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.

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.

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.

Association between asymptomatic carriage and sporadic (endemic) meningococcal disease in an open community.

We analysed a strain collection representative of the overall Neisseria meningitidis population circulating in an open community (46,000 inhabitants, Spain) during an endemic period (30 isolates from patients and 191 from throat cultures of healthy individuals) by both phenotypic and molecular techniques. Almost all patient isolates were assigned to three hyper-virulent lineages (ET-5 complex, ET-37 complex and cluster A4) by both multilocus enzyme electrophoresis (MEE) and pulsed-field gel electrophoresis (PFGE). In contrast, MEE and PFGE assigned 20% and 15% respectively of carrier isolates to the hyper-virulent clones (4% for both methods together). There was also a higher correlation between PFGE and phenotypes associated with virulent clones. These notable differences between the two molecular methods were further observed in more than half the carrier isolates, suggesting that the associations between these strains were distorted by recombination events. However, almost one-third of total endemic strains from symptom-free carriers and almost all patient strains belonged to clones defined by MEE and PFGE, with no known epidemiological connection. These data indicate low transmission and a weak clonal structure for N. meningitidis.

QSAR analysis of hypoglycemic agents using the topological indices.

The molecular topology model and discriminant analysis have been applied to the prediction of some pharmacological properties of hypoglycemic drugs using multiple regression equations with their statistical parameters. Regression analysis showed that the molecular topology model predicts these properties. The corresponding stability (cross-validation) studies performed on the selected prediction models confirmed the goodness of the fits. The method used for hypoglycemic activity selection was a linear discriminant analysis (LDA). We make use of the pharmacological distribution diagrams (PDDs) as a visualizing technique for the identification and selection of new hypoglycemic agents, and we tested on rats the predictive ability of the model.

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.

Análisis de la transmisión de la tuberculosis en la población penitenciaria

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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.

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.

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.

Translocated intestinal bacteria cause spontaneous bacterial peritonitis in cirrhotic rats: molecular epidemiologic evidence.

BACKGROUND/AIMS: Intestinal bacterial translocation is common in cirrhotic rats with spontaneous bacterial peritonitis, and it is thought to play a major pathogenic role. There has so far been no evidence for clonality between bacteria isolated from intestine and ascites. This study aimed to use molecular epidemiology techniques to show that spontaneous bacterial peritonitis is due to translocated intestinal bacteria. METHODS: Samples of ascitic fluid, portal blood, mesenteric lymph nodes and ileal contents from healthy (n=10) and ascitic cirrhotic rats with (n=12) or without (n=15) spontaneous bacterial peritonitis were cultured. In six infected rats, DNA macrorestriction fragments of 30 bacterial isolates [Escherichia coli (n=13), Enterococcus faecalis (n=12) and Proteus mirabilis (n=5)] from ascites (n=8), mesenteric lymph nodes (n=7), portal blood (n=6), and ileal flora (n=9) were compared. RESULTS: Bacterial translocation was more frequent in animals with (58%) than in those without spontaneous bacterial peritonitis (20%, p=0.049) or controls (10%, p=0.026). The same bacterial strain was simultaneously isolated in ascites and in mesenteric lymph nodes and/or ileum in 7/8 (87%) instances. The identity rate for bacteria present in both ascites and mesenteric lymph nodes was 80% (4/5). Likewise, identity was demonstrated in 3/4 instances of bacteria found in both ascites and portal blood. CONCLUSIONS: These results indicate that spontaneous bacterial peritonitis in cirrhotic rats is mainly due to intestinal bacteria translocated to mesenteric lymph nodes. Portal blood could be a less frequent route.

Recurrent Pseudomonas aeruginosa pneumonia in ventilated patients: relapse or reinfection?

A prospective observational study was performed to determine whether recurrent episodes of pneumonia caused by Pseudomonas aeruginosa in ventilated patients were due to a relapse of the previous clone or to reinfection with a new one. Diagnosis was based on quantitative cultures of secretions obtained by bronchoscopy. Comparison of strains was made by chromosomal fingerprinting based on pulsed field gel electrophoresis (PFGE). Thirty-three (89.1%) of 37 patients survived the initial week after pneumonia diagnosis; six survivors (18.1%) had multiple episodes caused by the same species. Presence of adult respiratory distress syndrome (83.3% versus 22.2%, p = 0.02) was the only factor significantly associated with clinical recurrences. The 16 isolates from five patients (nine recurrences) were analyzed by PFGE. All new isolates from recurrent episodes, excepting one, were considered as relapses. These data suggest that most recurrent episodes of P. aeruginosa pneumonia in ventilated patients occur due to persistence of strains present in a prior infection.

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.

Serial surveillance cultures of skin and catheter hub specimens from critically ill patients with central venous catheters: molecular epidemiology of infection and implications for clinical management and research.

A prospective study of 45 central venous catheters was conducted to assess, by strain delineation, the turnover of skin and catheter hub (superficial) colonization and the relative contributions of catheter hub and skin colonization to catheter tip colonization. Serial quantitative cultures of skin and catheter hub were performed. Catheter tip, blood, and specimens for culture from targeted superficial sites (TSSs) were also collected at the time of catheter removal. Strains from 17 tip-positive catheters were delineated by pulsed-field gel electrophoresis. Only 12 (28.6%) of 42 skin strains and 14 (31.1%) of 45 catheter hub strains were found to be present at the time of catheter removal. In addition, only 9 (29.0%) of the 31 tip-colonizing strains were present on TSSs. Moreover, 15 (48.4%) of the 31 tip-colonizing strains had a superficial origin, and the other 16 (51.6%) were of unknown origin. In catheters suspected of infection, cultures of TSSs had a negative predictive value for catheter-related bacteremia of 94.4% but a positive predictive value of 44.4%. When the causative agent was identified (to the strain level) these values dropped to 80.9 and 18.7%, respectively. The study shows that skin and catheter hub colonization is a common, dynamic phenomenon. Strains recovered from TSSs showed a low level of correlation with strains from previous cultures of specimens from superficial sites and catheter tip isolates. Consequently, TSSs cannot be recommended for use in determining the therapy. However, catheter-related bacteremia is uncommon when cultures of TSSs are negative.

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.