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.

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.

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.

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.

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.

Comparison of two selective media for the recovery of Clostridium difficile from environmental surfaces.

Two culture media were compared for their ability to isolate Clostridium difficile from environmental sites within a UK hospital. The media were cefoxitin-cycloserine-egg yolk agar plus lysozyme (CCEY/L) and chromID C. difficile. A wide range of environmental surfaces was sampled using sterile sponges (Polywipes) and these were inoculated on to both media. C. difficile was recovered from 105 of 496 sites (21%) using a combination of both media. The sensitivity of chromID C. difficile was 87.6% compared with 26.6% for CCEY/L (P < 0.0001). chromID C. difficile performed significantly better than CCEY/L for the recovery of C. difficile from the environment.

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.

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.

Family history of depression is associated with younger age of onset in patients with recurrent depression.

BACKGROUND: Genetic epidemiology data suggest that younger age of onset is associated with family history (FH) of depression. The present study tested whether the presence of FH for depression or anxiety in first-degree relatives determines younger age of onset for depression. METHOD: A sample of 1022 cases with recurrent major depressive disorder (MDD) was recruited at the Max Planck Institute and at two affiliated hospitals. Patients were assessed using the Schedules for Clinical Assessment in Neuropsychiatry and questionnaires including demographics, medical history, questions on the use of alcohol and tobacco, personality traits and life events. Survival analysis and the Cox proportional hazard model were used to determine whether FH of depression signals earlier age of onset of depression. RESULTS: Patients who reported positive FH had a significantly earlier age of onset than patients who did not report FH of depression (log-rank=48, df=1, p<0.0001). The magnitude of association of FH varies by age of onset, with the largest estimate for MDD onset before age 20 years (hazard ratio=2.2, p=0.0009), whereas FH is not associated with MDD for onset after age 50 years (hazard ratio=0.89, p=0.5). The presence of feelings of guilt, anxiety symptoms and functional impairment due to depressive symptoms appear to characterize individuals with positive FH of depression. CONCLUSIONS: FH of depression contributes to the onset of depression at a younger age and may affect the clinical features of the illness.

The application of chromogenic media in clinical microbiology.

Since 1990, a wide range of chromogenic culture media has been made commercially available providing useful tools for diagnostic clinical microbiology. By the inclusion of chromogenic enzyme substrates targeting microbial enzymes, such media are able to target pathogens with high specificity. Examples of target pathogens include Staphylococcus aureus, Streptococcus agalactiae, Salmonella spp. and Candida spp. The inclusion of multiple chromogenic substrates into culture media facilitates the differentiation of polymicrobial cultures, thus allowing for the development of improved media for diagnosis of urinary tract infections and media for the enhanced discrimination of yeasts. The purpose of this review is to provide some insight into how such media work and appraise their utility in routine clinical diagnostics, in comparison with conventional media.

Evaluation of novel chromogenic substrates for the detection of bacterial beta-glucosidase.

AIMS: To evaluate three previously unreported substrates for the detection of beta-glucosidase activity in clinically relevant bacteria and to compare their performance with a range of known substrates in an agar medium. METHODS AND RESULTS: The performance of 11 chromogenic beta-glucosidase substrates was compared using 109 Enterobacteriaceae strains, 40 enterococci and 20 strains of Listeria spp. Three previously unreported beta-glucosides were tested including derivatives of alizarin, 3',4'-dihydroxyflavone and 3-hydroxyflavone. These were compared with esculin and beta-glucoside derivatives of 3,4-cyclohexenoesculetin, 8-hydroxyquinoline and five indoxylics. All substrates yielded coloured precipitates upon hydrolysis in agar. Alizarin-beta-D-glucoside was the most sensitive substrate tested and detected beta-glucosidase activity in 72% of Enterobacteriaceae strains and all enterococci and Listeria spp. The two flavone derivatives showed poor sensitivity with Gram-negative bacteria but excellent sensitivity with enterococci and Listeria spp. CONCLUSIONS: Alizarin-beta-d-glucoside is a highly sensitive substrate for detection of bacterial beta-glucosidase and compares favourably with existing substrates. beta-glucosides of 3',4'-dihydroxyflavone and 3-hydroxyflavone are effective substrates for the detection of beta-glucosidase in enterococci and Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented allow for informed decisions to be made regarding the optimal choice of beta-glucosidase substrate for detection of pathogenic and/or indicator bacteria.

Evaluation of a new chromogenic agar medium for isolation and identification of Group B streptococci.

AIMS: To evaluate a new chromogenic agar as a screening medium for the isolation of Group B streptococci from high vaginal swabs from pregnant women. METHODS AND RESULTS: The medium was evaluated with 195 high vaginal swabs referred for antenatal screening and compared with blood agar and Granada medium. The new chromogenic medium showed 100% sensitivity for the detection of Group B streptococci, and also showed a positive predictive value of 100%. Granada medium also showed excellent sensitivity and specificity and both media were superior to blood agar. CONCLUSIONS: The new chromogenic medium showed excellent performance for the detection of Group B streptococci from clinical samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first chromogenic medium described for the detection of Group B streptococci. The medium offers an effective and convenient alternative to conventional media, currently used in clinical laboratories.

Evaluation of novel fluorogenic substrates for the detection of glycosidases in Escherichia coli and enterococci.

AIMS: Enzyme substrates based on 4-methylumbelliferone are widely used for the detection of Escherichia coli and enterococci in water, by detection of beta-glucuronidase and beta-glucosidase activity respectively. This study aimed to synthesize and evaluate novel umbelliferone-based substrates with improved sensitivity for these two enzymes. METHODS AND RESULTS: A novel beta-glucuronide derivative based on 6-chloro-4-methylumbelliferone (CMUG) was synthesized and compared with 4-methylumbelliferyl-beta-D-glucuronide (MUG) using 42 strains of E. coli in a modified membrane lauryl sulfate broth. Over 7 h of incubation, the fluorescence generated from the hydrolysis of CMUG by E. coli was over twice that from MUG, and all of the 38 glucuronidase-positive strains generated a higher fluorescence with CMUG compared with MUG. Neither substrate caused inhibition of bacterial growth in any of the tested strains. Four beta-glucosidase substrates were also synthesized and evaluated in comparison with 4-methylumbelliferyl-beta-D-glucoside (MU-GLU) using 42 strains of enterococci in glucose azide broth. The four substrates comprised beta-glucoside derivatives of umbelliferone-3-carboxylic acid and its methyl, ethyl and benzyl esters. Glucosides of the methyl, ethyl and benzyl esters of umbelliferone-3-carboxylic acid, were found to be superior to MU-GLU for the detection of enterococci, especially after 18 h of incubation, while umbelliferone-3-carboxylic acid-beta-D-glucoside was inferior. However, the variability in detectable beta-glucosidase activity among the different strains of enterococci in short-term assays using the three carboxylate esters (7 h incubation) may compromise their use for rapid detection and enumeration of these faecal indicator bacteria. CONCLUSIONS: The beta-glucuronidase substrate CMUG appears to be a more promising detection system than the various beta-glucosidase substrates tested. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel substrate CMUG showed enhanced sensitivity for the detection of beta-glucuronidase-producing bacteria such as E. coli, with a clear potential for application in rapid assays for the detection of this indicator organism in natural water and other environmental samples.

Guidelines for the prevention of endocarditis: report of the Working Party of the British Society for Antimicrobial Chemotherapy.

These guidelines have been produced following a literature review of the requirement for prophylaxis to prevent bacterial endocarditis following dental and surgical interventions. Recommendations are made based on the quality of available evidence and the consequent risk of morbidity and mortality for "at risk" patients.