Recovery of Legionella species from water samples using an internal method based on ISO 11731: suggestions for revision and implementation

The study aim was to determine retrospectively whether the parallel use of 2 media [buffered charcoal yeast extract (BCYE) and medium of Wadowsky and Yee (MWY)] to isolate Legionella spp. from water samples taken from hospital water supply systems increased the sensitivity of the culture method as compared with methods/protocols in which only seeding on a selective medium is used. We analyzed the results obtained from 931 positive water samples. In 484 of the 931 positive water samples, Legionella spp. was isolated in the presence of other microorganisms; in 83% (400/484), we used MWY to count suspected colonies, which gave a lower number of unreadable plates. In the 447 samples containing only Legionella spp., the highest frequency of positive samples (93%, 418/447) was obtained with BCYE, whereas seeding on MWY yielded 78% (348/447) (P < 0.001). Evaluation of the influence of the media on the Legionella spp. counts obtained by the 2 media showed that BCYE agar produced significantly higher counts than MWY (P < 0.001). The major conclusions that may be drawn from our data are as follows: 1) BCYE gives a high recovery rate of positive samples (93%) and a much greater yield of Legionella spp. than MWY; 2) BCYE was necessary for the detection of non-L. pneumophila spp. which grew poorly on selective media; 3) selective media [MWY or GVPC (glycine, vancomycin, polymyxin B, and cycloheximide)] were necessary for the recovery of Legionella spp. when the non-selective medium (BCYE) was difficult to interpret because of contaminating background flora. The use of different media is recommended for routine water tests in hospitals.     

Streptomyces pneumonia in an immunocompetent patient: a case report and literature review

We describe a case of Streptomyces lanatus pneumonia in an immunocompetent 52-year-old woman. Bronchoalveolar lavage culture grew Streptomyces, which was identified by 16S rRNA sequencing. The infection resolved completely after successful treatment with doxycycline for 6 months. We also review the literature on human invasive Streptomyces infections.     

Characterization and monitoring of linezolid-resistant clinical isolates of Staphylococcus epidermidis in an intensive care unit 4 years after an outbreak of infection by cfr-mediated linezolid-resistant Staphylococcus aureus

Resistance to linezolid is emerging among Staphylococcus epidermidis isolates. During the 4 years following an outbreak of cfr-mediated linezolid-resistant S. aureus in our intensive care unit in 2008, we analyzed the clinical context and characterized the resistance mechanisms of 100 linezolid-resistant strains of S. epidermidis. The prevalence of the cfr gene in our strains reached 50% alone or in combination with other mechanisms. The mutation G2576T in domain V was found in 22% of strains, and both the cfr gene and G2576T in 44%. We also found an association between the cfr gene and mutations in the ribosomal protein L3. All 3 mechanisms co-occurred in 1 strain. MICs in combinations rose to >256 μg/mL. 58% of colonized patients, and 90% of infected patients had previously received linezolid for at last 10 days. Vancomycin was the main antibiotic in these infections, most of which were bacteremia. We found a high prevalence of the cfr gene in our clinical S. epidermidis isolates after the 2008 outbreak, despite having implemented isolation and control measures. The potential transmissibility of this agent, even without prior exposure to linezolid, can have serious epidemiological repercussions.     

Nursing assistants' behaviour during morning care: effects of the implementation of snoezelen, integrated in 24-hour dementia care

AIM: This paper reports an investigation of the effects of the implementation of snoezelen, or multisensory stimulation, on the quality of nursing assistants' behaviour during morning care. BACKGROUND: Nursing assistants in long-term dementia care are often unaware of the impact of their behaviour on patient functioning. Snoezelen is a psychosocial intervention that might improve the quality of caregiver behaviour by combining a person-centred approach with the integration of sensory stimuli. METHODS: A quasi-experimental pre- and post-test design was implemented in 12 wards for older mentally infirm patients at six nursing homes. The experimental group intervention was a 4-day in-house 'snoezelen' training, stimulus preference screening and supervision meetings. The control group received usual nursing home care. The effectiveness of the intervention was studied by analysing 250 video recordings, which were assessed by independent observers using a 4-point measurement scale developed for this study and based on Kitwood's Dialectical Framework. RESULTS: The results showed a statistically significant increase in 'Positive Person Work' and decrease in 'Malignant Social Psychology' (total scores) after the implementation of snoezelen. Nursing assistants in the experimental group also improved by statistically significant amounts on all subitems of 'Positive Person Work'. The mean number of sensory stimuli, offered explicitly, increased. CONCLUSION: The implementation of snoezelen succeeded in effecting a change to a more person-centred approach during morning care. The results indicate that nursing assistants' behaviour can be positively changed provided that the new care model has been successfully implemented.     

Candida nivariensis as an etiologic agent of vulvovaginal candidiasis in a tertiary care hospital of New Delhi,India

Candida nivariensis is a cryptic species, phenotypically indistinguishable from Candida glabrata and identified by molecular methods. Aside its isolation from broncho-alveolar lavage, we report for the first time the etiologic role of C. nivariensis in 4 patients with vulvovaginal candidiasis. Of 100 phenotypically identified C. glabrata isolates originating from vaginal swabs, 4 were identified as C. nivariensis by polymerase chain reaction and confirmed by sequencing. All of the C. nivariensis isolates exhibited white colonies on CHROMagar. Phylogenetic analysis revealed genotypic diversity in the C. nivariensis isolates originating from within or outside of India. Barring a solitary C. nivariensis isolate with MIC, 16 μg/mL of fluconazole, the rest were susceptible to voriconazole, itraconazole, posaconazole, isavuconazole, amphotericin B, and echinocandins. The patient with high fluconazole MIC did not respond to fluconazole therapy. It is suggested that the prevalence of this species is likely to be much higher than apparent from the sporadic published reports.     

Epidemiology and molecular characterization of a clone of Burkholderia cenocepacia responsible for nosocomial pulmonary tract infections in a French intensive care unit

Clustered cases of nosocomial pulmonary infections were observed in a French intensive care unit. Biochemical tests showed the etiologic agents to be part of the Bcc (Bcc). recA polymerase chain reaction-restriction fragment length polymorphism analysis and molecular phylogeny positioned the isolates into Burkholderia cenocepacia. Their recA sequences were found identical to those of ET12 strains responsible of necrotic pneumonia in cystic fibrosis patients. Analyses of a multi locus sequence typing genes set confirmed this proximity and suggested a wide distribution among occidental countries but could not resolve their phylogenetic position unambiguously. A novel marker, ecfB, indicated a significant phylogenetic divergence from ET12 strains. Pulse field gel electrophoresis analysis of SpeI-restricted total genomic DNA of the strains showed a unique profile indicative of a clonal outbreak. Environmental hospital screenings indicated cross-contamination between staff and patients. Bcc strains from outdoor environments were not related to this clone but indicated the presence of Burkholderia multivorans and Burkholderia vietnamiensis.     

Identification of an erm(T) gene in strains of inducibly clindamycin-resistant group B Streptococcus

Five inducibly clindamycin (CLI)-resistant group B Streptococcus (GBS) isolates, all negative for erm(A) and erm(B) genes, were found to contain erm(T), a gene previously reported in erythromycin-resistant animal isolates of Lactobacillus spp. and human isolates of Streptococcus bovis. One additional GBS isolate, constitutively resistant to CLI, was also positive for the erm(T) gene in addition to erm(B). To our knowledge, this is the 1st report of erm(T) in GBS, the 2nd bacterial species from humans in which the erm(T) gene has been identified, and the 3rd erm gene to be found in GBS.     

Rapid identification of strains belonging to the Mycobacterium abscessus group through erm(41) gene pyrosequencing

Mycobacterium abscessus and Mycobacterium massiliense lung infections have different clarithromycin susceptibilities, making proper identification important; however, standard multi-gene sequencing in clinical laboratories is laborious and time consuming. We developed a pyrosequencing-based method for rapid identification of strains belonging to the M. abscessus group by targeting erm(41). We examined 55 isolates from new pulmonary M. abscessus infections and identified 28 M. abscessus, 25 M. massiliense, and 2 Mycobacterium bolletii isolates. Multi-gene sequencing of 16S rRNA, hsp65, rpoB, and the 16S–23S ITS region was concordant with the results of erm(41) pyrosequencing; thus, the M. abscessus group can be identified by single-nucleotide polymorphisms in erm(41). The method also enables rapid identification of polymorphic, inducible clarithromycin-resistant sequevars (T28 or C28). Pyrosequencing of erm(41) is a rapid, reliable, high-throughput alternative method for identifying and characterizing M. abscessus species. Further testing of a diverse collection of isolates is necessary to demonstrate the discriminatory power of erm(41) sequencing to differentiating species with this highly divergent group.     

Clinical characteristics and outcomes of patients with Burkholderia cepacia bacteremia in an intensive care unit

The purpose of this study was to investigate a cohort of patients with Burkholderia cepacia bacteremia in the intensive care unit (ICU) at our institution. A large outbreak of B. cepacia bacteremia involving 95 patients lasted for 4 years in an ICU in northern Taiwan. The clinical characteristics and antimicrobial treatment responses of these patients were analyzed. Minimal inhibitory concentrations were determined and pulse-field gel electrophoresis was performed for the 73 available isolates. Overall, the in-hospital mortality rate was 53.8% and the 14-day mortality rate was 16.8%. Most patients (95.6%) had several underlying diseases and all but 1 patient had tracheal intubation. Malignancy (37.5% versus 13.9%, P = 0.02) and higher Sequential Organ Failure Assessment (SOFA) scores at the onset of bacteremia (11.9 ± 4.7 versus 7.9 ± 3.6, P < 0.001) were significant risk factors for 14-day mortality. In contrast, treatment with ceftazidime (76.0% versus 43.7%, P = 0.02) and diabetes (51.9% versus 13.8%, P = 0.01) were associated with decreased mortality. In the multivariate analysis, malignancy and higher SOFA score were significant risk factors for mortality [odds ratio (OR) 12.45, 95% confidence interval (CI) 2.35-65.94; OR 1.20, 95% CI 1.00-1.45, respectively]. Meropenem, ceftazidime, and piperacillin-tazobactam were the most active agents (susceptible rate 100%, 97.3%, and 97.3%, respectively). Pulsed-field gel electrophoresis results indicated 49 of the 73 isolates could be classified as outbreak-related strains. There was no significant difference in the clinical characteristics and outcomes of patients with bacteremia due to outbreak-related and non-outbreak-related strains. In conclusion, malignancy and a higher SOFA score at onset of bacteremia predicted increased mortality, but the clinical presentation and outcome of patients with outbreak and non-outbreak strains were similar.     

Candidemia due to Candida tropicalis: clinical, epidemiologic, and microbiologic characteristics of 188 episodes occurring in tertiary care hospitals

Candida tropicalis is the 2nd most frequent agent of candidemia in Brazil (20-24%). We attempted to characterize the epidemiology, microbiology, and outcome of candidemia due to C. tropicalis by comparing patients with candidemia due to C. tropicalis with those with candidemia due to Candida albicans. Among the 924 episodes of candidemia, 188 (20%) were caused by C. tropicalis. These cases were compared with 384 candidemias due to C. albicans. C. tropicalis was the 2nd most frequent species in adults (21.6%) and elderly patients (23.2%), and 3rd in neonates (11.9%) and children (18.5%). Cancer was the most frequent underlying disease, and in adults and elderly patients, diabetes was the 2nd most frequent underlying disease. The only difference between C. tropicalis and C. albicans candidemia was a higher proportion of neutropenic patients in C. tropicalis candidemia. C. tropicalis is a leading cause of candidemia in Brazil, and its epidemiology is similar to that of C. albicans.     

Enhancement of antistaphylococcal activities of six antimicrobials against sasG-negative methicillin-susceptible Staphylococcus aureus: an in vitro biofilm model

This study was designed to evaluate antimicrobial activities against methicillin-susceptible Staphylococcus aureus in both sessile and planktonic forms and to detect genes associated with this biofilm phenotype. Minimal biofilm inhibition and eradication concentrations (MBIC and MBEC, respectively) were determined by an in vitro biofilm model, and icaA, atlA, and sasG genes were detected by polymerase chain reaction. Vancomycin and tigecycline presented better biofilm inhibitory activity (MBIC range: 4-8 μg/mL) (P ≤ 0.05) and lower MBEC/MIC ratios (P ≤ 0.001) than other antimicrobials. All isolates harbored icaA and atlA, whereas sasG was present only in strong biofilm formers (P ≤ 0.05). Interestingly, antimicrobial activities against sasG- weak biofilm formers were significantly higher than those against sasG+ strong biofilm formers (P ≤ 0.05), demonstrating that number of cells in a biofilm matrix affected the antimicrobial activity, which was also variable, and might be associated with specific genetic determinants. To our knowledge, this was the first study reporting the presence of sasG in clinical isolates of S. aureus in South America.     

An outbreak case of Clostridium difficile-associated diarrhea among elderly inpatients of an intensive care unit of a tertiary hospital in Rio de Janeiro, Brazil

The aim of this study was to investigate Clostridium difficile-associated diarrhea (CDAD) in an intensive care unit (ICU) of a tertiary hospital in Rio de Janeiro, Brazil, and to characterize epidemiologically C. difficile strains obtained from an outbreak of CDAD. Within almost a 4-year surveillance period, CDAD incidence was determined for the first time in Brazil, and a 3-fold increase was observed in the average rate of CDAD, featuring an outbreak. About 80% of the patients were over 65 years. The main antibiotic that could be probably associated to CDAD was piperacillin/tazobactam. Four toxigenic strains were isolated, 3 from stools and 1 from environmental samples. They were all resistant to clindamycin and fluoroquinolones. Fingerprinting analysis revealed their distribution between 2 different polymerase chain reaction ribotypes, with one of them being exclusively found in Brazil. It was possible to detect cross-infection and environmental contamination in the ICU. Our results highlight the importance of a continuous CDAD surveillance in the hospitals, especially when a risk group is exposed.     

Development of a diagnostic real-time polymerase chain reaction assay for the detection of invasive Haemophilus influenzae in clinical samples

Since the introduction of the Haemophilus influenzae serotype b vaccine, invasive H. influenzae disease has become dominated by nontypeable (NT) strains. Several widely used molecular diagnostic methods have been shown to lack sensitivity or specificity in the detection of some of these strains. Novel real-time assays targeting the fucK, licA, and ompP2 genes were developed and evaluated. The fucK assay detected all strains of H. influenzae tested (n = 116) and had an analytical sensitivity of 10 genome copies/polymerase chain reaction (PCR). This assay detected both serotype b and NT H. influenzae in 12 previously positive specimens (culture and/or bexA PCR) and also detected H. influenzae in a further 5 of 883 culture-negative blood and cerebrospinal fluid (CSF) samples. The fucK assay has excellent potential as a diagnostic test for detection of typeable and nontypeable strains of invasive H. influenzae in clinical samples of blood and CSF.     

Comparison of direct cultivation on a selective solid medium,polymerase chain reaction from an enrichment broth,and the BD GeneOhm™ VanR Assay for identification of vancomycin-resistant enterococci in screening specimens

Fast and reliable diagnostics of vancomycin-resistant enterococci (VRE) is an important prerequisite for containing VRE transmission rates and controlling VRE outbreaks among hospital patients. The BD GeneOhm™ VanR Assay (Becton Dickinson Diagnostics, Erembodegem, Belgium) is a real-time polymerase chain reaction (PCR) assay for screening perianal/rectal samples for the presence of vanA or vanB genes that can be associated with VRE. A set of 51 reference strains (vanA–G genotypes) were correctly identified. Performance of the assay was evaluated and compared with culture-based methods and subsequent PCR analysis in 2 university hospitals with a different VRE prevalence. A total of 1786 samples were analyzed. With the use of the BD GeneOhm™ VanR Assay, 88 of 102 vanA-positive specimens, 62 of 67 vanB-positive specimens, 3 of 4 vanA- and vanB-positive specimens, and 1403 of 1613 negative specimens were correctly identified. The overall sensitivity was 93.1%; the specificity was 87.0% mainly due to false-positive vanB results. Results did not differ between study institutions.     

FoxP3 demethylation is increased in human colorectal cancer and rat cholangiocarcinoma tissue

Objectives

FoxP3 expression is a marker for Tregs which are known to be involved in tumor immunity. We aimed to evaluate FoxP3 promoter demethylation in human colorectal cancer (CRC) and rat intrahepatic cholangiocarcinoma (ICC).

Design and methods

Bisulfite-treated genomic DNA templates of shock frozen paired samples were studied from 13 anonymous CRC patients and from 10 male rats (n = 6 ICC induced by thioacetamide and n = 4 age-matched controls). Real-time PCR was carried out using a LightCycler 480 system. Human FoxP3 and CD3 promoter demethylations were estimated using previously described assays; and rat FoxP3 promoter demethylation using a newly developed assay.

Results

A significant 3.5-fold increase of the demethylation in FoxP3 promoter region was found in human CRC and rat ICC (P < 0.05). The average frequency of cells with FoxP3 demethylation in patients suffering from CRC was 0.26% in normal tissue and 0.92% in tumor tissue (n = 11 paired samples). Although, no significant difference was found between the mean frequency of CD3 demethylation in normal tissue (4.80%, n = 6) and in tumor tissue (4.14%, n = 6) from CRC patients, the ratio of demethylated CD3/FoxP3 promoter areas was significantly lower in tumor specimens (P < 0.05). Using our novel assay, we found a significant increase in mean frequencies of cells with FoxP3 demethylation in rats with ICC (7.42%, n = 6) in comparison to controls (2.14%, n = 4).

Conclusion

FoxP3 seems to be an interesting biomarker for immune response to epithelial tumors. Functional consequences from the increase of Tregs remain to be demonstrated. Further studies with outcome data are necessary.     

Diagnosis of the infection by the Helicobacter pylori through stool examination: Method standardization in adults

Objectives

Infection caused by Helicobacter pylori (H. pylori) is one of the most common causes of chronic infection in the world. The presence of the infection is strongly associated with the neoplasia of the gastrointestinal tract, and its diagnosis is easily made by means of invasive or non-invasive methods. Among such methods, the H. pylori antigen detection in stool through ELISA technique is easily performed and it is an alternative to endoscopy in children, since this exam is not usually indicated in this age group. The aim of the current study is to establish the standardization of the ELISA method for the detection of H. pylori in stool specimens in Brazil.

Design and methods

Patients between 18 and 70 years of age were randomly selected in the gastroenterology ambulatory center at Faculdade de Medicina do ABC between 2007 and 2009. They all answered a questionnaire to investigate possible dyspeptic symptoms and then underwent endoscopy and detection of H. pylori through no more than 4 methods. Besides the gastric biopsy, established as the gold standard test, the urease test, the stool ELISA test and serology were also methods applied.

Results

The sensitivity and specificity of the exams in this sample were respectively 87.2% and 44% for the stool ELISA test, 41.9% and 64% for serology, 65.6% and 58.8% for the urease test and 100% and 80.8% for the clinical analysis.

Conclusions

The ROC curve showed a good correlation between the compared methods. In Brazil the standardization of the ELISA test for the detection of H. pylori in stool specimens constitutes a non-invasive diagnostic alternative.     

Advantages of the high resolution melting in the detection of BRCA1 or BRCA2 mutation carriers

Objective

The aim of the study is to explore the reliability of the high resolution melting (HRM) analysis for the identification of BRCA1/BRCA2 mutation carriers among the family members of index patient (IP) and for distinguishing the presence of two or more genetic variants within the same amplicon.

Design and methods

We studied 27 different BRCA1/BRCA2 pathogenic mutations detected in 35 families with 194 subjects. HRM was performed in the LightCycler 480 (Roche).

Results

HRM method detected 110 BRCA1/BRCA2 mutations among the 192 relatives studied (57%). No false negative results were observed in any of the family members and all of them were in agreement with sequencing analysis, therefore the method might help to avoid unnecessary sequencing of wild type (WT) genotypes. The HRM method also allows the detection of other alterations that we initially had not searched (three unclassified variants and several polymorphisms). Furthermore, HRM has also been capable of distinguishing the presence of two or more genetic variants in the same amplicon of the same sample.

Conclusions

HRM is a rapid, sensitive, specific, cost-effective and reliable screening method that in less than 2 h allows the easy identification of BRCA1 and BRCA2 genetic variations and also avoids the unnecessary sequencing of WT genotypes. Furthermore the method is also capable of detecting new genetic variants and allows the simultaneous detection of the presence of more than one genetic variant.     

Five-year report of national surveillance of antimicrobial resistance in Pseudomonas aeruginosa isolated from non-tertiary care hospitals in Korea (2002-2006)

A nationwide surveillance of the antimicrobial resistance of Pseudomonas aeruginosa isolates from non-tertiary care hospitals was conducted in Korea from 2002 to 2006. Resistance to almost all antimicrobial agents decreased significantly from 2003 (P < 0.01). Resistance rates to the major antipseudomonal agents, ceftazidime, imipenem, meropenem, and aztreonam, were 18.8%, 20.5%, 18.7%, and 19.7%, respectively, in 2003. However, they had all decreased to below 10% in 2006. The proportion of multidrug-resistant isolates that were resistant to at least 3 of 5 major antipseudomonal agent decreased from 33.5% in 2003 to 23.1% in 2006 (P < 0.05). In this study, we found a decreasing trend in resistance rates and low resistance rates in P. aeruginosa from non-tertiary care hospitals compared with those from general hospitals, including tertiary care hospitals, in Korea. Our data provide valuable information for the selection of reliable empiric therapies for P. aeruginosa infections in non-tertiary care hospital patients, including outpatients.     

Identification of genetic recombination between Acinetobacter species based on multilocus sequence analysis

During multilocus sequence analysis of Acinetobacter calcoaceticus-Acinetobacter baumannii complex, we identified the evidence of recent genetic recombination between 2 Acinetobacter species. While 3 isolates belonged to A. nosocomialis based on 16S rRNA, gyrB, fusA, gdhB, and rplB gene sequences, they showed close relationships with Acinetobacter genomic species 'close to 13TU' in rpoB, recA, cpn60, rpoD, and gltA gene trees.