Implication of Candida parapsilosis FKS1 and FKS2 mutations in reduced echinocandin susceptibility.

We evaluated FKS1 and FKS2 mutations in Candida parapsilosis bloodstream isolates and correlated them with the echinocandin MIC values determined by guidelines in CLSI document M27-A3 and the YeastOne panel. All mutations detected were outside hot spot (HS) regions. The F1386S mutation detected in an isolate that was resistant by the YeastOne panel but not by the M27-A3 guidelines might be implicated in echinocandin resistance. Further studies are needed to confirm the implication of the F1386S mutation and to elucidate the capability of the M27-A3 guidelines to detect echinocandin resistance.

Two unusual hepatitis C virus subtypes, 2j and 2q, in Spain: Identification by nested-PCR and sequencing of a NS5B region.

Many studies have reported the use of the NS5B gene to subtype hepatitis C virus (HCV). Other HCV genes, such as HCV-5' UTR, Core (C) and E1, have also been used. In some studies, NS5B have been used together with 5'-UTR or C genes to improve genotyping results obtained using commercial procedures. Only two studies in Spain have compared molecular techniques versus commercial procedures regarding the efficacy of HCV subtyping. The aim of this study was to determine whether nested PCR and sequencing of a NS5B region was more reliable than commercial procedures to subtype HCV. We analyzed the results of HCV genotyping in [726] serum specimens collected from 2001 to 2013. From 2001 to 2011, we used PCR and INNO-LiPA hybridization or its new version Versant HCV Genotype 2.0 assay (471 samples). From 2012 to 2013, we used nested PCR and sequencing of a NS5B region (255 cases). This method used two pairs of primers to amplify the RNA of the sample converted to DNA by retrotranscription. The amplification product of 270 base pairs was further sequenced. To identify the subtype, the sequences obtained were compared to those in the international database: http://hcv.lanl.gov./content/sequence/, HCV/ToolsOutline.html and Geno2pheno[hcv] http://hcv.bioinf.mpi-inf.mpg.de/index.php. Nested PCR of a NS5B region and sequencing identified all but one subtype (0.4%, 1/255), differentiated all 1a subtypes from 1b subtypes, and characterized all HCV 2-4 subtypes. This approach also distinguished two subtypes, 2j and 2q, that had rarely been detected previously in Spain. However, commercial procedures failed to subtype 12.7% (60/471) of samples and to genotype 0.6% of specimens (3/471). Nested PCR and sequencing of a NS5B region improved the subtyping of HCV in comparison with classical procedures and identified two rare subtypes in Spain: 2j and 2q. However, full length genome sequencing is recommended to confirm HCV 2j and 2q subtypes.

Predictors of tuberculosis transmission in prisons: an analysis using conventional and molecular methods.

OBJECTIVE: To determine the tuberculosis (TB) transmission patterns within the prison system in Catalonia, conventional epidemiological techniques were combined with DNA fingerprinting of Mycobacterium tuberculosis. METHODS: IS6110- and polymorphic GC-rich repeat sequence (PGRS)-based restriction fragment length polymorphism (RFLP) were combined with epidemiological studies to assess the relatedness of isolates from all patients with confirmed TB at five prisons in the province of Barcelona (Catalonia, Spain), between 1 July 1994 and 31 December 1996. Risk factors for transmission were analysed to a logistic regression. The extent of drug-resistant TB was also assessed. RESULTS: The incidence of TB during the study period was 2775 cases per 100,000 inmate years. Of the 247 culture-positive cases, 126 (51%) appeared to have active TB as a result of recent transmission. Using conventional epidemiological methods, 14 active chains of transmission were identified in prison involving 65 isolates (52% of clustered patients). A lengthy history of imprisonment [odds ratio (OR) 2.8, 95% confidence interval (CI) 1.52-5.11] and pulmonary TB (OR 2.36, 95% CI 1.17-4.75) were independently associated with clustering. Low rates of both initial (2.9%) and acquired drug resistance (5.8%) were identified and there was no evidence of the transmission of drug-resistant TB. CONCLUSION: In the prison system studied, the recent transmission of TB contributes substantially to the overall incidence of the disease. Both lengthy incarcerations and delays in identifying inmates with pulmonary symptoms play a key role in this recent transmission. Directly observed therapy (DOT) is a critical control strategy for reducing the emergence of drug resistance and for avoiding the transmission of resistant organisms.

Artificial neural network applied to prediction of fluorquinolone antibacterial activity by topological methods.

A new topological method that makes it possible to predict the properties of molecules on the basis of their chemical structures is applied in the present study to quinolone antimicrobial agents. This method uses neural networks in which training algorithms are used as well as different concepts and methods of artificial intelligence with a suitable set of topological descriptors. This makes it possible to determine the minimal inhibitory concentration (MIC) of quinolones. Analysis of the results shows that the experimental and calculated values are highly similar. It is possible to obtain a QSAR interpretation of the information contained in the network after the training has been carried out.

Epidemiologic evidence of transmission of donor-related bacterial infection through a transplanted heart.

This study describes a patient who had fulminant infectious myocarditis as a result of methicillin-resistant Staphylococcus aureus after receiving a heart transplant from an infected donor. There was complete concordance of typing results between donor and recipient strains that were different from the 20 isolates with which they were compared. Molecular epidemiologic study provided compelling evidence that a transplanted organ can transmit a bacterial infection from the donor to the recipient.

Comparison of conventional ribotyping and PCR-RFLP ribotyping for the analysis of endemic strains of Neisseria meningitidis isolated in a local community over 7 years.

Conventional ribotyping was compared with the PCR amplification of the intergenic spacer region between 16S and 23S rRNA genes (PCR-RFLP ribotyping) when applied to the subtyping of sporadic Neisseria meningitidis strains. Thirty isolates out of a total of 36 meningococcal disease cases, reported as having occurred in a particular community over a 7-year endemic period, were analyzed by each of the methods. Only ribotyping with three restriction enzymes (EcoRI, ClaI and XhoI) gave acceptable discriminatory power for short-term epidemiological purposes. We conclude that conventional ribotyping is a suitable method for typing sporadic meningococcal strains and that it cannot be replaced by the more straightforward PCR-RFLP ribotyping method.

Comparison of IS1245 restriction fragment length polymorphism and pulsed-field gel electrophoresis for typing clinical isolates of Mycobacterium avium subsp avium.

SETTING: Little is still known about the epidemiology and pathogenesis of Mycobacterium avium subsp avium (MASA) infection. OBJECTIVE: Examination of the reproducibility and the stability over time of pulsed-field gel electrophoresis (PFGE) and IS1245 restriction fragment length polymorphism (IS1245-RFLP) techniques. The ability of these typing systems for differentiating clinical isolates of MASA was also assessed. DESIGN: Clinical isolates recovered from 63 patients (59 human immunodeficiency virus [HIV] positive and four HIV-negative) were studied by insertion sequence IS1245 and PFGE. For the study of in vivo and in vitro stability, strains collected over time from four patients and five strains chosen at random, respectively, were used. RESULTS: The stability of PFGE and IS1245-RFLP in vitro was excellent. PFGE was also stable in vivo, but IS1245-RFLP patterns showed some variation. The discriminatory power of IS1245-RFLP and PFGE was 0.995 and 0.989, respectively. The cluster analysis did not reveal differences between strains recovered from HIV-negative and HIV-positive patients or between patients with colonisation, local infection or disseminated disease. CONCLUSION: IS1245-RFLP and PFGE are useful tools for typing MASA strains. However, IS1245 variations in vivo may complicate the analysis of epidemiological relationships.

Molecular and conventional epidemiology of tuberculosis in an inner city district.

SETTING: Molecular epidemiology has underlined the importance of recent tuberculosis (TB) transmission and has uncovered notable discrepancies compared with conventional epidemiology. OBJECTIVES: 1) To determine, by RFLP analysis, the percentage of clustered cases in an inner city district with a high incidence of TB (163/100,000) and the groups at risk of being clustered; and 2) to compare the role of conventional contact tracing (CCT) with that of RFLP. DESIGN: RFLP was carried out using the IS6110 and pTBN12 (PGRS) sequences of 165 cultures positive for Mycobacterium tuberculosis in the Ciutat Vella district of Barcelona during 1997-1998. Contact tracing was carried out in 171 of 251 declared cases (68.1%). Associations were assessed by calculating odds ratios (OR) with 95% confidence intervals (CI). Logistic regression was used for multivariate analysis. RESULTS: Using RFLP, 76 (46.0%) strains were found to be clustered by IS6110 and PGRS. From CCT, 30 new patients were found among 858 contacts (3.5%) and 57 patients were linked. In terms of RFLP and CCT, the main risk factor was intravenous drug use (IVDU). In 44 cases who lived alone and were not involved in CCT, 50% were in RFLP clusters. The concordance rate between RFLP and CCT was 8/13 (61.5%); the disagreement corresponded to a cluster of five recent immigrants from Africa. Subsequent to RFLP, an epidemiological connection was found in 15/55 cases (27.2%). CONCLUSIONS: The percentage of clustered cases is very high. CCT was useful for identifying new cases, but it was insufficient for detecting the pathways of transmission. CCT coverage needs to be improved in marginalized individuals, and the results correlated with those of RFLP.

Pharmacological studies of 1-(p-chlorophenyl)propanol and 2-(1-hydroxy-3-butenyl)phenol: two new non-narcotic analgesics designed by molecular connectivity.

Molecular topology has been applied to the design of new analgesic drugs. Linear discriminant analysis and connectivity functions were used to design two potentially suitable drugs which were synthesized and tested for analgesic properties by the acetic acid-induced abdominal constriction test in mice and the tail-flick test in rats. In mice, the compound 1-(p-chlorophenyl)propanol showed higher analgesic activity, both intraperitoneally and orally, than acetylsalicylic acid. 2-(1-Hydroxy-3-butenyl)phenol exhibited a lesser protective effect (70% of that shown by acetylsalicylic acid). In rats, acetylsalicylic acid gave the greatest protection against pain when administered intraperitoneally, while 1-(p-chlorophenyl)propanol was the most active orally. The 2-(1-hydroxy-3-butenyl)phenol, both intraperitoneally and orally, showed the least protective effect. These results demonstrated the peripheral analgesic properties of the selected compounds, thus confirming the validity of the molecular design method.

Correlation of pharmacological properties of a group of beta-blocker agents by molecular topology.

The molecular connectivity method has been applied to the study of pharmacological properties, among which are found the angor treatment dose, alpha-distribution half-life and intravenous LD50 in mouse, of a group of beta-blocker agents, verifying its application in the prediction of theoretic values for said pharmacological properties. To do this, the obtained multiple regression functions of the corresponding connectivity indices were used in relation with the experimental values of the properties, which are accompanied by the statistical parameters used in their selection criteria, as well as the corresponding random and cross-validation studies of said functions, which corroborate the good correlation of the selected equations.

Correlation of pharmacological properties of a group of hypolipaemic drugs by molecular topology.

This investigation was undertaken to test the ability of the molecular connectivity model to predict the percentage of plasma protein binding, the percentage of total cholesterol reduction and oral LD50 in rats of a group of hypolipaemic drugs using multi-variable regression equations with multiple correlation coefficients, standard error of estimate, degrees of freedom, F-Snedecor function values, Mallow's CP and Student's t-test as criteria of fit. Regression analyses showed that the molecular connectivity model predicts these properties. Corresponding stability (cross validation) studies were made on the selected prediction models which confirmed their goodness of fit. The results also demonstrated that the presence of substituents and molecular volume, determine the value of these properties in hypolipaemic drugs.

[Application of molecular epidemiology techniques in the study of food poisoning caused by Staphylococcus aureus]. / Aplicación de las técnicas de epidemiología molecular en el estudio de una toxiinfección alimentaria por Staphylococcus aureus.

BACKGROUND: The use of molecular epidemiology techniques has provided better knowledge as to the clonal organization of bacterian populations and thus allows better follow up of epidemics. An alimentary toxiinfection in a Barcelona school produced by Staphylococcus aureus was analyzed by the combination of epidemiologic, phenotype and genotype markers with the aim of determining the source of the alimentary contamination. METHODS: Nine strains of Staphylococcus aureus isolated in 6 food manipulators and 3 patients were studied with the following markers: biotype, antibiotype, phagotype, plasmid profile, polymorphism of the size of the restriction fragments of total DNA and ribotype. RESULTS: Epidemiologic study of the strains analyzed showed that both the phenotype markers and the plasmid profile are thecniques of little discriminatory value. The only clearly discriminatory technique used was ribotyping which defined 3 clones in the 9 strains of Staphylococcus aureus studied. CONCLUSIONS: Molecular study of isolated strains of Staphylococcus aureus was able to identify the causal origin of the alimentary toxiinfection in one of the 6 food manipulators studied.

Outbreak of Pseudomonas fluorescens bloodstream infection in a coronary care unit.

An outbreak of Pseudomonas fluorescens infection in six patients in a coronary care unit was associated with a source not previously reported, namely the ice bath used for cardiac output determinations. Outbreaks of pseudobacteraemia caused by P. fluorescens and occasional blood transfusion-associated bloodstream infection (BSI) have been described. However, during the last two decades, two outbreaks of P. fluorescens BSI have been described and this article reports a third. Isolation of P. fluorescens in blood cultures must alert clinicians to the possibility of contamination of infusate, lock solutions or catheter flush.

Nosocomial outbreak of Blastoschizomyces capitatus associated with contaminated milk in a haematological unit.

In July 2002, Blastoschizomyces capitatus was isolated from four neutropenic patients in a haematology unit. Two patients died due to disseminated infection while the other two had oropharyngeal colonisation. Nosocomial acquisition of the fungus was suspected and epidemiological and environmental studies were undertaken. To determine the potential source for the acquisition of the fungus, epidemiological relationships between the patients were investigated. We performed surveillance cultures on all patients and took environmental cultures of air, inanimate surfaces, food samples, blood products and chemotherapy drugs. No direct contact transmission between patients was found and B. capitatus was isolated only in vacuum flasks used for breakfast milk distribution. All isolates were compared by four independent molecular typing methods: pulsed-field gel electrophoresis, genomic DNA restriction endonuclease analysis, randomly amplified polymorphic DNA, and polymerase chain reaction fingerprinting using a single primer specific for one minisatellite or two microsatellite DNAs. Milk vacuum flasks and clinical strains were genetically indistinguishable by all typing techniques. Milk vacuum flasks were withdrawn from all hospital units and no further B. capitatus infection was detected. Our findings suggest that clonal dissemination of a single strain of B. capitatus from vacuum flasks used for milk distribution was responsible for this nosocomial outbreak in the haematological unit.

Tuberculosis transmission patterns among Spanish-born and foreign-born populations in the city of Barcelona.

During a 2-year period (2003-2004), tuberculosis (TB) transmission in Barcelona and the factors related to transmission among the Spanish- and foreign-born populations were studied by molecular epidemiology. Data were obtained from TB cases and Conventional Contact Tracing registries and genotyping was performed using restriction fragment length polymorphism (RFLP)-IS6110 and MIRU12 as a secondary typing method. Of the 892 TB cases reported, 583 (65.3%) corresponded to Spanish-born and 309 (34.6%) to foreign-born. Six hundred and eighty-seven cases (77%) were confirmed by culture. RFLP typing of 463/687 (67.4%) isolates was performed, revealing 280 (60.5%) unique and 183 (39.5%) shared patterns, which were grouped into 65 clusters. Spanish-born individuals were significantly more clustered than foreign-born individuals (44.6% vs. 28.8%; p 0.016). Clustering in foreign-born individuals was associated with HIV (p 0.051, odds ratio = 3.1, 95% confidence interval 1-10.9) and alcohol abuse (p 0.022), whereas, in the Spanish-born individuals, clustering was associated with age in the range 21-50 years, (p 0.024). Of the total clusters, 36/65 (55.3%) included only Spanish-born patients, whereas 22/65 (33.8%) included individuals from both populations. In mixed clusters, the index case was Spanish-born in 53% and foreign-born in 47%. Among the foreign-born, 2.8% were ill on arrival, 30% developed TB within the first year and 50.3% developed TB within the first 2 years; 58.3% were from South America. In conclusion, half of the foreign-born TB patients developed the disease during the first 2 years after arrival, which, in most cases, was the result of endogenous reactivation. Recent TB transmission among Spanish-born and foreign-born populations, as well as bidirectional transmission between communities, contributed significantly to the burden of TB in Barcelona, suggesting the need to improve Public Health interventions in both populations.

[IS6110 for typing strains of Mycobacterium tuberculosis: application of a standardized protocol with computerized image analysis]. / IS6110 en la tipificación de cepas de Mycobacterium tuberculosis: aplicación de un protocolo estandarizado con análisis informatizado de las imágenes.

BACKGROUND: For the epidemiological control of tuberculosis it is necessary to use specific markers for strain differentiation. The analysis of the restriction fragment length polymorphism (RFLP) by using the insertion sequence IS6110 as a probe, allows us to establish accurately the strains which are around in a population. Our study evaluates the efficiency of this marker in 136 strains of Mycobacterium tuberculosis isolated from our surroundings. A standardized protocol is used, and a computerized system to analyze the images is described. METHODS: DNA restriction was done with PvuII. The filters were revealed by following a chemiluminescence method using a 867 bp probe, which is located to the right of the PvuII site IS6110. The comparison of the patterns obtained was done in a computer. The coefficient of Dice was calculated to express the similarity of the profiles given by two strains. The strains were clustered by the UPGMA method. The graphic representation of the clusters was done by means of homology dendrograms. RESULTS: 124 different patterns were obtained from which 118 strains had a unique pattern while 18 shared it. The index of discrimination was 0.996. Ninety two percent of the strains had more than 6 bands and none had no IS6110. The median of homology was 27% in 8 of the patients, 2 consecutive isolates were studied with the result of no variation seen on the patterns. CONCLUSION: IS6110 has shown very effective because of its stability and high discriminative power. The standardized protocol together with a computerized analysis of the images obtained allows comparison of results, furthermore, to establish centralized data base. This would be very useful for epidemiological studies of tuberculosis and the chase of strains with special characteristics.

Acquired drug resistance in Mycobacterium tuberculosis isolates recovered from compliant patients with human immunodeficiency virus-associated tuberculosis.

We describe five compliant patients with human immunodeficiency virus (HIV)-associated tuberculosis (TB) that relapsed, with acquisition of resistance by the original Mycobacterium tuberculosis strains. Both the first and second isolates from each patient had the same IS (insertion sequence) 6110-based DNA fingerprint patterns. Three of the five patients developed TB that was resistant to rifampin alone; no mutation in the region of the rpoB gene was detected by a line probe assay in two of the isolates from these patients. We discuss several factors presumably associated with acquired drug resistance in HIV-infected patients, including exogenous reinfection, drug interactions, malabsorption of drugs, and the presence of a large organism burden.

[Usefulness of DR, PGRS, and spoligotyping in the typing of Mycobacterium tuberculosis. Comparison with IS6110]. / Utilidad de DR, PGRS y spoligotyping en la tipificación de Mycobacterium tuberculosis. Comparación con IS6110.

BACKGROUND: To evaluate 4 markers (IS6110, DR, PGRS and spoligotyping) to differentiate the strains of Mycobacterium tuberculosis isolated in our surroundings, most specially in those which contain a reduced number of IS6110 copies. In addition, to confirm the identity of the strains that share the same IS6110 restriction-hybridization pattern. METHODS: We selected 37 strains from a previous study: 25 had a unique IS6110 pattern and 12 grouped in 3 clusters (8 strains with 11 bands, 2 with 17 bands and 2 which shared the same six-band pattern). The PGRS and DR-RFLPs were obtained by AluI restriction and synthetic oligonucleotides specific to these sequences. The polymorphism of the DR region spacers was analyzed by spoloigotyping. For the amplification of the spacers we used the DRa and DRb primers. Detection was done hybridizing the PCR products on a membrane in which 43 specific spacers had been previously immobilized. RESULTS: Twenty-three different PGRS patterns and 18 spolygotyping patterns were obtained from 25 strains with unique IS6110 pattern. Eight patterns resulted from the 10 strains studied by DR. The 8 strains which shared an 11-band pattern, as well as the 2 strains which shared a 17-band pattern, resulted identical by the other markers. However, 2 strains which shared a 6-band pattern were different by both PGRS and DR or spoligotyping. CONCLUSIONS: 1) IS6110 resulted the most discriminative marker of all. 2) The clonality of clusters with a low number of bands has to be confirmed with alternative markers.

[Clinical relevance of gram-negative bacteria having inducible chromosomic beta-lactamase at an intensive care unit]. / Relevancia clínica de las bacterias gramnegativas que poseen betalactamasa cromosómica inducible en una unidad de cuidados intensivos.

BACKGROUND: The aim of the study was to determine the frequency of third-generation cephalosporins and aztreonam resistance in gram-negative bacteria with inducible chromosomal beta-lactamase (beta Lac-ind) after beta-lactam therapy in the medical-surgical intensive care unit (ICU) at a university-affiliated hospital. PATIENTS AND METHODS: We studied 34 infections in 29 patients admitted to the ICU. All were infected by strains with beta Lac-ind and all were treated with beta-lactam antibiotics. Susceptibility was determined by disc-diffusion. The beta-lactamase activity of those strains showing constitutive beta-lactamase overproduction were characterized by isoelectrofocusing. When this derepression occurred during the therapy, the strains were compared by genomic macrorestriction (PGFE). RESULTS: In 29 out of 34 infections the initial strains was susceptible. In 11 cases, the culture were not negativized in spite of their susceptible pattern. In 4 cases there was derepression during therapy. In 5 cases the initial strains were derepressed. The microorganisms isolated more frequently were Pseudomonas aeruginosa (22 cases) and Enterobacter cloacae (5 cases). The beta-lactamase activity detected correspond well with a betaLac-ind. In those cases with derepression during therapy, the initial susceptible strain and the resistant strain were identical by PGFE.