of-the-art of the use of MALDI-TOF MS for the detection of human clinical fungal pathogens in the laboratory and discusses the future applications of this technology, which will further improve routine mycological diagnosis.

Microsatellite (STRAf) genotyping cannot differentiate between invasive and colonizing Aspergillus fumigatus isolates.

We studied whether short tandem repeats of Aspergillus fumigatus (STRAf) can differentiate between invasive and colonizing genotypes of A. fumigatus. Of the 395 genotypes detected (n = 1,373 isolates), 50 were clusters and 24 (6% of all genotypes) involved the patients with invasive aspergillosis and those colonized with A. fumigatus, indicating that genotyping cannot discriminate between invasive and colonizing isolates.

Aspergillus citrinoterreus, a new species of section Terrei isolated from samples of patients with nonhematological predisposing conditions.

The use of molecular identification techniques has revealed an increasing number of new species within Aspergillus section Terrei. We phenotyped a set of 26 clinical isolates that showed genetic differences from Aspergillus terreus sensu stricto by analyzing sequences from PCR-amplified ß-tubulin and calmodulin genes and the internal transcribed spacer region. Since the isolates were phylogenetically and morphologically different from all of the members of Aspergillus section Terrei, they are described here as a new species, Aspergillus citrinoterreus, so named because it produces a diffusible yellowish pigment in agar. A. citrinoterreus isolates were significantly more susceptible to itraconazole, voriconazole, and posaconazole than A. terreus sensu stricto isolates were; in contrast, the amphotericin B MICs for both species were high. A. citrinoterreus was found in clinical samples from patients with proven or probable invasive aspergillosis and colonized patients, none of whom had hematological malignancies as predisposing conditions. However, they did have other underlying conditions such as chronic obstructive pulmonary disease, cirrhosis, and cancer or had received a solid organ transplants and presented not only with invasive pulmonary aspergillosis but also with mediastinitis. A. citrinoterreus isolates were detected for the first time in 2002. In all cases of invasive aspergillosis, A. citrinoterreus was found to be a copathogen, mostly with A. fumigatus.

Sputum and bronchial secretion samples are equally useful as bronchoalveolar lavage samples for the diagnosis of invasive pulmonary aspergillosis in selected patients.

In the absence of histopathology studies of lung biopsies, the bronchoalveolar lavage (BAL) sample is preferred for the diagnosis of invasive pulmonary aspergillosis. Isolation of Aspergillus fumigatus from sputum and bronchial secretion samples are commonly interpreted as colonization or laboratory contamination, particularly in nonneutropenic patients. We studied if sputum/bronchial secretions and BAL samples are equally useful for the diagnosis of invasive pulmonary aspergillosis. We retrospectively selected 14 patients with proven (n = 1) or probable (n = 13) invasive pulmonary aspergillosis from whose samples A. fumigatus had been simultaneously isolated in BAL and sputum/bronchial secretions between 2006 and 2012. The isolates were identified by sequencing the ß-tubulin gene and genotyped using the STRAf assay. Matches between BAL and sputum/bronchial secretions were observed in patients with identical genotypes in BAL and sputum/bronchial secretions. All patients had clinically suspected pneumonia, before the diagnosis of invasive pulmonary aspergillosis. The sample from which A. fumigatus was initially isolated was collected as a result of the presence of fever (50%), abnormal radiological findings (100%), and/or pneumonia that did not respond to antibiotics (36%). The underlying conditions varied, although the most common predisposing factors were hematological malignancies (21.5%) and COPD (43%). In 13 of the 14 patients (93%), we found matching genotypes in the BAL and the sputum/bronchial secretion samples. Genotyping showed that samples of sputum or bronchial secretions were equally useful as samples of BAL for the diagnosis of invasive pulmonary aspergillosis.

In vitro amphotericin B susceptibility of Malassezia pachydermatis determined by the CLSI broth microdilution method and Etest using lipid-enriched media.

We determined the in vitro amphotericin B susceptibility of 60 Malassezia pachydermatis isolates by the CLSI broth microdilution method and the Etest using lipid-enriched media. All isolates were susceptible at MICs of ≤ 1 µg/ml, confirming the high activity of amphotericin B against this yeast species. Overall, the essential agreement between the tested methods was high (80% and 96.7% after 48 h and 72 h, respectively), and all discrepancies were regarded as nonsubstantial.

Is biofilm production a predictor of catheter-related candidemia?

Catheter-related candidemia (CRC) is typically a biofilm related disease, but it is mostly unknown if the production of biofilm is a feature exclusively shown by Candida spp. isolates causing CRC. We performed an in vitro biofilm assay using Candida isolates obtained from the blood of patients with candidemia. We demonstrated that biofilm production was not a good predictor of catheter-related candidemia. Also, we demonstrated that there was no difference in the mortality of candidemia patients infected by biofilm-forming isolates and those in which the infection is caused by nonbiofilm-forming species.

Is it feasible to diagnose catheter-related candidemia without catheter withdrawal?

Many bloodstream infections (BSI) in patients with central venous catheters (CVC) are not catheter-related (CR). Assessment of catheter involvement without catheter withdrawal has not been studied in candidemia. We assessed the value of conservative techniques to evaluate catheters as the origin of candidemia in patients with CVC in a prospective cohort study (superficial Gram stain and culture, Kite technique (Gram stain and culture of the first 1 cm blood drawn from the CVC), proportion of positive blood cultures (PPBCs), differential time to positivity (DTP), and minimal time to positivity (MTP)). All catheters were cultured at withdrawal. From June 2008 to January 2012, 22 cases fulfilled the inclusion criteria. CR-candidemia (CRC) was confirmed in 10. Validity values for predicting CRC were: superficial Gram stain (S, 30%; Sp, 81.83%; PPV, 60%; NPV, 56.3%; Ac, 57.1%), superficial cultures (S, 40%; Sp, 75%; PPV, 57.1%; NPV, 60%; Ac, 59.1%), Kite Gram stain (S, 33.3%; Sp, 66.7%; PPV, 50%; NPV, 50%; Ac, 50%), Kite culture (S, 80%; Sp, 66.7%; PPV, 66.7%; NPV, 80%; Ac, 72.7%), PPBC (S, 50%; Sp, 41.7%; PPV, 41.7%; NPV, 50.0%; Ac, 45.5%), DTP (S, 100%; Sp, 33.3%; PPV, 55.6%; NPV, 100%; Ac, 63.6%), and MTTP (S, 70%; Sp, 58.3%; PPV, 58.3%; NPV, 70%; Ac, 63.6%). While combinations of two tests improved sensitivity and NPV, more than two tests did not improve validity values. Classic tests to assess CR-BSI caused by bacteria cannot be reliably used to diagnose CRC. Combinations of tests could be useful, but more and larger studies are required.

First case of autochthonous Clostridium difficile PCR ribotype 027 detected in Spain.

INTRODUCTION: Clostridium difficile ribotype 027 (Cd027) has caused outbreaks in the United States, Canada, and Europe since 2001. In Spain, the importance of Cd027 is still unknown. In 2007, we began active surveillance of Cd027 to determine its incidence in our hospital. METHODS: From January 2007 to April 2012, isolates of C. difficile by multiplex PCR were studied to detect toxin genes. Binary toxin-positive isolates were characterized using PCR-ribotyping. Cd027 were further characterized by toxino-typing, sequencing of tcdC gene, and MLVA (multilocus-variable-number-tandem-repeat-analysis). RESULTS: Only 8 strains were Cd027 from 3666 isolates of C. difficile analyzed during the study period. These strains were isolated from 4 patients: a Spanish patient previously hospitalized in the UK, a pregnant laboratory technician, a British tourist, and a Spanish patient without epidemiological antecedents for acquiring Cd027. MLVA typing of Cd027 isolates revealed 4 different patterns. The first patient had 2 episodes of diarrhea caused by different Cd027. The strains from the first episode of patient 1 and the strain from patient 2 were grouped in the same clonal cluster (these cases were previously published as laboratory transmission), while strains from patients 3 and 4 were genetically unrelated to each other, and to the strains from patients 1 and 2. CONCLUSION: We report the first finding of an autochthonous case of non-severe Cd027 infection. Our results indicate that Cd027 diarrhea is uncommon in our area, and it appears mainly as imported cases. MLVA typing enables us to distinguish different genotypes among our Cd027 isolates.

Is azole resistance in Aspergillus fumigatus a problem in Spain?

Aspergillus fumigatus complex comprises A. fumigatus and other morphologically indistinguishable cryptic species. We retrospectively studied 362 A. fumigatus complex isolates (353 samples) from 150 patients with proven or probable invasive aspergillosis or aspergilloma (2, 121, and 6 samples, respectively) admitted to the hospital from 1999 to 2011. Isolates were identified using the ß-tubulin gene, and only 1 isolate per species found in each sample was selected. Antifungal susceptibility to azoles was determined using the CLSI M38-A2 procedure. Isolates were considered resistant if they showed an MIC above the breakpoints for itraconazole, voriconazole, or posaconazole (>2, >2, or >0.5 µg/ml). Most of the samples yielded only 1 species (A. fumigatus [n = 335], A. novofumigatus [n = 4], A. lentulus [n = 3], A. viridinutans [n = 1], and Neosartorya udagawae [n = 1]). The remaining samples yielded a combination of 2 species. Most of the patients were infected by a single species (A. fumigatus [n = 143] or A. lentulus [n = 2]). The remaining 5 patients were coinfected with multiple A. fumigatus complex species, although A. fumigatus was always involved; 4 of the 5 patients were diagnosed in 2009 or later. Cryptic species were less susceptible than A. fumigatus. The frequency of resistance among A. fumigatus complex and A. fumigatus to itraconazole, voriconazole, and posaconazole was 2.5 and 0.3%, 3.1 and 0.3%, and 4.2 and 1.8%, respectively, in the per-isolate analysis and 1.3 and 0.7%, 2.6 and 0.7%, and 6 and 4% in the per-patient analysis. Only 1 of the 6 A. fumigatus isolates in which the cyp51A gene was sequenced had a mutation at position G448. The proportion of patients infected by azole-resistant A. fumigatus isolates was low.

Does identification to species level provide sufficient evidence to confirm catheter-related fungemia caused by Candida albicans?

We retrospectively studied 22 patients with catheter-related candidemia caused by Candida albicans. Strains isolated simultaneously from blood and catheter tips were genotyped using six microsatellite markers. Matches between genotypes of isolates recovered from both sample sources were found in 20/22 (91%) patients. Consequently, identification of the same species from both the catheter tip and blood could be used to confirm catheter-related candidemia.

Characterization of swine isolates of Clostridium difficile in Spain: a potential source of epidemic multidrug resistant strains?

Clostridium difficile is an emerging pathogen for humans and animals and there is concern about the possibility that livestock might serve as a reservoir of epidemic strains. In Spain, ribotype 078 is one of the most prevalent in human episodes of C. difficile infection, but the distribution of this and other ribotypes in animals is yet unknown. We present the first report on the ribotype distribution and antimicrobial susceptibility of C. difficile in swine in Spain. A total of 144 isolates were PCR ribotyped, and their MIC values for 13 antimicrobial agents were determined using the Etest. Toxins A and B production was assessed using a commercial immunoassay and, in the case of toxin B, a specific cytotoxicity test. Our results show a high prevalence of the toxigenic 078 ribotype (94.4%) and multidrug resistance (49.3%) among the studied isolates. A minority of isolates (5.6%) belonged to a mostly non-toxinogenic ribotype. All isolates were resistant to the fluoroquinolone ciprofloxacin, but susceptible to daptomycin, linezolid, meropenem, rifampicin, teicoplanin, tigecycline, metronidazole and vancomycin. Resistance to clindamycin, ertapenem, erythromycin and moxifloxacin was common (≥27.8% in all cases). Resistance rates for the different antibiotics tested were in all cases independent from the ribotype of isolates and the host's condition (diarrheic or non-diarrheic), but erythromycin and moxifloxacin resistance was associated with the geographic origin of isolates. Metronidazole heteroresistance was found among animal isolates of C. difficile. Our results highlight the role of livestock as a potential source of epidemic multidrug resistant strains in Spain.