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1.  Multidisciplinary Analysis of a Nontoxigenic Clostridium difficile Strain with Stable Resistance to Metronidazole 
Stable resistance to metronidazole in a nontoxigenic Clostridium difficile strain was investigated at both the genomic and proteomic levels. Alterations in the metabolic pathway involving the pyruvate-ferredoxin oxidoreductase were found, suggesting that reduction of metronidazole, required for its activity, may be less efficient in this strain. Proteomic studies also showed a cellular response to oxidative stress.
doi:10.1128/AAC.02350-14
PMCID: PMC4135993  PMID: 24913157
2.  Antifungal Resistance to Fluconazole and Echinocandins Is Not Emerging in Yeast Isolates Causing Fungemia in a Spanish Tertiary Care Center 
Accurate knowledge of fungemia epidemiology requires identification of strains to the molecular level. Various studies have shown that the rate of resistance to fluconazole ranges from 2.5% to 9% in Candida spp. isolated from blood samples. However, trends in antifungal resistance have received little attention and have been studied only using CLSI M27-A3 methodology. We assessed the fungemia epidemiology in a large tertiary care institution in Madrid, Spain, by identifying isolates to the molecular level and performing antifungal susceptibility testing according to the updated breakpoints of European Committee for Antimicrobial Susceptibility Testing (EUCAST) definitive document (EDef) 7.2. We studied 613 isolates causing 598 episodes of fungemia in 544 patients admitted to our hospital (January 2007 to December 2013). Strains were identified after amplification and sequencing of the ITS1-5.8S-ITS2 region and further tested for in vitro susceptibility to amphotericin B, fluconazole, posaconazole, voriconazole, micafungin, and anidulafungin. Resistance was defined using EUCAST species-specific breakpoints, and epidemiological cutoff values (ECOFFs) were applied as tentative breakpoints. Most episodes were caused by Candida albicans (46%), Candida parapsilosis (28.7%), Candida glabrata (9.8%), and Candida tropicalis (8%). Molecular identification enabled us to better detect cryptic species of Candida guilliermondii and C. parapsilosis complexes and episodes of polyfungal fungemia. The overall percentage of fluconazole-resistant isolates was 5%, although it was higher in C. glabrata (8.6%) and non-Candida yeast isolates (47.4%). The rate of resistance to echinocandins was 4.4% and was mainly due to the presence of intrinsically resistant non-Candida species. Resistance mainly affected non-Candida yeasts. The rate of resistance to fluconazole and echinocandins did not change considerably during the study period.
doi:10.1128/AAC.02670-14
PMCID: PMC4136060  PMID: 24867979
4.  Use of rapid diagnostic techniques in ICU patients with infections 
BMC Infectious Diseases  2014;14(1):593.
Background
Infection is a common complication seen in ICU patients. Given the correlation between infection and mortality in these patients, a rapid etiological diagnosis and the determination of antimicrobial resistance markers are of paramount importance, especially in view of today’s globally spread of multi drug resistance microorganisms. This paper reviews some of the rapid diagnostic techniques available for ICU patients with infections.
Methods
A narrative review of recent peer-reviewed literature (published between 1995 and 2014) was performed using as the search terms: Intensive care medicine, Microbiological techniques, Clinical laboratory techniques, Diagnosis, and Rapid diagnosis, with no language restrictions.
Results
The most developed microbiology fields for a rapid diagnosis of infection in critically ill patients are those related to the diagnosis of bloodstream infection, pneumonia –both ventilator associated and non-ventilator associated–, urinary tract infection, skin and soft tissue infections, viral infections and tuberculosis.
Conclusions
New developments in the field of microbiology have served to shorten turnaround times and optimize the treatment of many types of infection. Although there are still some unresolved limitations of the use of molecular techniques for a rapid diagnosis of infection in the ICU patient, this approach holds much promise for the future.
doi:10.1186/s12879-014-0593-1
PMCID: PMC4247221  PMID: 25430913
Rapid diagnosis; Clinical laboratory techniques; Intensive care unit; Microbiology
5.  New Automated Chemiluminescence Immunoassay for Simultaneous but Separate Detection of Human Immunodeficiency Virus Antigens and Antibodies 
Journal of Clinical Microbiology  2014;52(5):1467-1470.
The recently launched Liaison XL Murex HIV Ab/Ag assay (DiaSorin S.p.A) uses chemiluminescence immunoassay technology for the combined qualitative determination of p24 antigen of HIV-1 and specific antibodies to both HIV-1 and HIV-2. We studied 571 serum samples from those submitted to our laboratory for HIV screening. The samples were divided into 3 subsets: subset A, 365 samples collected prospectively during 1 week; subset B, 158 samples from confirmed HIV-positive patients; and subset C, 48 samples with a positive screening result but a negative or indeterminate confirmatory test result. Our standard screening/confirmatory algorithm was used as a reference. In subset A (prospective), 5 samples were positive and 360 negative by the standard procedure. Liaison XL Murex HIV Ab/Ag correctly identified all 5 positive samples (100%) and 357 negative samples (99.2%). In subset B (confirmed positive), all 158 positive samples were in total agreement in both procedures. In subset C (screen positive only), Liaison XL Murex HIV Ab/Ag yielded accurate results in 42 out of 48 samples (87.5%). Global sensitivity and specificity for Liaison XL Murex HIV Ab/Ag (all subsets included) were 98.3% and 98.5%, respectively. Considering only nonselected prospective samples and confirmed positive samples (subsets A and B), the corresponding sensitivity and specificity values were 100% and 99.2%, respectively. The new fully automated HIV screening test showed high sensitivity and specificity compared to our standard algorithm. Its added advantage of being able to detect HIV-1 and HIV-2 antibodies and p24 antigen separately could prove useful in the diagnosis of early infections.
doi:10.1128/JCM.03486-13
PMCID: PMC3993662  PMID: 24574285
6.  Spread of Streptococcus pneumoniae Serotype 8-ST63 Multidrug-Resistant Recombinant Clone, Spain 
Emerging Infectious Diseases  2014;20(11):1848-1856.
This clone has spread throughout this country and caused invasive pneumococcal disease.
Since 2004, a total of 131 isolates of Streptococcus pneumoniae multidrug-resistant invasive serotype 8 have been detected in Spain. These isolates showed resistance to erythromycin, clindamycin, tetracycline, and ciprofloxacin. All isolates were obtained from adult patients and shared a common genotype (sequence type [ST]63; penicillin-binding protein 1a [pbp1a], pbp2b, and pbp2x gene profiles; ermB and tetM genes; and a ParC-S79F change). Sixty-eight isolates that required a ciprofloxacin MIC ≥16 μg/mL had additional gyrA gene changes. Serotype 8-ST63 pbp2x sequences were identical with those of antimicrobial drug–susceptible serotype 8-ST53 isolates. Serotype 8-ST63 pbp2b sequences were identical with those of the multidrug-resistant Sweden 15A-ST63 clone. Recombination between the capsular locus and flanking regions of an ST53 isolate (donor) and an ST63 pneumococcus (recipient) generated the novel 15A-ST63 clone. One recombination point was upstream of pbp2x and another was within pbp1a. A serotype 8-ST63 clone was identified as a cause of invasive disease in Spain.
doi:10.3201/eid2011.131215
PMCID: PMC4214286  PMID: 25340616
Streptococcus pneumoniae; bacteria; antimicrobial resistance; multidrug-resistant recombinant clone; fluoroquinolone resistance; Sweden 15A-ST63 clone; serotype 8-ST63; dissemination; spread; Spain
8.  Qualitative Analysis To Ascertain Genotypic Identity of or Differences between Mycobacterium tuberculosis Isolates in Laboratories with Limited Resources 
Journal of Clinical Microbiology  2013;51(12):4230-4233.
Mycobacterium tuberculosis is currently genotyped using mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing, although the high cost of this technique restricts its implementation in resource-limited settings. We designed a MIRU-VNTR format, MLP3 (MIRU-VNTR length polymorphism triplex), that is based on the qualitative comparison of 5 nonfluorescent 3-band fingerprints in conventional electrophoresis and minimizes costs and technical demands. MLP3 successfully resolved cross-contamination alerts, discriminated reinfections from reactivations, clarified suspected microepidemics, and tracked transmission events of high epidemiological interest.
doi:10.1128/JCM.02183-13
PMCID: PMC3838068  PMID: 24088847
9.  Clinical Practice Guidelines for the Diagnosis and Management of Intravascular Catheter-Related Infection: 2009 Update by the Infectious Diseases Society of Americaa 
These updated guidelines replace the previous management guidelines published in 2001. The guidelines are intended for use by health care providers who care for patients who either have these infections or may be at risk for them.
doi:10.1086/599376
PMCID: PMC4039170  PMID: 19489710
10.  Comparison between the EUCAST Procedure and the Etest for Determination of the Susceptibility of Candida Species Isolates to Micafungin 
Antimicrobial Agents and Chemotherapy  2013;57(11):5767-5770.
We compared the ability of the EUCAST EDef 7.2 and the Etest to detect the susceptibility to micafungin of 160 Candida and non-Candida clinical isolates. Agreement was higher when Etest MICs were obtained after 24 h of incubation; essential agreement was 90%, and categorical agreement was >90%. False susceptibility was seen only for Candida krusei (10%), and false resistance was observed in 6% of the isolates, ranging from 2.6% (C. glabrata) to 13% (C. albicans).
doi:10.1128/AAC.01032-13
PMCID: PMC3811256  PMID: 23979756
11.  Ethanol Lock Therapy (E-Lock) in the Prevention of Catheter-Related Bloodstream Infections (CR-BSI) after Major Heart Surgery (MHS): A Randomized Clinical Trial 
PLoS ONE  2014;9(3):e91838.
Background
Lock-therapy with antimicrobials has been used for the treatment and prevention of catheter-related bloodstream infections (CR-BSI). Experiences with Ethanol-Locks (E-locks) have included therapeutic interventions with variable results. Patients undergoing Major Heart Surgery (MHS) are a high-risk population for CR-BSI.The aim of this study was to assess the efficacy and tolerance to E-Locks in the prevention of CR-BSI of patients undergoing MHS.
Methods and Findings
This is an academic, prospective, randomized, non-blinded and controlled clinical trial assessing the incidence of CR-BSI of patients with E-locks (E-lock) and the tolerance to the procedure in comparison with patients receiving conventional catheter-care (CCC). Patients undergoing MHS with intravascular catheters for more than 48 hours were randomly assigned into treatment or control group by a computer-generated list of randomly assigned numbers. In the treatment group, all their catheter lumens were locked with an ethanol solution at 70% for two hours, every three days (E-Locks). The control group received conventional catheter-care (CCC).
Overall, 200 patients with 323 catheters were included in the study, which was stopped after 10 months due to adverse events. Of them, 179 catheters (113 patients) had E-Locks and 144 catheters (87 patients) were CCC. Euroscore Surgical Risk in both groups was 4.04 vs 4.07 p = 0.94 respectively. The results for the E-Locks and CCC were as follows: Incidence of CR-BSI/1000 days of exposure 2.1 vs 5.2 (p = 0.33), catheter tip colonization 14 (7.8%) vs 6 (4.2%) patients (p = 0.17), median length of hospital stay, 15 vs 16 days (p = 0.77). Seven patients (6.19%), all in the ethanol branch, had to discontinue the trial due to intolerance or adverse events.
Conclusions
We do not recommend prophylaxis of CR-BSI with ethanol-lock on a routine basis in patients undergoing Major Heart Surgery.
Trial Registration
Clinical Trials.gov NCT01229592
doi:10.1371/journal.pone.0091838
PMCID: PMC3967996  PMID: 24675993
12.  Impact of four sequential measures on the prevention of ventilator-associated pneumonia in cardiac surgery patients 
Critical Care  2014;18(2):R53.
Introduction
Ventilator-associated pneumonia (VAP) is the most frequent infection in patients admitted to intensive care units.
The efficacy of individual measures for the prevention of VAP is well documented, and data on the impact of implementing bundle measures have usually been reported from studies in which several measures are implemented simultaneously in the general intensive care unit (ICU).
The objective of our work was to evaluate the impact of four sequentially implemented measures for preventing VAP in a major heart surgery ICU. The measures were a specific training program, aspiration of subglottic secretions (ASSs), introduction of an inclinometer to improve the semirecumbent position, and reinforcement of oral care with chlorhexidine.
Methods
We compared rates of VAP, days on mechanical ventilation (MV), and cost of antimicrobial agents before and during implementation.
Results
We collected data from 401 patients before the intervention and from 1,534 patients during the intervention. Both groups were comparable. No significant differences in EuroSCORE were observed between the patients of both periods (6.4 versus 6.3; P = 0.7). The rates of VAP (episodes/1,000 days of ventilation) were, respectively, 23.9 versus 13.5 (P = 0.005). Mean number of days of MV/1,000 days of stay was 507 versus 375 (P = 0.001), and the cost of antimicrobial therapy (Euros/1,000 days of stay) was €70,612 versus €52,775 (P = 0.10). The main effect of sequential application of preventive measures in time achieved a relative-rate reduction of VAP of 41% (IRR, 0.41; 95% CI, 0.28 to 0.62). The mortality rate before and during the intervention was 13.0% and 10.2%, respectively.
VAP rate was most significantly reduced by training and the use of the inclinometer.
Conclusions
A sequentially applied bundle of four preventive measures reduces VAP rates, days of MV, and the cost of antimicrobial therapy in patients admitted to the major heart surgery ICU.
Trial registration
Clinical Trials.gov: NCT02060045. Registered 4 February 2014.
doi:10.1186/cc13799
PMCID: PMC4056787  PMID: 24667011
13.  PCR for Detection of Herpes Simplex Virus in Cerebrospinal Fluid: Alternative Acceptance Criteria for Diagnostic Workup 
Journal of Clinical Microbiology  2013;51(9):2880-2883.
The determination of herpes simplex virus (HSV) infection using a PCR assay is one of the most commonly requested tests for analysis of cerebrospinal fluid (CSF), although only a very low proportion of results are positive. A previously reported study showed that selecting only those CSF samples with >5 leukocytes/mm3 or a protein level of >50 mg/dl was adequate for the diagnostic workup. The aim of the present study was to assess the reliability of alternative acceptance criteria based on elevated CSF white blood cell counts (>10 cells/mm3). We analyzed all requests for HSV PCR received between January 2008 and December 2011. CSF samples were accepted for analysis if they had >10 cells/mm3 or if the sample was from an immunocompromised patient or a child aged <2 years. In order to evaluate our selection criteria, we identified those CSF samples with a leukocyte count of 5 to 10 cells/mm3 or protein levels of >50 mg/dl in order to test them for HSV type 1 and 2 (HSV-1 and HSV-2) DNA. During the study period, 466 CSF samples were submitted to the microbiology laboratory for HSV PCR. Of these, 268 (57.5%) were rejected, and 198 (42.5%) were tested according to our routine criteria. Of the tested samples, 11 (5.5%) were positive for HSV DNA (7 for HSV-1 and 4 for HSV-2). Of the 268 rejected specimens, 74 met the criteria of >5 cells/mm3 and/or protein levels of >50 mg/dl. Of these, 70 (94.6%) were available for analysis. None of the samples yielded a positive HSV PCR result. Acceptance criteria based on CSF leukocyte counts, host immune status, and age can help to streamline the application of HSV PCR without reducing sensitivity.
doi:10.1128/JCM.00950-13
PMCID: PMC3754672  PMID: 23804382
14.  Prospective Study of BK Virus Infection in Patients with Inflammatory Bowel Disease 
The Scientific World Journal  2014;2014:970528.
Patients with inflammatory bowel disease (IBD) have an immune-deficient baseline status further modulated by immunosuppressive therapy that may promote the reactivation of latent viruses such as BK virus (BKV). The aim of this prospective study was to determine the prevalence of BKV infection in IBD patients and its potential relationship with the immunosuppressive treatment. Paired urine and plasma samples from 53 consecutive patients with IBD and 53 controls were analyzed. BKV detection was performed by conventional PCR and positive samples were further quantified by real-time PCR. No viremia was detected. BKV viruria was significantly more common in IBD patients than among the controls (54.7% versus 11.3%; P < 0.0001). The only risk factor for BKV viruria in IBD was age (47.2 ± 16.3 versus 37.8 ± 15.2; P = 0.036), and there was a trend towards higher rate of viruria in outpatients (61.5% versus 38.5%; P = 0.096) and in those not receiving ciprofloxacin (59.5% versus 40.5%; P = 0.17). A clear impact of the immunosuppressive regimen on BKV infection could not be demonstrated.
doi:10.1155/2014/970528
PMCID: PMC3947848  PMID: 24696669
15.  Gram-Stain Plus MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry) for a Rapid Diagnosis of Urinary Tract Infection 
PLoS ONE  2014;9(1):e86915.
Microbiological confirmation of a urinary tract infection (UTI) takes 24–48 h. In the meantime, patients are usually given empirical antibiotics, sometimes inappropriately. We assessed the feasibility of sequentially performing a Gram stain and MALDI-TOF MS mass spectrometry (MS) on urine samples to anticipate clinically useful information. In May-June 2012, we randomly selected 1000 urine samples from patients with suspected UTI. All were Gram stained and those yielding bacteria of a single morphotype were processed for MALDI-TOF MS. Our sequential algorithm was correlated with the standard semiquantitative urine culture result as follows: Match, the information provided was anticipative of culture result; Minor error, the information provided was partially anticipative of culture result; Major error, the information provided was incorrect, potentially leading to inappropriate changes in antimicrobial therapy. A positive culture was obtained in 242/1000 samples. The Gram stain revealed a single morphotype in 207 samples, which were subjected to MALDI-TOF MS. The diagnostic performance of the Gram stain was: sensitivity (Se) 81.3%, specificity (Sp) 93.2%, positive predictive value (PPV) 81.3%, negative predictive value (NPV) 93.2%, positive likelihood ratio (+LR) 11.91, negative likelihood ratio (−LR) 0.20 and accuracy 90.0% while that of MALDI-TOF MS was: Se 79.2%, Sp 73.5, +LR 2.99, −LR 0.28 and accuracy 78.3%. The use of both techniques provided information anticipative of the culture result in 82.7% of cases, information with minor errors in 13.4% and information with major errors in 3.9%. Results were available within 1 h. Our serial algorithm provided information that was consistent or showed minor errors for 96.1% of urine samples from patients with suspected UTI. The clinical impacts of this rapid UTI diagnosis strategy need to be assessed through indicators of adequacy of treatment such as a reduced time to appropriate empirical treatment or earlier withdrawal of unnecessary antibiotics.
doi:10.1371/journal.pone.0086915
PMCID: PMC3899310  PMID: 24466289
16.  Endemic Genotypes of Candida albicans Causing Fungemia Are Frequent in the Hospital 
Journal of Clinical Microbiology  2013;51(7):2118-2123.
Genotyping of Candida albicans strains causing candidemia can uncover the presence of endemic genotypes in the hospital. Using a highly reproducible and discriminatory microsatellite marker panel, we studied the genetic diversity of 217 C. albicans isolates from the blood cultures of 202 patients with candidemia (from January 2007 to December 2011). Each isolate represented 1 candidemia episode. Multiple episodes were defined as the isolation of C. albicans in further blood cultures taken ≥7 days after the last isolation in blood culture. Of the 202 patients, 188 had 1 episode, 13 had 2 episodes, and 1 had 3 episodes. Identical genotypes showed the same alleles for all 6 markers. The genotypes causing both episodes were identical in most patients with 2 episodes (11/13; 84.6%). In contrast, 2 different genotypes were found in the patient with 3 episodes, one causing the first and second episodes and the other causing the third episode (isolated 6 months later). We found marked genetic diversity in 174 different genotypes: 155 were unique, and 19 were endemic and formed 19 clusters (2 to 6 patients per cluster). Up to 25% of the patients were infected by endemic genotypes that infected 2 or more different patients. Some of these endemic genotypes were found in the same unit of the hospital, mainly neonatology, whereas others infected patients in different wards.
doi:10.1128/JCM.00516-13
PMCID: PMC3697706  PMID: 23616451
17.  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 β-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.
doi:10.1128/AAC.02487-12
PMCID: PMC3716172  PMID: 23629706
18.  The Value of Combining Blood Culture and SeptiFast Data for Predicting Complicated Bloodstream Infections Caused by Gram-Positive Bacteria or Candida Species 
Journal of Clinical Microbiology  2013;51(4):1130-1136.
Management of complicated bloodstream infections requires more aggressive treatment than uncomplicated bloodstream infections. We assessed the value of follow-up blood culture in bloodstream infections caused by Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Candida spp. and studied the value of persistence of DNA in blood (using SeptiFast) for predicting complicated bloodstream infections. Patients with bloodstream infections caused by these microorganisms were enrolled prospectively. After the first positive blood culture, samples were obtained every third day to perform blood culture and SeptiFast analyses simultaneously. Patients were followed to detect complicated bloodstream infection. The study sample comprised 119 patients. One-third of the patients developed complicated bloodstream infections. The values of persistently positive tests to predict complicated bloodstream infections were as follows: SeptiFast positive samples (sensitivity, 56%; specificity, 79.5%; positive predictive value, 54%; negative predictive value, 80.5%; accuracy, 72.3%) and positive blood cultures (sensitivity, 30.5%; specificity, 92.8%; positive predictive value, 64%; negative predictive value, 75.5%; accuracy, 73.9%). Multivariate analysis showed that patients with a positive SeptiFast result between days 3 and 7 had an almost 8-fold-higher risk of developing a complicated bloodstream infection. In S. aureus, the combination of both techniques to exclude endovascular complications was significantly better than the use of blood culture alone. We obtained a score with variables selected by the multivariate model. With a cutoff of 7, the negative predictive value for complicated bloodstream infection was 96.6%. Patients with a positive SeptiFast result between days 3 and 7 after a positive blood culture have an almost 8-fold-higher risk of developing complicated bloodstream infections. A score combining clinical data with the SeptiFast result may improve the exclusion of complicated bloodstream infections.
doi:10.1128/JCM.02882-12
PMCID: PMC3666801  PMID: 23363819
19.  Rapid Detection of Staphylococcus aureus in Lower Respiratory Tract Secretions from Patients with Suspected Ventilator-Associated Pneumonia: Evaluation of the Cepheid Xpert MRSA/SA SSTI Assay 
Journal of Clinical Microbiology  2012;50(12):4095-4097.
A preclinical evaluation was conducted to evaluate the performance of the Cepheid Xpert assay on 135 lower respiratory tract secretions for detection of methicillin-resistant Staphylococcus aureus and S. aureus. Compared with the quantitative culture, the sensitivity, specificity, and positive and negative predictive values were 99.0%, 72.2%, 90.7%, and 96.3%, respectively.
doi:10.1128/JCM.02409-12
PMCID: PMC3502952  PMID: 22993185
20.  HACEK Infective Endocarditis: Characteristics and Outcomes from a Large, Multi-National Cohort 
PLoS ONE  2013;8(5):e63181.
The HACEK organisms (Haemophilus species, Aggregatibacter species, Cardiobacterium hominis, Eikenella corrodens, and Kingella species) are rare causes of infective endocarditis (IE). The objective of this study is to describe the clinical characteristics and outcomes of patients with HACEK endocarditis (HE) in a large multi-national cohort. Patients hospitalized with definite or possible infective endocarditis by the International Collaboration on Endocarditis Prospective Cohort Study in 64 hospitals from 28 countries were included and characteristics of HE patients compared with IE due to other pathogens. Of 5591 patients enrolled, 77 (1.4%) had HE. HE was associated with a younger age (47 vs. 61 years; p<0.001), a higher prevalence of immunologic/vascular manifestations (32% vs. 20%; p<0.008) and stroke (25% vs. 17% p = 0.05) but a lower prevalence of congestive heart failure (15% vs. 30%; p = 0.004), death in-hospital (4% vs. 18%; p = 0.001) or after 1 year follow-up (6% vs. 20%; p = 0.01) than IE due to other pathogens (n = 5514). On multivariable analysis, stroke was associated with mitral valve vegetations (OR 3.60; CI 1.34–9.65; p<0.01) and younger age (OR 0.62; CI 0.49–0.90; p<0.01). The overall outcome of HE was excellent with the in-hospital mortality (4%) significantly better than for non-HE (18%; p<0.001). Prosthetic valve endocarditis was more common in HE (35%) than non-HE (24%). The outcome of prosthetic valve and native valve HE was excellent whether treated medically or with surgery. Current treatment is very successful for the management of both native valve prosthetic valve HE but further studies are needed to determine why HE has a predilection for younger people and to cause stroke. The small number of patients and observational design limit inferences on treatment strategies. Self selection of study sites limits epidemiological inferences.
doi:10.1371/journal.pone.0063181
PMCID: PMC3656887  PMID: 23690995
21.  Evaluation of MycAssay™ Aspergillus for Diagnosis of Invasive Pulmonary Aspergillosis in Patients without Hematological Cancer 
PLoS ONE  2013;8(4):e61545.
Methods based on real-time polymerase chain reaction (PCR) can speed up the diagnosis of invasive aspergillosis but are limited by a lack of standardization. We evaluated the commercially available MycAssay™ Aspergillus test for the diagnosis of invasive aspergillosis in patients without hematological cancer. We prospectively collected 322 lower respiratory tract samples (November 2009–January 2011) from 175 patients with lower respiratory tract infection and the following predisposing conditions: solid cancer (16.8%), cirrhosis (16.8%), corticosteroid therapy (71.7%), HIV infection (15.6%), chronic obstructive pulmonary disease (COPD, 52.6%), solid organ transplantation (kidney [1.2%], heart [3%], liver [4.6%]), or none (3.5%). Specimens were obtained when clinically indicated and analyzed in the microbiology laboratory. Aspergillus DNA was extracted and amplified by means of MycXtra® and MycAssay™ Aspergillus. Aspergillus spp. was isolated from 65 samples (31 patients). According to the European Organization for Research and Treatment of Cancer and Bulpa's criteria (for patients with COPD), 15 had probable invasive aspergillosis. MycAssay™ Aspergillus results were negative (n = 254), positive (n = 54), or indeterminate (n = 14). The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic odds ratio of the MycAssay™ (first sample/any sample) were 86.7/93, 87.6/82.4, 34.1/34.1, 92.2/100, and 48/68.75. The differences between the proportion of samples with positive PCR determinations (63%) and the proportion of samples with Aspergillus spp. isolation (75%) did not reach statistical significance (P = 0.112). The median time from sample culture to visualization of fungal growth was 3 days, compared with ∼4 hours for MycAssay™ Aspergillus PCR. MycAssay™ Aspergillus showed high sensitivity for the diagnosis of invasive aspergillosis in patients without hematological cancer. Sensitivity increased when multiple samples were used. Compared with fungal culture, PCR significantly reduced the time to diagnosis.
doi:10.1371/journal.pone.0061545
PMCID: PMC3631214  PMID: 23620764
22.  Rapid Detection and Identification of Aspergillus from Lower Respiratory Tract Specimens by Use of a Combined Probe–High-Resolution Melting Analysis 
Journal of Clinical Microbiology  2012;50(10):3238-3243.
Diagnosis of invasive aspergillosis (IA) requires increasingly rapid molecular methods that enable sensitive detection and discrimination between species. We designed and evaluated a real-time PCR-based method that combined melting temperature (Tm) calling analysis of a specific probe with high-resolution melting analysis of the full amplicon. The test correctly identified 78 isolates of Aspergillus section Fumigati and non-Fumigati sections of Aspergillus with a limit of detection of 102 conidia/ml (102 fg/ml). No cross-reactivity with other fungi was found. The assay was further validated on lower respiratory tract specimens containing Aspergillus or not. It successfully identified Aspergillus to section level in 56 of 59 specimens. With culture as the gold standard, our assay shows 100% sensitivity and specificity and constitutes an efficient alternative for identification of Aspergillus in lower respiratory tract samples.
doi:10.1128/JCM.00176-12
PMCID: PMC3457424  PMID: 22837320
23.  Resistance to Voriconazole Due to a G448S Substitution in Aspergillus fumigatus in a Patient with Cerebral Aspergillosis 
Journal of Clinical Microbiology  2012;50(7):2531-2534.
A voriconazole-resistant isolate of Aspergillus fumigatus was recovered from an immunocompetent patient receiving long-term antifungal therapy for cerebral aspergillosis. A G448S amino acid substitution in the azole target (Cyp51A) was identified as the cause of the resistance phenotype. This article describes the first isolation of a voriconazole-resistant A. fumigatus isolate from an immunocompetent patient in Spain.
doi:10.1128/JCM.00329-12
PMCID: PMC3405584  PMID: 22573589
24.  Real-Time Molecular Epidemiology of Tuberculosis by Direct Genotyping of Smear-Positive Clinical Specimens 
Journal of Clinical Microbiology  2012;50(5):1755-1757.
We applied MIRU-VNTR (mycobacterial interspersed repetitive-unit–variable-number tandem-repeat typing) to directly analyze the bacilli present in 61 stain-positive specimens from tuberculosis patients. A complete MIRU type (24 loci) was obtained for all but one (no amplification in one locus) of the specimens (98.4%), and the allelic values fully correlated with those obtained from the corresponding cultures. Our study is the first to demonstrate that real-time genotyping of Mycobacterium tuberculosis can be achieved, fully transforming the way in which molecular epidemiology techniques can be integrated into control programs.
doi:10.1128/JCM.00132-12
PMCID: PMC3347103  PMID: 22378907
25.  In Vitro Acquisition of Secondary Azole Resistance in Aspergillus fumigatus Isolates after Prolonged Exposure to Itraconazole: Presence of Heteroresistant Populations 
Secondary resistance to azoles in Aspergillus fumigatus isolates from patients taking long-term itraconazole therapy has been described. We studied the acquisition of secondary azole resistance in 20 A. fumigatus isolates with no mutations at codon 54, 98, 138, 220, 432, or 448 in the cyp51A gene. Adjusted conidium inocula (3 × 107 CFU/ml) of each isolate were prepared and progressively or directly exposed to increasing itraconazole concentrations, ranging from 0.5 μg/ml to 16 μg/ml. Itraconazole, voriconazole, and posaconazole MICs were determined using the CLSI M38-A2 procedure before (MICinitial) and after (MICfinal) exposure to itraconazole. In both procedures, the MICfinal was significantly higher than the MICinitial. However, after progressive exposure to itraconazole, the MICs of the three azoles were higher than after direct exposure. No mutations were found at codon 54, 98, 138, 220, 432, or 448 in the cyp51A gene of isolates growing at the highest concentration of itraconazole. More concentrated conidium inocula (2 × 109 CFU/ml) plated in itraconazole at 4 μg/ml revealed the presence of heteroresistant populations in two initially wild-type isolates. These isolates became resistant to itraconazole and posaconazole only after use of the concentrated inoculum. These heteroresistant isolates harbored a mutation at codon G54, and the MICs of itraconazole and posaconazole were >16 μg/ml. In all procedures, A. fumigatus short tandem repeat (STRAf) typing was used to demonstrate that the genotype did not change before or after exposure to itraconazole.
doi:10.1128/AAC.00301-11
PMCID: PMC3256017  PMID: 22006000

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