Evaluation of whole genome sequencing and software tools for drug susceptibility testing of Mycobacterium tuberculosis.

OBJECTIVES: Culture-based assays are currently the reference standard for drug susceptibility testing for Mycobacterium tuberculosis. They provide good sensitivity and specificity but are time consuming. The objective of this study was to evaluate whether whole genome sequencing (WGS), combined with software tools for data analysis, can replace routine culture-based assays for drug susceptibility testing of M. tuberculosis. METHODS: M. tuberculosis cultures sent to the Finnish mycobacterial reference laboratory in 2014 (n = 211) were phenotypically tested by Mycobacteria Growth Indicator Tube (MGIT) for first-line drug susceptibilities. WGS was performed for all isolates using the Illumina MiSeq system, and data were analysed using five software tools (PhyResSE, Mykrobe Predictor, TB Profiler, TGS-TB and KvarQ). Diagnostic time and reagent costs were estimated for both methods. RESULTS: The sensitivity of the five software tools to predict any resistance among strains was almost identical, ranging from 74% to 80%, and specificity was more than 95% for all software tools except for TGS-TB. The sensitivity and specificity to predict resistance to individual drugs varied considerably among the software tools. Reagent costs for MGIT and WGS were €26 and €143 per isolate respectively. Turnaround time for MGIT was 19 days (range 10-50 days) for first-line drugs, and turnaround time for WGS was estimated to be 5 days (range 3-7 days). CONCLUSIONS: WGS could be used as a prescreening assay for drug susceptibility testing with confirmation of resistant strains by MGIT. The functionality and ease of use of the software tools need to be improved.

Clostridium difficile infections in Finland, 2008-2015: trends, diagnostics and ribotypes.

We evaluated Clostridium difficile (CD) diagnostics in Finnish clinical microbiology laboratories during 2006-2011, with an update in 2015, in relation to CD surveillance data of the National Infectious Disease Register (NIDR) and ribotyping data from the national reference laboratory during the years 2008-2015. In 2011, diagnostic activity varied regionally more than three-fold and the positivity rate ranged between 7 and 21%. Nucleic acid amplification testing (NAAT) was implemented in the regions with high activity and NAAT users tested 30% more patients and found 15% more cases per population than those not using it. Culture was performed in 79% of laboratories, primary toxin testing by enzyme immunoassay (EIA) in 83% and by NAAT in 17%. In 2014, 12/19 laboratories used NAAT as the primary detection method and four as the secondary method, and ten cultured. Increasing usage of NAAT was not systematically related to various trends detected regionally in annual CD rates. Polymerase chain reaction (PCR) ribotyping of 1771 CD isolates (4.1% of CD cases) identified 146 distinct profiles, of which 37% were binary toxin positive. The most common ribotype was 027, but its proportion decreased, while 078 slightly increased. Transition from culture to NAAT in CD infection (CDI) diagnostics did not cause a significant increase in the observed CDI incidence. Major differences between diagnostic activity, methods and strategies in different regions have persisted over the years, which should be considered when comparing the regional epidemiology of CDI.

Reduction in Clostridium difficile infections in Finland, 2008-2010.

BACKGROUND: In January 2008, laboratory-based surveillance of Clostridium difficile was initiated as a part of the Finnish National Infectious Disease Register (NIDR) and enhanced surveillance of hospitalized patients with C. difficile-associated infections (CDI) by the Finnish Hospital Infection Programme (SIRO). AIM: To present data from the first three years. METHODS: All laboratories reported C. difficile findings positive for toxin production from stools to NIDR. Surveillance of hospitalized patients with CDI was conducted using the interim case definitions of the European Centre for Disease Prevention and Control for CDI, origin and severe case of CDI. In all, 16 acute care hospitals from 10 of the 21 healthcare districts (HDs) participated in SIRO during 2008-2010. Clinical microbiology laboratories were asked to send isolates from severe cases and persistent outbreaks to the national reference laboratory for genotyping. FINDINGS: The annual incidence rate of CDIs decreased by 24%, from 119 per 100,000 population in 2008 to 90 per 100,000 in 2010. The decrease occurred in 13/21 (62%) HDs (range of decrease by HD: 2-51%). The nosocomial rate decreased 26%, from 0.31 to 0.23 per 1000 patient-days, and occurred in about half of the hospitals that participated in SIRO. During 2008-2010, 17 HDs sent C. difficile specimens for typing. Ribotype 027 was found in eight HDs, all showing values above the mean or increasing population-based incidence rates of CDIs. CONCLUSIONS: Population-based surveillance of CDIs and enhanced surveillance of nosocomial cases showed reduction in CDIs, but success in controlling the disease varied between regions.

Factors associated with ileal mucosal morphology and inflammation in patients with ileal pouch-anal anastomosis for ulcerative colitis.

PURPOSE: Pouchitis has been associated with abnormal bacterial flora responding to antibiotics. Dietary factors may play a role in modifying the qualitative and quantitative components of the microflora. We evaluated interactions between nutritional factors, fecal and mucosal bacterial flora, and mucosal morphology in patients with a history of pouchitis compared with patients with optimal outcome at least five years after ileal pouch-anal anastomosis for ulcerative colitis. METHODS: Thirty-two patients were enrolled in the study: 11 (7 males; mean age, 49.8 years) with optimal outcome and 21 (11 males; mean age, 47.3 years) with pouchitis history. A seven-day food diary was recorded, endoscopy performed, and biopsies taken from the pouch for histology, mucin staining, and bacterial culture. Fresh fecal samples were quantitatively cultured, and fecal bile acids analyzed by gas-liquid chromatography. RESULTS: No differences existed in mean nutrient intake, composition of fecal bile acids, or microbial tissue biopsy cultures between the groups with and without pouchitis. Those with optimal outcome tended to have more benign disease course of ulcerative colitis than patients with pouchitis. In those patients, fecal concentrations (log10 colony-forming unit/g) of anaerobes and aerobes were significantly higher (P = 0.007). Degree of villous atrophy and colonic metaplasia were both associated with fecal anaerobic flora. Low intake of lactose was associated with sulfomucin predominance. A negative correlation existed between fecal aerobes and dietary lactose consumption. CONCLUSIONS: A higher total load of fecal anaerobic bacterial flora is strongly associated with degree of colonic metaplasia, villous atrophy, and inflammation activity after surgery for ulcerative colitis. An association existed between dietary lactose, fecal bacteria, and pouch morphology. Lactose may have prebiotic properties.

Effect of Lactobacillus rhamnosus GG on ileal pouch inflammation and microbial flora.

BACKGROUND: Preliminary trials of probiotics in preventing recurrent chronic pouchitis have been encouraging. AIM: To investigate the efficacy of Lactobacillus GG supplementation as primary therapy for ileal pouch inflammation, and its effect on the microbial flora. METHODS: Twenty patients, with a previous history of pouchitis and endoscopic inflammation, were recruited for a prospective, randomized, double-blind, placebo-controlled trial of Lactobacillus GG supplementation (10 LGG, 10 placebo) in two gelatine capsules [(0.5-1) x 10(10) colony-forming units/capsule] b.d. for 3 months. Quantitative bacterial culture of fresh faecal samples and biopsies taken from the pouch and afferent limb was performed before and after supplementation. RESULTS: Lactobacillus GG supplementation changed the pouch intestinal flora by increasing the ratio of total faecal lactobacilli to total faecal anaerobes (P = 0.03) and enhancing the frequency of lactobacilli-positive cultures in the pouch and afferent limb mucosal biopsy samples. However, only 40% of patients were colonized with Lactobacillus GG. No differences were observed between the groups with regard to the mean pouchitis disease activity index or the total anaerobes or aerobes of faecal or tissue biopsy samples. CONCLUSIONS: A single-strain probiotic bacterium supplement of Lactobacillus GG changed the pouch intestinal bacterial flora, but was ineffective as primary therapy for a clinical or endoscopic response. More clinical trials are needed to evaluate the right placement and dosage of probiotics within a treatment regimen for pouchitis.

Pan-European study on culture-proven Legionnaires' disease: distribution of Legionella pneumophila serogroups and monoclonal subgroups.

This pan-European study included unrelated strains of Legionella pneumophila obtained from 1335 cases of Legionnaires' disease. The isolates were serotyped into the serogroups 1 to 15 by monoclonal antibodies (MAb) and/or rabbit antisera. Additionally, MAb subgrouping was undertaken for isolates belonging to serogroups 1, 4, and 5. Monoclonal types of serogroup 1 were subdivided as having, or not having, the virulence-associated epitope recognized by the MAb 3/1 (Dresden Panel). This epitope is not present on strains belonging to any other serogroups. Taking all Legionella incidents together, MAb 3/1-positive cases were most frequent (66.8%); 11.7% of the isolates belonged to MAb 3/1-negative serogroup 1 subgroups and 21.5% to other serogroups, with serogroups 3 and 6 predominating. Among all serotypes discriminated in this study, monoclonal subtype Philadelphia was the most frequent. If categories of infection were considered, the proportion of MAb 3/1-negative strains differed significantly ( P<0.0005) between community-acquired cases (139/510; 27.3%) and travel-associated (42/295; 14.2%) or hospital-acquired infections (176/329; 53.5%). Moreover, taking distribution in different European areas into account, the proportion of MAb 3/1-negative strains was significantly higher in the Scandinavian region than in the Mediterranean countries or the UK for both community-acquired (48.7% vs. 18.6% or 12.0%; P<0.0005) and nosocomial cases (87.7% vs. 32.6% or 52.6%; P

Designation of the European Working Group on Legionella Infection (EWGLI) amplified fragment length polymorphism types of Legionella pneumophila serogroup 1 and results of intercentre proficiency testing Using a standard protocol.

The utility of amplified fragment length polymorphism (AFLP) analysis as a genotyping method for the epidemiological typing of Legionella pneumophila serogroup 1 has been previously demonstrated. This study (i). reports recommendations for the designation of the European Working Group on Legionella Infections (EWGLI) AFLP types, (ii). describes the EWGLI AFLP types identified for the 130 strains in the EWGLI culture collection, and (iii). reports the results of a newly introduced international programme of proficiency testing. Following preliminary analysis of 20 epidemiologically unrelated isolates, 16 major AFLP types were identified. A coded proficiency panel, comprising 12 additional isolates representing 9 of these 16 AFLP types, was sent to 17 centres in 14 European countries where it was analysed following a previously determined standard protocol. The identity of each coded strain (recorded as AFLP type 001-016 or untypeable) was determined by participants with reference to these 16 AFLP types, either visually or using gel analysis software where available, and reported to the coordinating centre. Nine of the 12 strains, including an epidemiologically related pair and two pairs of unrelated isolates of the same type, were correctly identified to the correct AFLP type by all or all but one of the participants. Seven laboratories correctly identified all 12 isolates, and a further seven laboratories correctly identified 11. Type identification scores ranged from 75% (1 centre), 83% (2 centres), and 92% (7 centres) to 100% (7 centres). The AFLP method as described is robust and rapid and allows the genotypic comparison of isolates of Legionella pneumophila between different testing centres without the need for exchange of the strains studied.

Ethanol-derived microbial production of carcinogenic acetaldehyde in achlorhydric atrophic gastritis.

BACKGROUND: Acetaldehyde is a local carcinogen in the digestive tract in humans. Atrophic gastritis leads to microbial colonization of the stomach, which could enhance microbial production of acetaldehyde from ethanol. The aim of the study was to study microbial ethanol metabolism and acetaldehyde production in the stomach of achlorhydric atrophic gastritis patients. METHODS: For the in vivo study, glucose or ethanol was infused via a nasogastric tube to the stomach of seven achlorhydric atrophic gastritis patients and five healthy controls. Gastric juice samples for ethanol and acetaldehyde determinations and microbial analysis were obtained at 30 and 60 min after the infusions. For the in vitro study, gastric juice samples from 14 atrophic gastritis patients and 16 controls were obtained during gastroscopy, whereafter the samples were incubated for 2 h with 1% ethanol at 37 degrees C and acetaldehyde was determined. RESULTS: Minor endogenous ethanol and acetaldehyde concentrations were detected after glucose infusion in the gastric juice of four atrophic gastritis patients. After ethanol infusion, the mean intragastric acetaldehyde level of the atrophic gastritis patients was 4.5-fold at 30 min and 6.5-fold at 60 min compared to controls. In vitro, the difference between the study groups was even higher, 7.6-fold. A vast selection of oral bacterial species and some Enterobacteriaceae and yeasts were presented in the gastric juice of atrophic gastritis patients. CONCLUSIONS: Microbial ethanol metabolism leads to high intragastric acetaldehyde levels after ethanol drinking in achlorhydric atrophic gastritis patients. This could be one of the factors responsible for enhanced gastric cancer risk among atrophic gastritis patients.