An evaluation of methods for the isolation of nontuberculous mycobacteria from patients with cystic fibrosis, bronchiectasis and patients assessed for lung transplantation.

BACKGROUND: RGM medium is an agar-based, selective culture medium designed for the isolation of nontuberculous mycobacteria (NTM) from the sputum of patients with cystic fibrosis (CF). We evaluated RGM medium for the detection of NTM in patients with CF (405 samples), bronchiectasis (323 samples) and other lung diseases necessitating lung transplantation (274 samples). METHODS: In total, 1002 respiratory samples from 676 patients were included in the study. Direct culture on RGM medium, with incubation at two temperatures (30 °C and 37 °C), was compared with conventional culture of decontaminated samples for acid-fast bacilli (AFB) using both a solid medium (Löwenstein-Jensen medium) and a liquid medium (the Mycobacterial Growth Indicator Tube; MGIT). RESULTS: For all three patient groups, significantly more isolates of NTM were recovered using RGM medium incubated at 30 °C than by any other method (sensitivity: 94.6% vs. 22.4% for conventional AFB culture; P < 0.0001). Significantly more isolates of Mycobacterium abscessus complex were isolated on RGM at 30 °C than by AFB culture (sensitivity: 96.1% vs. 58.8%; P < 0.0001). The recovery of Mycobacterium avium complex was also greater using RGM medium at 30 °C compared to AFB culture (sensitivity: 83% vs. 70.2%), although this difference was not statistically significant and a combination of methods was necessary for optimal recovery (P = 0.21). CONCLUSIONS: In the largest study of RGM medium to date, we reaffirm its utility for isolation of NTM from patients with CF. Furthermore; we show that it also provides an effective tool for culture of respiratory samples from patients with bronchiectasis and other lung diseases.

Evaluation of a new chromogenic medium, chromID OXA-48, for recovery of carbapenemase-producing Enterobacteriaceae from patients at a university hospital in Turkey.

The purpose of this study was to evaluate a new chromogenic medium, chromID OXA-48, for the isolation of carbapenemase-producing Enterobacteriaceae (CPE) directly from rectal swabs. chromID CARBA and chromID OXA-48 are two chromogenic media that have been commercialized for the isolation of CPE directly from clinical samples. Both media were evaluated alongside a broth enrichment method recommended by the CDC for isolation of CPE, with rectal swabs from 302 unique hospitalized patients at the Hacettepe University Hospital, Ankara, Turkey. A total of 33 patients (11 %) were found to be colonized with CPE using a combination of all methods, and all CPE produced OXA-48 carbapenemase. Klebsiella pneumoniae was by far the most dominant species of CPE and was isolated from 31 patients. Culture on chromID OXA-48 offered the highest sensitivity (75.8 %) for detection of CPE compared with the other two methods (sensitivity for both other methods was 57.6 %) and also offered the highest specificity (99.3 %). However, a combination of methods (either chromID OXA-48 plus CDC method or chromID OXA-48 plus chromID CARBA) was necessary to achieve an acceptable sensitivity (90.9 %). For isolation of CPE, in a setting where OXA-48 carbapenemase is the dominant type of carbapenemase, chromID OXA-48 is a highly useful medium but using a combination of methods is optimal for adequate detection. The combined use of two chromogenic media offered acceptable sensitivity (90.9 %) and the highest specificity (98.5 %) and also allowed for isolation of CPE within 18-20 h.

Development of a novel method for detection of Clostridium difficile using HS-SPME-GC-MS.

AIMS: A novel method has been developed that allows successful differentiation between Clostridium difficile culture-positive and culture-negative stool samples based on volatile organic compound (VOC) evolution and detection by headspace solid-phase microextraction coupled with gas chromatography mass spectrometry (HS-SPME-GC-MS). METHODS AND RESULTS: The method is based on the activation of p-hydroxyphenylacetate decarboxylase produced by Cl. difficile and the detection of a specific VOC, that is 2-fluoro-4-methylphenol from an enzyme substrate. In addition, other VOCs were good indicators for Cl. difficile, that is isocaproic acid and p-cresol, although they could not be used alone for identification purposes. One hundred stool samples were tested, of which 77 were positive by culture. Detection using HS-SPME-GC-MS allowed confirmation of the presence of Cl. difficile within 18 h with a sensitivity and specificity of 83·1 and 100%, respectively. CONCLUSIONS: It is recommended that this new approach could be used alongside conventional methods for Cl. difficile detection, including toxin detection methods, which would allow any false-negative results to be eliminated. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to identify Cl. difficile-positive stool samples by the analysis of VOCs could allow the development of a VOC detection device which could allow rapid diagnosis of disease and hence prompt treatment with appropriate antibiotics.

TNFα from classically activated macrophages accentuates epithelial to mesenchymal transition in obliterative bronchiolitis.

Bronchiolitis obliterans syndrome is characterized by fibrotic obliteration of small airways which severely impairs graft function and survival after lung transplantation. Bronchial epithelial cells from the transplanted lung can undergo epithelial to mesenchymal transition and this can be accentuated by activated macrophages. Macrophages demonstrate significant plasticity and change phenotype in response to their microenvironment. In this study we aimed to identify secretory products from macrophages that might be therapeutic targets for limiting the inflammatory accentuation of epithelial to mesenchymal transition in bronchiolitis obliterans syndrome. TNFα, IL-1ß and IL-8 are elevated in bronchoalveolar lavage from lung transplant patients prior to diagnosis of bronchiolitis obliterans syndrome. Classically activated macrophages secrete more TNFα and IL-1ß than alternatively activated macrophages and dramatically accentuate TGF-ß1-driven epithelial to mesenchymal transition in bronchial epithelial cells isolated from lung transplant patients. Blocking TNFα, but not IL-1ß, inhibits the accentuation of epithelial to mesenchymal transition. In a pilot unblinded therapeutic intervention in five patients with progressive bronchiolitis obliterans syndrome, anti-TNFα treatment improved forced expiratory volume in 1 second and 6-min walk distances in four patients. Our data identify TNFα as a potential new therapeutic target in bronchiolitis obliterans syndrome deserving of a randomized placebo controlled clinical trial.

Prevalence of NDM-1 carbapenemase in patients with diarrhoea in Pakistan and evaluation of two chromogenic culture media.

AIMS: To evaluate two chromogenic media, Brilliance CRE and chromID CARBA, with stool samples referred to the Public Health Laboratories Division of the National Institute of Health in Islamabad, and assess the prevalence of carbapenemase-producing Enterobacteriaceae (CPE) in this population. METHODS AND RESULTS: One hundred and fifty-two stool samples from patients with diarrhoea were referred to the Microbiology Department and were investigated for the presence of CPE using two chromogenic culture media, Brilliance CRE and chromID CARBA. Thirteen patients (8·6%) were found to be colonized with CPE and all produced NDM-1 carbapenemase. Twelve of these patients (92%) were found to be colonized by culture on chromID CARBA compared with seven (54%) using Brilliance CRE. CONCLUSIONS: If only coloured colonies were considered as presumptive CPE, the sensitivity, specificity and positive predictive value were 54, 23 and 6% for Brilliance CRE and 85, 85 and 36% for chromID CARBA, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: We conclude that Enterobacteriaceae that produce NDM-1 carbapenemase can be found in patients from all major provinces of Pakistan and that chromID CARBA was the most effective of the two chromogenic media in this setting.

The potential impact of washing machines on laundry malodour generation.

A multidisciplinary approach has been adopted to investigate and identify the source of malodour in washing machines and the potential for cross-contamination of laundry. Four washing machines were olfactively graded, and the number of colony-forming units (CFUs) bacteria was determined in four specific locations. Then, samples of terry-towel and fleece were washed, without the use of detergent, in the machines, and the occurrence of malodour over a 52-h period was assessed. Analysis of the scrapings from the four locations in the two malodorous machines identified a plethora of volatile organic compounds (VOCs) by either olfactory detection or mass spectral identification post-gas chromatographic separation. In addition, microbiological analysis from the swabs from the four locations within all four washing machines was carried out. Quantitative analysis of VOCs from 66 microbiological isolates from either the washing machines or fabrics was carried out. In total, 10 VOCs were identified: dimethyl disulfide, 3-methyl-1-butanol, 2,4-dithiapentane, dimethyl trisulfide, 2-tridecanone, indole, 2-phenylethanol, isovaleric acid, isobutyric acid and 1-undecene.

The preterm gut microbiota: changes associated with necrotizing enterocolitis and infection.

AIM: To describe gut colonization in preterm infants using standard culture and 16S gene rRNA profiling, exploring differences in healthy infants and those who developed NEC/late onset sepsis (LOS). METHODS: Ninety-nine stools from 38 infants of median 27-week gestation were cultured; 44 stools from 27 infants had their microbial profiles determined by 16S. Ordination analyses explored effects of patient variables on gut communities. RESULTS: Standard microbiological culture identified a mean of two organisms (range 0-7), DGGE 12 (range 3-18) per patient. Enterococcus faecalis and coagulase negative staphylococci (CONS) were most common by culture (40% and 39% of specimens). Meconium was not sterile. No fungi were cultured. Bacterial community structures in infants with NEC and LOS differed from healthy infants. Infants who developed NEC carried more CONS (45% vs 30%) and less Enterococcus faecalis (31% vs 57%). 16S identified Enterobacter and Staphylococcus presence associated with NEC/LOS, respectively. CONCLUSIONS: Important differences were found in the gut microbiota of preterm infants who develop NEC/LOS. The relationship of these changes to current practices in neonatal intensive care requires further exploration.

Volatile biomarkers of Pseudomonas aeruginosa in cystic fibrosis and noncystic fibrosis bronchiectasis.

AIMS: The purpose of this study was to determine whether volatile organic compounds specific to Pseudomonas aeruginosa could be detected in clinical sputum specimens. METHODS AND RESULTS: Patients were recruited from specialist bronchiectasis and cystic fibrosis clinics. The gold standard for diagnosing Ps. aeruginosa infection was a positive sputum culture. About 72 sputum headspace samples taken from patients at risk of or known to have prior Ps. aeruginosa infection were analysed by solid phase micro-extraction mass spectrometry. 2-nonanone was a marker in Ps. aeruginosa in sputum headspace gas with sensitivity of 72% and specificity of 88%. A combination of volatile compounds, a sputum library of 17 compounds with 2-nonanone, increased sensitivity in the detection of Ps. aeruginosa to 91% with specificity of 88%. CONCLUSIONS: In contrast to the 48-hour turnaround for classical microbiological culture, these results were available within 1-2 h. These data demonstrate the potential for rapid and accurate diagnosis of Ps. aeruginosa infection from sputum samples. SIGNIFICANCE AND IMPACT OF THE STUDY: 2-Nonanone is a compound requiring further study in the exhaled breath as it may improve diagnostic of Ps. aeruginosa infection when combined with other reported volatile markers.

Assessment of sample handling practices on microbial activity in sputum samples from patients with cystic fibrosis.

AIM: The aim of this study was to quantitatively and qualitatively assess the effect of sample storage on the metabolically active microbial community found in sputum samples from patients with cystic fibrosis (CF). METHODS: Sputum samples were collected and split in two equal aliquots one of which was immersed in RNAlater and refrigerated immediately, the second stored at room temperature for 24 h and RNAlater was subsequently added. mRNA was extracted, and RT-PCR-DGGE and qPCR analysis of the bacterial and fungal communities was carried out. RESULTS: Significant differences in the bacterial communities between the two protocols were observed but there were no significant difference seen in the fungal community analyses. Analysis by qPCR demonstrated that room temperature storage gave statistically significant increases in eubacteria and Pseudomonas spp. and a statistically significant decrease in those of Haemophilus influenzae. CONCLUSIONS: The analysis of metabolically active microbial communities from CF sputum using molecular techniques indicated that samples should be stored at 4 degrees C upon addition of RNAlater to obtain an accurate depiction of the CF lung microbiota. Also, storing respiratory samples at room temperature may cause an over representation of Pseudomonas aeruginosa and mask the presence of other clinically significant organisms.

Impact of rapid microbial identification on clinical outcomes in bloodstream infection: the RAPIDO randomized trial.

OBJECTIVES: Bloodstream infection has a high mortality rate. It is not clear whether laboratory-based rapid identification of the organisms involved would improve outcome. METHODS: The RAPIDO trial was an open parallel-group multicentre randomized controlled trial. We tested all positive blood cultures from hospitalized adults by conventional methods of microbial identification and those from patients randomized (1:1) to rapid diagnosis in addition to matrix-assisted desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) performed directly on positive blood cultures. The only primary outcome was 28-day mortality. Clinical advice on patient management was provided to members of both groups by infection specialists. RESULTS: First positive blood culture samples from 8628 patients were randomized, 4312 into rapid diagnosis and 4136 into conventional diagnosis. After prespecified postrandomization exclusions, 2740 in the rapid diagnosis arm and 2810 in the conventional arm were included in the mortality analysis. There was no significant difference in 28-day survival (81.5% 2233/2740 rapid vs. 82.3% 2313/2810 conventional; hazard ratio 1.05, 95% confidence interval 0.93-1.19, p 0.42). Microbial identification was quicker in the rapid diagnosis group (median (interquartile range) 38.5 (26.7-50.3) hours after blood sampling vs. 50.3 (47.1-72.9) hours after blood sampling, p < 0.01), but times to effective antimicrobial therapy were no shorter (respectively median (interquartile range) 24 (2-78) hours vs. 13 (2-69) hours). There were no significant differences in 7-day mortality or total antibiotic consumption; times to resolution of fever, discharge from hospital or de-escalation of broad-spectrum therapy or 28-day Clostridioides difficile incidence. CONCLUSIONS: Rapid identification of bloodstream pathogens by MALDI-TOF MS in this trial did not reduce patient mortality despite delivering laboratory data to clinicians sooner.

Evaluation of chromogenic media for the isolation of vancomycin-resistant enterococci from stool samples.

AIMS: This study sought to evaluate the performance of two chromogenic media designed for the isolation of vancomycin-resistant enterococci (VRE) and compare them with a traditional bile-esculin medium for the isolation of VRE from stool samples. METHODS AND RESULTS: A total of 285 stool samples were inoculated onto Chromogenic VRE Agar (AES VRE agar; AES Chemunex), chromID VRE (bioMérieux) and VRE Agar (Oxoid) both directly and also following broth enrichment. In total 18 strains of vancomycin-resistant Enterococcus faecium were recovered, including 17 harbouring the vanA gene and one with vanB. On direct culture, the sensitivity of the three media was 66.7%, 77.8% and 44.4% and after broth enrichment 66.7%, 83.3% and 77.8% using AES VRE Agar, chromID VRE and Oxoid VRE Agar respectively. CONCLUSIONS: All three media are useful tools for the isolation of VRE from stool samples. AES VRE Agar and bioMérieux chromID VRE are easier to use than Oxoid VRE Agar due to diffusion of black coloration from the latter. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to evaluate the performance of AES VRE Agar and the first to compare two media containing synthetic chromogens for the isolation of VRE.

Visual dysfunction in patients receiving vigabatrin: clinical and electrophysiologic findings.

BACKGROUND: Vigabatrin is an antiepileptic drug that, although relatively well tolerated, is associated with visual field constriction and other visual disturbances of unclear origin. METHODS: We performed a complete neuroophthalmologic examination and electrophysiologic studies on 39 patients receiving vigabatrin and on 11 control patients. RESULTS: Nearly 50% of patients receiving vigabatrin had constricted visual fields compared with control patients. Some of the vigabatrin patients also had reduced visual acuity and abnormal color vision. In addition, most vigabatrin patients had abnormal electroretinographic results, the severity of which correlated strongly with the degree of visual field constriction. CONCLUSIONS: Vigabatrin can cause electrophysiologic evidence of retinal dysfunction and clinically detectable disturbances of visual sensory function.

The effects of adherent cells on measurement of the hyper-responsiveness of rheumatoid B lymphocytes to Epstein-Barr virus.

Rheumatoid peripheral blood mononuclear cells show an increased responsiveness to superinfection with Epstein-Barr virus (EBV). We have investigated the role of adherent cells in this hyperresponsiveness using two different methods of adherent cell depletion. Depletion of adherent cells from both rheumatoid and normal mononuclear cells, using either dextran bead columns or plastic petri dishes, produced inconsistent changes in the response of autologous non-adherent cells to EBV. The addition of supernatants of cultured rheumatoid adherent cells also produced an inconsistent change in response although normal adherent cell supernatants increased the responsiveness of autologous non-adherent cells to EBV. The inconsistencies observed are discussed with respect to adherent cell subpopulations present in rheumatoid and normal peripheral blood mononuclear cell preparations when using recognized methods of adherent cell depletion.

Centrifugation of normal and rheumatoid arthritis blood on Ficoll-Hypaque and Ficoll-Nycodenz solutions.

Attempts to use the rapid single-step Ficoll-Hypaque centrifugation procedure for the purification of mononuclear and polymorphonuclear leucocytes from the blood of normal individuals and rheumatoid arthritis patients have sometimes been unsuccessful, largely because the erythrocytes would not sediment through the centrifugation medium. Re-evaluation of the factors (e.g. Ficoll concentration, temperature, and ratio of the diatrizoate salts) which affect these separations showed that under our conditions it was advantageous to use a medium with a lower viscosity (Ficoll concentration) and/or a higher osmotic strength (increased sodium diatrizoate: meglumine diatrizoate) than had been recommended previously (Ferrante and Thong, 1978; 1980; Ferrante et al., 1982). Higher osmotic strength media must be used for separating the components of blood from rheumatoid arthritis patients than from normal individuals because rheumatoid arthritis erythrocytes have a lower buoyant density than normal erythrocytes.

Rapid conventional scheme for biochemical identification of antibiotic resistant enterobacteriaceae isolates from urine.

Six conventional biochemical tests were combined to produce an identification scheme. These tests included decarboxylation of lysine and ornithine, fermentation of glucose and cellobiose, indole production and urease production. Three hundred antibiotic resistant coliforms from urine specimens were tested by this scheme and also by the API 20E for comparison. Two hundred and seventy nine (93%) of organisms were correctly identified using the six tests, 17 (5.7%) were referred for further study, and four (1.3%) were misidentified. It is concluded that this combination of tests provides an inexpensive, accurate, and rapid tool for identification.

Evaluation of a new chromogenic medium, Uriselect 4, for the isolation and identification of urinary tract pathogens.

AIMS: To compare the performance of a new chromogenic medium, Uriselect 4, with cystine lactose electrolyte deficient (CLED) agar and an established chromogenic agar, CPS ID 2 medium, for detection of urinary tract pathogens. METHODS: Using a semiquantitative culture method, 777 samples were inoculated on to the three test media in duplicate. All bacterial strains that yielded a potentially significant growth were observed for colony colour and identified using standard methods. RESULTS: Of the 777 samples tested, 589 urine samples yielded potentially significant growth of at least one strain. A total of 811 strains were isolated on at least one of the three media. A total of 168 urine samples yielded a mixture of at least two strains. Uriselect 4 medium showed the best sensitivity of the three media and only failed to recover 14 strains (1.7%). CPS ID 2 medium failed to recover 22 strains (2.7%). CLED medium showed the worst recovery and failed to recover 74 strains (9.1%). Both chromogenic media allowed for identification of Escherichia coli with a high degree of specificity (98% for Uriselect 4, 99.7% for CPS ID 2). Inclusion of a spot indole test increased the specificity of both chromogenic media to 100% for E coli. CONCLUSIONS: Uriselect 4 and CPS ID 2 were superior to CLED medium for the isolation of urinary tract pathogens mainly because of their ability to discriminate mixed cultures. Both chromogenic media were also useful for the preliminary identification of the most common urinary tract pathogens.

Routine use of a one minute trehalase and maltase test for the identification of Candida glabrata in four laboratories.

AIMS: To evaluate the rapid identification of Candida glabrata using a one minute trehalase and maltase test in four clinical laboratories. METHOD: The test was evaluated with 944 freshly isolated yeasts comprising 572 C glabrata and 372 non-C glabrata strains. These strains were isolated on one of three differential media-Candida ID, CHROMagar Candida, or Albicans ID2 medium-and all strains were fully identified using standard methods. RESULTS: The trehalase and maltase test allowed the overall identification of 550 of 572 C glabrata strains (sensitivity, 96.2%) and only 11 of 372 isolates of other yeast species yielded a false positive result (specificity, 96.8 %). Sensitivity and specificity were consistent from one laboratory to another. Using Candida ID medium, the rapid trehalase and maltase test showed a sensitivity of 95% and specificity of 96.2%. Using CHROMagar Candida, sensitivity and specificity were 95.6% and 98.1%, respectively. Using Albicans ID2 medium (tested by two laboratories), the sensitivity was 100% and 98.5% and specificity was 98.1% and 98.2%. In 60% of cases, the test could be performed directly from the primary isolation medium, thus reducing the time for identification. CONCLUSION: The rapid trehalase and maltase test was highly reliable for the presumptive identification of C glabrata on primary isolation using three different chromogenic media. Direct recognition of C albicans by means of their characteristic colour on chromogenic media coupled with one minute trehalase maltase testing performed only on suspect colonies of C glabrata allowed for rapid presumptive identification of the two yeast species most commonly encountered in clinical samples.

Balloon catheter dilation for treatment of adults with partial nasolacrimal duct obstruction: a preliminary report.

PURPOSE: The purpose of this study is to determine the efficacy and morbidity of balloon catheter dilation for treatment of partial acquired nasolacrimal duct obstruction in adults with epiphora. METHODS: We performed balloon dacryocystoplasty prospectively on a series of 15 partial nasolacrimal duct obstructions in 13 adults with epiphora. Partial obstruction was diagnosed by a negative Jones-1 test and canalicular irrigation revealing simultaneous reflux through the opposing punctum and drainage into the nose. Balloon dacryocystoplasty was performed under local anesthesia using an antegrade insertion technique. Silicone intubation of the nasolacrimal system was performed immediately after balloon catheter dilation, and the tubes were removed 2 months postoperatively. RESULTS: Success was measured objectively and subjectively at follow-up examinations 2 months and 6 months after the procedure. At 2 months, 11 (73%) of 15 obstructions were open on irrigation, with subjective success (Munk, grade 0 or grade 1) reported in 13 (87%) of 15 obstructions. At 6 months, 11 (73%) of 15 obstructions were open on irrigation, with subjective success (Munk, grade 0 or grade 1) reported in 9 (60%) of 15 obstructions. CONCLUSIONS: Balloon dacryocystoplasty may be a satisfactory primary treatment for adults with acquired partial nasolacrimal duct obstruction who exhibit no clinical signs of chronic infection. Additional long-term observations following balloon dacryocystoplasty are required.