Antibacterial efficacy of R-type pyocins against Pseudomonas aeruginosa on biofilms and in a murine model of acute lung infection.

BACKGROUND: The appearance of MDR strains and the development of biofilms make Pseudomonas aeruginosa infections a therapeutic challenge. To overcome this scenario, bacteriocins have been proposed as a potential adjuvant or alternative to antibiotic treatment. OBJECTIVES: To study the activity of R-pyocins on biofilms and in a murine model of pneumonia using a high-risk clone of P. aeruginosa. METHODS: The activity of R-pyocins on P. aeruginosa biofilms was tested on bacteria attached to a silicone surface, before and after biofilm formation. The effectiveness of R1-pyocin was studied in a murine model of pneumonia using ST175, a high-risk clone of P. aeruginosa. RESULTS: R-pyocins attacked adherent bacteria, preventing biofilm formation, and penetrated into the biofilm, killing P. aeruginosa within it, resulting in a dramatic reduction in bacterial load. R1-pyocin was active in a murine model of P. aeruginosa lung infection, administered before infection as a preventive treatment, and in acute pneumonia, with efficiency higher than standard colistin treatment. In addition, this work is the first to describe histopathological lung changes after administration of R-pyocins, contributing to the resolution of P. aeruginosa pneumonia in a murine model. CONCLUSIONS: This work highlights the potential use of the R-pyocins as therapeutic agents, alone or as adjuvants, due to its effectiveness on biofilms and in a murine model of pneumonia using ST175, a high-risk clone of P. aeruginosa. It may thus be feasible to consider R-pyocins as a possible therapeutic alternative in XDR infections, where treatment alternatives are limited.

Extended-spectrum resistance to ß-lactams/ß-lactamase inhibitors (ESRI) evolved from low-level resistant Escherichia coli.

OBJECTIVES: Escherichia coli is characterized by three resistance patterns to ß-lactams/ß-lactamase inhibitors (BLs/BLIs): (i) resistance to ampicillin/sulbactam and susceptibility to amoxicillin/clavulanic acid and piperacillin/tazobactam (RSS); (ii) resistance to ampicillin/sulbactam and amoxicillin/clavulanic acid, and susceptibility to piperacillin/tazobactam (RRS); and (iii) resistance to ampicillin/sulbactam, amoxicillin/clavulanic acid and piperacillin/tazobactam (RRR). These resistance patterns are acquired consecutively, indicating a potential risk of developing resistance to piperacillin/tazobactam, but the precise mechanism of this process is not completely understood. METHODS: Clinical isolates incrementally pressured by piperacillin/tazobactam selection in vitro and in vivo were used. We determined the MIC of piperacillin/tazobactam in the presence and absence of piperacillin/tazobactam pressure. We deciphered the role of the blaTEM genes in the new concept of extended-spectrum resistance to BLs/BLIs (ESRI) using genomic analysis. The activity of ß-lactamase was quantified in these isolates. RESULTS: We show that piperacillin/tazobactam resistance is induced in E. coli carrying blaTEM genes. This resistance is due to the increase in copy numbers and transcription levels of the blaTEM gene, thus increasing ß-lactamase activity and consequently increasing piperacillin/tazobactam MICs. Genome sequencing of two blaTEM-carrying representative isolates showed that piperacillin/tazobactam treatment produced two types of duplications of blaTEM (8 and 60 copies, respectively). In the clinical setting, piperacillin/tazobactam treatment of patients infected by E. coli carrying blaTEM is associated with a risk of therapeutic failure. CONCLUSIONS: This study describes for the first time the ESRI in E. coli. This new concept is very important in the understanding of the mechanism involved in the acquisition of resistance to BLs/BLIs.

Susceptibility to R-pyocins of Pseudomonas aeruginosa clinical isolates from cystic fibrosis patients.

Background: The appearance and dissemination of MDR among pathogenic bacteria has forced the search for new antimicrobials. Bacteriocins have been proposed as potential alternatives for the treatment of infections due to multiresistant strains. Objectives: To analyse the activity of R-pyocins against clinical isolates of Pseudomonas aeruginosa from patients with cystic fibrosis and other sources and evaluate them as a potential adjuvant or alternative to the current antibiotic treatment. Methods: The activity of R-pyocins against 150 strains of P. aeruginosa isolated from patients with cystic fibrosis or bacteraemia was studied through spot assay. Interactions between R-pyocins and antipseudomonal agents were quantitatively studied by the chequerboard method. Results: The proportion of P. aeruginosa isolates susceptible to R-pyocins was found to be higher in cystic fibrosis isolates compared with bacteraemia isolates (79.41% versus 50%). Moreover, no interactions were found between common antipseudomonal agents and R-pyocin susceptibility, except for the ST175 high-risk clone. Conclusions: Our results highlight the possibility of using R-pyocins as therapeutic agents, alone or as adjuvants, against P. aeruginosa in cystic fibrosis.

Identification of clinical isolates of Aspergillus, including cryptic species, by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

An expanded library of matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been constructed using the spectra generated from 42 clinical isolates and 11 reference strains, including 23 different species from 8 sections (16 cryptic plus 7 noncryptic species). Out of a total of 379 strains of Aspergillus isolated from clinical samples, 179 strains were selected to be identified by sequencing of beta-tubulin or calmodulin genes. Protein spectra of 53 strains, cultured in liquid medium, were used to construct an in-house reference database in the MALDI-TOF MS. One hundred ninety strains (179 clinical isolates previously identified by sequencing and the 11 reference strains), cultured on solid medium, were blindy analyzed by the MALDI-TOF MS technology to validate the generated in-house reference database. A 100% correlation was obtained with both identification methods, gene sequencing and MALDI-TOF MS, and no discordant identification was obtained. The HUVR database provided species level (score of ≥2.0) identification in 165 isolates (86.84%) and for the remaining 25 (13.16%) a genus level identification (score between 1.7 and 2.0) was obtained. The routine MALDI-TOF MS analysis with the new database, was then challenged with 200 Aspergillus clinical isolates grown on solid medium in a prospective evaluation. A species identification was obtained in 191 strains (95.5%), and only nine strains (4.5%) could not be identified at the species level. Among the 200 strains, A. tubingensis was the only cryptic species identified. We demonstrated the feasibility and usefulness of the new HUVR database in MALDI-TOF MS by the use of a standardized procedure for the identification of Aspergillus clinical isolates, including cryptic species, grown either on solid or liquid media.

Time to positivity of blood cultures in patients with bloodstream infections: A useful prognostic tool.

OBJECTIVE: The time to positivity (TTP) of blood cultures in patients with bloodstream infections (BSIs) has been considered to be a possible prognostic tool for some bacterial species. However, notable differences have been found between sampling designs and statistical methods in published studies to date, which makes it difficult to compare results or to derive reliable conclusions. Our objective was to evaluate the clinical and microbiological implications of TTP among patients with BSI caused by the most common pathogens. METHODS: A total of 361 episodes of BSI were reported for 332 patients. The survival of the entire cohort was measured from the time of blood culture sampling. In order to compare our results with those of previous studies, TTP was divided in three different groups based on log rank (short TTP <12h; medium TTP ≥12h to ≤27h, and long TTP >27h). Cox proportional hazard models were used to calculate crude and adjusted hazard ratios (HR). RESULTS: The Cox proportional hazard model revealed that TTP is an independent predictor of mortality (HR=1.00, p=0.031) in patients with BSIs. A higher mortality was found in the group of patients with the shortest TTP (<12h) (HR=2.100, p=0.047), as well as those with longest TTP (>27h) (HR=3.277, p=0.031). CONCLUSIONS: It seems that TTP may provide a useful prognostic tool associated with a higher risk of mortality, not only in patients with shorter TTP, but also in those with longer TTP.

Urinary Tract Physiological Conditions Promote Ciprofloxacin Resistance in Low-Level-Quinolone-Resistant Escherichia coli.

Escherichia coli isolates carrying chromosomally encoded low-level-quinolone-resistant (LLQR) determinants are frequently found in urinary tract infections (UTIs). LLQR mutations are considered the first step in the evolutionary pathway producing high-level fluoroquinolone resistance. Therefore, their evolution and dissemination might influence the outcome of fluoroquinolone treatments of UTI. Previous studies support the notion that low urine pH decreases susceptibility to ciprofloxacin (CIP) in E. coli However, the effect of the urinary tract physiological parameters on the activity of ciprofloxacin against LLQR E. coli strains has received little attention. We have studied the activity of ciprofloxacin under physiological urinary tract conditions against a set of well-characterized isogenic E. coli derivatives carrying the most prevalent chromosomal mutations (ΔmarR, gyrA-S83L, gyrA-D87N, and parC-S80R and some combinations). The results presented here demonstrate that all the LLQR strains studied became resistant to ciprofloxacin (according to CLSI guidelines) under physiological conditions whereas the control strain lacking LLQR mutations did not. Moreover, the survival of some LLQR E. coli variants increased up to 100-fold after challenge with a high concentration of ciprofloxacin under UTI conditions compared to the results seen with Mueller-Hinton broth. These selective conditions could explain the high prevalence of LLQR mutations in E. coli Furthermore, our data strongly suggest that recommended methods for MIC determination produce poor estimations of CIP activity against LLQR E. coli in UTIs.

A six-month Serratia marcescens outbreak in a Neonatal Intensive Care Unit.

OBJECTIVE: To investigate a Serratia marcescens (S. marcescens) outbreak in a Neonatal Unit in a tertiary university hospital. METHODS: Descriptive study of children admitted to the Unit with S. marcescens infection from November 2012 to March 2013. Conventional microbiological methods for clinical and environmental samples were used. The clonal relationship between all available isolates was established by molecular methods. A multidisciplinary team was formed, and preventive measures were taken. RESULTS: S. marcescens was isolated from 18 children. The overall attack rate was 12%, and the case fatality rate in the Intensive Care Unit was 23.5%. The most prevalent types of infections were pneumonia (6), conjunctivitis (6), and bloodstream infection (5). Clinical isolates and environmental isolates obtained from an incubator belonged to a unique clone. The clonal relationship between all S. marcescens strains helped us to identify the possible source of the outbreak. CONCLUSION: Isolation of S. marcescens from stored water in a container, and from the surface of an incubator after cleaning, suggests a possible environmental source as the outbreak origin, which has been perpetuated due to a failure of cleaning methods in the Unit. The strict hygiene and cleaning measures were the main factors that contributed to the end of the outbreak.

Evaluation and optimization of the Sysmex UF1000i system for the screening of urinary tract infection in primary health care elderly patients.

OBJECTIVE: Urinary tract infections (UTIs) are a common problem in the elderly population. Urine culture is still considered the "gold standard" to diagnose infection in this population. However, urine cultures are laborious and costly, and most samples will yield no growth. METHODS: An evaluation was made of the Sysmex UF-1000i flow cytometer as a screening tool for UTI in an elderly population older than 65 years who lived in the community, using 346 urine samples submitted for culture. RESULTS: The Receiver Operating Characteristic (ROC) analysis showed a significant difference (P<0.01) between 0.98 bacteria area under the curve value and 0.82 of white blood cells (WBC). The combination of both counts for screening did not show any improvement in specificity or sensitivity. According to our data, the use of a single cut-off point of 200bacteria/µL is suggested, in which the sensitivity and specificity were 99.11% and 91.59%, respectively, with a NPV of 99.49%. Moreover, this cut-off value could avoid 60.24% of the samples to be cultured, with a minimal false negative results rate of 0.87%. CONCLUSIONS: The stratification of age groups stratification helps in selecting a more adjusted Sysmex UF1000i cut-off limit, leading to an improvement in the screening parameters that would imply a better management of these infections, as well as a high reduction in the workload and cost savings.

Determining accurate vancomycin MIC values for methicillin-resistant Staphylococcus aureus by the microdilution method.

OBJECTIVES: To model the standard broth microdilution method, based on a modified Gompertz function, to obtain accurate vancomycin MIC values for methicillin-resistant Staphylococcus aureus (MRSA). The effect of these MIC values on the vancomycin therapeutic target of AUC(0-24)/MIC ≥ 400 was evaluated. METHODS: Three clinical isolates of MRSA with different vancomycin MIC values were used in this model. The optical densities (OD) of each MIC determination were modelled by a non-linear regression method using an F-test. The OD data were adjusted to the Gompertz equation to obtain the MIC values. The mean vancomycin AUC(0-24) obtained with a 30 mg/kg/day dosing schedule was calculated using a Monte Carlo simulation over 5000 subjects, using the pharmacokinetic data obtained in vancomycin-treated patients in our hospital. RESULTS: Although the MIC values obtained with this model were lower than those of the diffusion method (Etest) in all three cases, this did not affect the AUC(0-24)/MIC ratio for the strains with MICs of 1 mg/L by Etest. However, in those strains with MIC values >1 mg/L, the confidence intervals obtained for this ratio included values <400. CONCLUSIONS: The inherent variability of the broth microdilution method could explain the differences in the clinical outcome in MRSA-infected patients treated with vancomycin, mainly in those due to strains with MIC values of 1.5-2 mg/L by Etest, because the corresponding MIC values would range from 0.84 to 1.52 mg/L by the microdilution method, which could affect the therapeutic target.

Cinematographic continuity edits across shot scales and camera angles: an ERP analysis.

Film editing has attracted great theoretical and practical interest since the beginnings of cinematography. In recent times, the neural correlates of visual transitions at edit cuts have been at the focus of attention in neurocinematics. Many Event Related Potential (ERP) studies studies have reported the consequences of cuts involving narrative discontinuities, and violations of standard montage rules. However, less is known about edits that are meant to induce continuity. Here, we addressed the neural correlates of continuity editing involving scale, and angle variations across the cut within the same scene, two of the most popular devices used for continuity editing. We recorded the electroencephalographic signal obtained from 20 viewers as they watched four different cinematographic excerpts to extract ERPs at edit points. First, we were able to reproduce the general time and scalp distribution of the typical ERPs to filmic cuts in prior studies. Second, we found significant ERP modulations triggered by scale changes (scale out, scale in, or maintaining the same scale). Edits involving an increase in scale (scale out) led to amplification of the ERP deflection, and scale reduction (scale in) led to decreases, compared to edits that kept scale across the cut. These modulations coincide with the time window of the N300 and N400 components and, according to previous findings, their amplitude has been associated with the likelihood of consciously detecting the edit. Third, we did not detect similar modulations as a function of angle variations across the cut. Based on these findings, we suggest that cuts involving reduction of scale are more likely to go unnoticed, than ones that scale out. This relationship between scale in/out and visibility is documented in film edition manuals. Specifically, in order to achieve fluidity in a scene, the edition is designed from the most opened shots to the most closed ones.

Molecular epidemiology of fluoroquinolone resistance in invasive clinical isolates of Streptococcus pneumoniae in Seville.

INTRODUCTION: Due to the emergence of drug-resistant pneumococcal isolates, new fluoroquinolones have been recommended for the treatment of pneumococcal infections. The purpose of this study was to establish surveillance, and to conduct molecular characterization, of fluoroquinolone-resistant Streptococcus pneumoniae in Seville. METHOD: Norfloxacin-resistant S. pneumoniae isolates were characterized by quinolone resistance-determining region (QRDR) substitutions, reserpine-sensitive efflux, serotype and by pulsed-field gel electrophoresis (PFGE) patterns. RESULTS: Fourteen isolates (5.1%) showed an MIC>16 µg/ml to norfloxacin. Eight of 10 adult isolates were susceptible to levofloxacin. The 4 infant isolates with norfloxacin MIC>16 µg/ml were susceptible to levofloxacin. Seven of these 12 low-level-resistant isolates had mutations in ParC, while mutations both in ParC and GyrA genes were only detected in one of the two high-level-resistant isolates. All the isolates without QRDR substitutions that remained norfloxacin-resistant were positive for reserpine-inhibited efflux. The serotyping and PFGE revealed significant heterogeneity. We obtained 9 different profiles, 3 of which had two isolates each. Two of the isolates with the same pulsotype were from the same patient. The first isolate showed a mutation in the QRDR of ParC, and the second one had an additional GyrA mutation. CONCLUSION: In our study a levofloxacin resistance rate of 0.7% was found among invasive isolates. Although resistance level is low, surveillance is necessary, especially to prevent cases of in vivo resistance development as reported.

[Microbiological characterisation of Listeria monocytogenes isolates from human cases in Andalusia]. / Caracterización microbiológica de los aislados de Listeria monocytogenes procedentes de casos humanos en Andalucía.

OBJECTIVE: The aim of this study was to perform a retrospective study by genotyping 154 isolates from human listeriosis cases occurred in the region of Andalusia (southern Spain) in the period 2005-2009. MATERIAL AND METHODS: Serotyping was performed for 1 and 4 somatic antigens using commercial Listeria antisera, and by multiplex-PCR serogrouping according to the method described by Doumith et al. (2004). The antimicrobial susceptibility was performed by Epsilon test and interpreted by CLSI criteria. PFGE was performed according to the PulseNet protocol with the ApaI enzyme. The similarity of PFGE profiles was evaluated using the Bionumerics software. The multiplex PCR protocol described by Chen and Knabel (2007) was used for the identification of isolates belonging to L. monocytogenes ECI, ECII, and ECIII epidemic clones. RESULTS: The 154 isolates were grouped into four serotypes: 4b [94 (61%)] strains, 1/2b [30 (19%)] strains, 1/2a [27 (18%)] strains, and 1/2c [3 (2%)] strains, with 100% of susceptibility to ampicillin and cotrimoxazole. A further sixty-two ApaI distinct pulsotypes were recognized. Thirty-seven isolates (24%) showed unique ApaI pulsotypes, and the remaining 117 strains (76%) were assigned to 25 ApaI clusters (60% in clusters of more than two isolates). The EC markers were found in 62 (40.3%) of the L. monocytogenes isolates tested. The ECI marker was present in 43 (46.2%) 4b serotype isolates, ECII in 10 (10.7%) 4b serotype isolates, and ECIII in 9 (33,3%) 1/2a serotype isolates. DISCUSSION: A large proportion of the human listeriosis cases under investigation could be grouped into molecular subtype clusters, and our cases could be related to international food-borne outbreaks.

Fine-tuning the space, time, and host distribution of mycobacteria in wildlife.

BACKGROUND: We describe the diversity of two kinds of mycobacteria isolates, environmental mycobacteria and Mycobacterium bovis collected from wild boar, fallow deer, red deer and cattle in Doñana National Park (DNP, Spain), analyzing their association with temporal, spatial and environmental factors. RESULTS: High diversity of environmental mycobacteria species and M. bovis typing patterns (TPs) were found. When assessing the factors underlying the presence of the most common types of both environmental mycobacteria and M. bovis TPs in DNP, we evidenced (i) host species differences in the occurrence, (ii) spatial structuration and (iii) differences in the degree of spatial association of specific types between host species. Co-infection of a single host by two M. bovis TPs occurred in all three wild ungulate species. In wild boar and red deer, isolation of one group of mycobacteria occurred more frequently in individuals not infected by the other group. While only three TPs were detected in wildlife between 1998 and 2003, up to 8 different ones were found during 2006-2007. The opposite was observed in cattle. Belonging to an M. bovis-infected social group was a significant risk factor for mycobacterial infection in red deer and wild boar, but not for fallow deer. M. bovis TPs were usually found closer to water marshland than MOTT. CONCLUSIONS: The diversity of mycobacteria described herein is indicative of multiple introduction events and a complex multi-host and multi-pathogen epidemiology in DNP. Significant changes in the mycobacterial isolate community may have taken place, even in a short time period (1998 to 2007). Aspects of host social organization should be taken into account in wildlife epidemiology. Wildlife in DNP is frequently exposed to different species of non-tuberculous, environmental mycobacteria, which could interact with the immune response to pathogenic mycobacteria, although the effects are unknown. This research highlights the suitability of molecular typing for surveys at small spatial and temporal scales.

Neural responses to shot changes by cut in cinematographic editing: An EEG (ERD/ERS) study.

In order to analyze and detect neural activations and inhibitions in film spectators to shot changes by cut in films, we developed a methodology based on comparisons of recorded EEG signals and analyzed the event-related desynchronization/synchronization (ERD/ERS). The aim of the research is isolating these neuronal responses from other visual and auditory features that covary with film editing. This system of comparing pairs of signals using permutation tests, the Spearman correlation, and slope analysis is implemented in an automated way through sliding windows, analyzing all the registered electrodes signals at all the frequency bands defined. Through this methodology, we are able to locate, identify, and quantify the variations in neuronal rhythms in specific cortical areas and frequency ranges with temporal precision. Our results detected that after a cut there is a synchronization in theta rhythms during the first 188 ms with left lateralization, and also a desynchronization between 250 ms and 750 ms in the delta frequency band. The cortical area where most of these neuronal responses are detected in both cases is the parietal area.

[Microbiology laboratory management: an (almost) pending matter]. / La gestión de los laboratorios de microbiología: una asignatura (casi) pendiente.

Many professionals from distinct disciplines work in health centers and consequently these workplaces should be considered as service companies, involving human, material and financial resources aimed at providing a service required by society. Hospitals are one of the most complex types of company, in which diverse goods and services are produced and consumed. Like the hospital as a whole, the various units and departments in which the hospital is divided, including the microbiology department, are sufficiently different to be called distinct, but related, branches of the same company, given that none can perform their function without the others. Viewing a hospital service as a branch of a large company (the hospital as a whole) allows its production, the resources used in this production, and its clients to be identified more clearly. The healthcare model based on clinical management units aims to constitute a new organizational model for public health systems in which health strategies are performed that allow innovation and decentralization of the healthcare network. Clinical management provides the framework for attending to the population's healthcare needs through a person-centered approach and involves all the professionals in any of the settings in which healthcare is provided. Among the aims of this model is to guarantee continuity of care, facilitate comprehensive health promotion and deliver daily healthcare effectively. The main instruments of clinical management are structured knowledge of the population's health needs, the use of the best scientific knowledge available, and a comprehensive and participatory practical model, together with assessment tools. Three possible clinical management models are proposed for the work of specialists in microbiology and parasitology: a) a microbiology clinical management unit; b) a biological diagnosis clinical management unit, and c) a multidisciplinary clinical management unit with cross-competencies with affiliated specialties. The latter two models have strengths and weaknesses and the choice of any model must be based on mutual trust, respect for areas of knowledge and the search for synergy among the units and services forming these models. Any of the proposed models could be valid, although the selection of a particular model should consider the working environment, the size of the hospital, and the interpersonal relations within its components, which should be based on complementariness, dialogue and the search for consensus in decision-making to achieve a synergistic environment. The model that might best satisfy clinical microbiologists' expectations and promote their future development and survival in the era of automatization of most microbiological diagnostic procedures could be the multidisciplinary model.

Effect of subinhibitory concentrations of ampicillin on Listeria monocytogenes.

Listeria monocytogenes is an important cause of meningoencephalitis associated with high mortality. The treatment of choice for listeriosis is ampicillin alone or in combination with gentamicin or penicillin G. However, only low ampicillin concentrations are recorded in the central nervous system (CNS). In this study, we analysed the effect of subinhibitory concentrations of ampicillin on the morphology, growth and survival of L. monocytogenes. The non-inhibitory concentration (NIC), the minimum inhibitory concentration (MIC) and the MIC/NIC ratio were determined. Gram and Live/Dead staining showed aggregates of L. monocytogenes cells when grown at subinhibitory concentrations of ampicillin, with >90% of viable cells. The L. monocytogenes strains tested showed an intermediate heteroresistance to ampicillin, characterised by a MIC/NIC ratio of 4. Our results seem to indicate that both intermediate heteroresistance and the formation of aggregates could play a role in the clinical failure of ampicillin in the treatment of CNS infections caused by L. monocytogenes. However, more studies are necessary to elucidate this question.

Molecular detection and identification of Aspergillus spp. from clinical samples using real-time PCR.

The definite and rapid diagnosis of invasive aspergillosis is necessary because of the high mortality caused. The objective of this study was to evaluate a real-time PCR assay to detect Aspergillus spp. in clinical samples, based on the Light Cycler technology. Specificity was assessed by using DNA extracted from pathogenic and non-pathogenic bacteria/fungi from Spanish Collection including: two Aspergillus flavus, four Aspergillus fumigatus, two Aspergillus nidulans, two Aspergillus niger and two Aspergillus terreus isolates. The analytical sensitivity was evaluated with different inocula (10(1)-10(5) conidia ml(-1)), and serially diluted DNA of A. fumigatus. To assess clinical applicability, samples from patients at risk were analysed. Species identification was determined by analysing the melting curves. Reactions using genomic DNA from other species of different genera than Aspergillus were negative (specificity: 100%). Analytical sensitivity was 60 fg using DNA and 5-20 conidia using conidial suspensions. The linear range was from 60 to 6 x 10(7) fg. The Tm ranged from 67.34 to 70.7 degrees C for the different Aspergillus spp. studied. Nine hundred and forty-eight consecutive blood samples from 127 patients were processed. In total, 10 (1%) of 948 samples from blood samples were PCR-positive. The real-time PCR assay provides a high sensitivity and specificity for detection of fungal DNA and rapidly identifies most of clinically relevant Aspergillus species.

Prevalence and in vitro antifungal susceptibility of cryptic species of the genus Aspergillus isolated in clinical samples.

INTRODUCTION: The genus Aspergillus contains more than 300 species, which are divided into closely related groups called sections. Molecular studies have revealed numerous cryptic species within different sections of this genus, which have different profiles of antifungal susceptibility and lack diagnostic morphological features. However, there are few studies on the prevalence and in vitro antifungal susceptibility of the cryptic species of this genus. The aim of this study was to investigate the distribution of Aspergillus spp. among clinical samples, and to study their in vitro susceptibility to different antifungal drugs. METHOD: Over a period of 2-years (2014-2015), a total of 379 strains of the genus Aspergillus were isolated. Most of the isolates were classified as respiratory colonizations; no cases of invasive aspergillosis were found. The strains were identified by MALDI-TOF mass spectrometry, and susceptibility testing was performed by the EUCAST reference procedure. RESULTS: Twenty species belonging to 8 sections were identified, being A. fumigatus the most prevalent (44.1%). The prevalence of cryptic species was 15.3%, with a clear predominance of A. tubingensis. Among the tested antifungal drugs, amphotericin B was the less active in vitro, followed by triazole drugs and echinocandins. The cryptic species had minimun inhibitory concentrations (MICs) higher than the corresponding type species. CONCLUSIONS: Accurate identification of the genus Aspergillus at the species level and in vitro antifungal susceptibility testing are necessary because, as it has been shown, some species of this genus may show resistance profiles against available antifungal drugs.