Evaluation of the STANDARD M10 MDR-TB and MTB/NTM assays for the detection of Mycobacterium tuberculosis, rifampicin and isoniazid resistance, and nontuberculous mycobacteria in a low-incidence setting.

Rapid detection is crucial for tuberculosis (TB) control. GeneXpert (Cepheid) is a widely used PCR system, known for its simplicity, random access, and point-of-care compatibility. SD BIOSENSOR recently introduced a similar system, STANDARD M10, including a Mycobacterium tuberculosis (MTB) and rifampicin (RIF) and isoniazid resistance (herein, MDR-TB) assay and an MTB/nontuberculous mycobacteria (NTM) assay. We evaluated these assays for the potential to replace the established Xpert MTB/RIF Ultra assay in a low-TB incidence setting. We analyzed 160 clinical respiratory samples (45 MTB-positive and 35 NTM-positive) and further 24 drug-resistant MTB, 30 mycobacterial species (2 MTB, 28 NTM), and 37 non-mycobacterial isolates. Compared with culture, clinical sensitivities and specificities for MTB detection were 88.9% (95% confidence interval [CI] = 76.1-95.6%) and 97.4% (CI = 92.3-99.4%) with Xpert Ultra, 88.9% (95% CI = 76.1-95.6%) and 98.3% (CI = 93.5-99.9%) with M10 MDR-TB, and 84.4% (CI = 70.9-94.4%) and 98.3% (CI = 93.5-99.9%) with M10 MTB/NTM, respectively. For NTM detection, M10 MTB/NTM showed sensitivity and specificity of 65.7% (CI = 49.1-79.2%) and 96.8% (CI = 91.8-99.0%). Compared with phenotypic drug susceptibility testing (DST), sensitivity and specificity for detecting RIF resistance were 100% (CI = 77.3-100%) and 95.6% (CI = 84.4-99.6%) with Xpert Ultra, and 100% (CI = 74.9-100%) and 95.5% (CI = 84.0-99.6%) with M10 MDR-TB. M10 MDR-TB showed 92.3% sensitivity (CI = 74.7-99.0%) and 100% specificity (CI = 87.3-100%) for detecting isoniazid resistance. All discrepancies in DST by PCR were concordant with whole-genome sequencing. While M10 MDR-TB demonstrated great potential as an alternative to Xpert Ultra, M10 MTB/NTM had limitations in NTM screening. Additionally, the M10 sputum pretreatment did not inactivate MTB efficiently, which should be considered in process risk assessment. IMPORTANCE: The molecular diagnostic STANDARD M10 system is highly analogous to the widely established GeneXpert system, which significantly increases the relevance of this evaluation study in the field of rapid detection of M. tuberculosis. To our knowledge, this is the first clinical evaluation describing the performance of the STANDARD M10 MDR-TB and MTB/NTM assays, including an extensive analytical specificity panel (inclusivity and exclusivity) for the detection of M. tuberculosis, drug resistance, and nontuberculous mycobacteria.

Transmission of drug-resistant Mycobacterium tuberculosis isolates between Finnish- and foreign-born cases, 2014-2021: A molecular epidemiological study.

BACKGROUND: Data on the molecular epidemiology and transmission of drug-resistant Mycobacterium tuberculosis (MTB) in low-incidence settings with immigration from high-incidence settings is limited. METHOD: We included 115 drug-resistant (DR) MTB isolates with whole-genome sequencing data isolated in Finland between 2014 and 2021. Potential transmission clusters were identified using a threshold of 12 single-nucleotide polymorphisms (SNPs). Highly related clusters were identified using a threshold of 5 SNPs. RESULT: Of the 115 DR MTB isolates, 31 (27.0%) isolates were from Finnish-born cases and 84 (73.0%) were from foreign-born cases. The proportion of multidrug-resistant (MDR) MTB isolates (30/84, 35.7%) from foreign-born cases was higher than that of MDR MTB isolates from Finnish-born cases (8/31, 25.8%). Lineage 2 (40/115, 34.8%) and lineage 4 (40/115, 34.8%) were the most prevalent lineages. A total of 25 (21.7%) isolates were classified into eight potential transmission clusters (≤12 SNPs). Furthermore, five highly related clusters (≤5 SNPs) were identified, including three DR MTB isolates from Finnish-born cases and 14 DR isolates from foreign-born cases. CONCLUSION: The risk of DR MTB transmission between Finnish- and foreign-born persons is not negligible. Further research on clustering analysis in drug-susceptible MTB is worth to inform tuberculosis management and control in low-incidence settings with increasing immigration.

Tap water as the source of a Legionnaires' disease outbreak spread to several residential buildings and one hospital, Finland, 2020 to 2021.

In Finland, all microbiology laboratories notify Legionella findings and physicians notify Legionnaires' disease (LD) cases to the National Infectious Disease Register. All cases are interviewed, and water samples obtained from potential places of exposure. Legionella isolates from humans and water are compared by whole genome sequencing (WGS). In March 2021, Legionella pneumophila serogroup 1 (Lp 1) pneumonia cases increased in one Finnish city (120,000 inhabitants) where single LD cases are detected annually. We identified 12 LD cases, nine living in different residential buildings and three nosocomial, linked by identical human and/or water isolates. Three of these cases were from January 2020, October 2020 and February 2021 and identified retrospectively. Eleven were diagnosed by urinary antigen test, 10 by PCR and five by culture; age ranged between 52 and 85 years, and 10 had underlying diseases. Nine of 12 homes of LD cases and 15 of 26 water samples from the hospital were positive for Lp 1, with concentrations up to 640,000 cfu/L. Water samples from regional storage tanks were negative. Positivity in homes and the hospital suggested inadequate maintenance measures. Enhanced surveillance combined with WGS was crucial in detecting this unusual LD outbreak related to domestic and hospital water systems.

External quality assessment by European mycobacterial laboratories: results of AFB microscopy and identification rounds.

We analyzed mycobacterial stain, culture and identification EQA data from altogether 134 laboratories in 15 mainly European countries over a 4-year period. The aim was to get an overview of the performance and methods and identify diagnostic challenges. The overall success rates for staining and identification were 94% and 91%. The false negative rate for staining was significantly higher for the medium positive than the strong positive slides (11% vs 4%). The false positive rate on negative slides was 10%, indicating contamination issues. The overall success of M. tuberculosis detection was high with error rates ranging from 0.7% to 1.2%. Pre- or postanalytical errors accounted for most of the unsuccessful responses. The detection of nontuberculous mycobacteria (NTM) was less consistent; accurate species identification depended on the assays used. Only 19% of participants performed species level identification for NTMs, 47% detected the presence NTMs while 21% focused on ruling out TB.

Emerging source of infection - Mycobacterium tuberculosis in rescue dogs: a case report.

Rescue dog activity is a heavily increasing form of dog charity. Imported homeless dogs represent a reservoir of zoonotic diseases putting owners, veterinarians and pathologists repeatedly at risk. The clinical signs of tuberculosis in a dog are non-specific and diagnosis is often delayed or dismissed. We present a case of 9 months of possible exposure at home and definite exposure at laparotomy and autopsy to intestinal tuberculosis in a family dog imported from Romania to Finland. Persistent gastrointestinal symptoms started 2 years after the import. Abdominal pain, diarrhoea and vomiting proceeded and led to spontaneous death. Mycobacterium tuberculosis was identified in the liver, lymph nodes and intestine at autopsy. Exposed persons were notified and follow-up was provided, and no further infections were identified within 12 months of follow-up. The heavily increasing import of companion animals presents unexpected public health risks, such as prolonged exposure to tuberculosis, of which the general public is not aware. The dramatic consequences and high costs of tuberculosis could be reduced through accessible information of the risks of imported animals to both the general public and veterinarians, in addition to availability of rapid diagnostics and proper personal protection.

Prospective Evaluation of the mariPOC Test for Detection of Clostridioides difficile Glutamate Dehydrogenase and Toxins A/B.

The objective of this study was to evaluate a novel automated random-access test, mariPOC CDI (ArcDia Ltd., Finland), for the detection of Clostridioides difficile glutamate dehydrogenase (GDH) and toxins A and B directly from fecal specimens. The mariPOC test was compared with both the GenomEra C. difficile PCR assay (Abacus Diagnostica Oy, Finland) and the TechLab C. diff Quik Chek Complete (Alere Inc.; now Abbot) membrane enzyme immunoassay (MEIA). Culture and the Xpert C. difficile assay (Cepheid Inc., USA) were used to resolve discrepant results. In total, 337 specimens were tested with the mariPOC CDI test and GenomEra PCR. Of these specimens, 157 were also tested with the TechLab MEIA. The sensitivity of the mariPOC test for GDH was slightly lower (95.2%) than that obtained with the TechLab assay (100.0%), but no toxin-positive cases were missed. The sensitivity of the mariPOC test for the detection of toxigenic C. difficile by analyzing toxin expression was better (81.6%) than that of the TechLab assay (71.1%). The analytical specificities for the mariPOC and the TechLab tests were 98.3% and 100.0% for GDH and 100.0% and 99.2% for toxin A/B, respectively. The analytical specificity of the GenomEra method was 100.0%. The mariPOC and TechLab GDH tests and GenomEra PCR had high negative predictive values of 99.3%, 98.3%, and 99.7%, respectively, in excluding infection with toxigenic C. difficile The mariPOC toxin A/B test and GenomEra PCR had an identical analytical positive predictive value of 100%, providing highly reliable information about toxin expression and the presence of toxin genes, respectively.

Lactobacillus reuteri to Treat Infant Colic: A Meta-analysis.

CONTEXT: Lactobacillus reuteri DSM17938 has shown promise in managing colic, but conflicting study results have prevented a consensus on whether it is truly effective. OBJECTIVE: Through an individual participant data meta-analysis, we sought to definitively determine if L reuteri DSM17938 effectively reduces crying and/or fussing time in infants with colic and whether effects vary by feeding type. DATA SOURCES: We searched online databases (PubMed, Medline, Embase, the Cumulative Index to Nursing and Allied Health Literature, the Database of Abstracts of Reviews of Effects, and Cochrane), e-abstracts, and clinical trial registries. STUDY SELECTION: These were double-blind randomized controlled trials (published by June 2017) of L reuteri DSM17398 versus a placebo, delivered orally to infants with colic, with outcomes of infant crying and/or fussing duration and treatment success at 21 days. DATA EXTRACTION: We collected individual participant raw data from included studies modeled simultaneously in multilevel generalized linear mixed-effects regression models. RESULTS: Four double-blind trials involving 345 infants with colic (174 probiotic and 171 placebo) were included. The probiotic group averaged less crying and/or fussing time than the placebo group at all time points (day 21 adjusted mean difference in change from baseline [minutes] -25.4 [95% confidence interval (CI): -47.3 to -3.5]). The probiotic group was almost twice as likely as the placebo group to experience treatment success at all time points (day 21 adjusted incidence ratio 1.7 [95% CI: 1.4 to 2.2]). Intervention effects were dramatic in breastfed infants (number needed to treat for day 21 success 2.6 [95% CI: 2.0 to 3.6]) but were insignificant in formula-fed infants. LIMITATIONS: There were insufficient data to make conclusions for formula-fed infants with colic. CONCLUSIONS: L reuteri DSM17938 is effective and can be recommended for breastfed infants with colic. Its role in formula-fed infants with colic needs further research.

The recognition and characterisation of Finnish Clostridium difficile isolates resembling PCR-ribotype 027.

PURPOSE: To characterise and compare twenty-eight Finnish Clostridium difficile RT027-like isolates, selected based on the presence of 18 bp deletion in the tcdC gene and toxin gene profile (A, B, binary), with eleven RT027 isolates from different Finnish geographical areas and time periods. METHODS: Twenty-eight C. difficile RT027-like isolates and 11 RT027 comparative strains were characterised by capillary-electrophoresis (CE) ribotyping, multi-locus variable tandem-repeats analysis (MLVA), multi-locus sequence typing (MLST), and sequencing of tcdC and gyrA gene fragments. Susceptibility to moxifloxacin was determined by E-test. RESULTS: Of 28 RT027-like isolates, seven RTs (016, 034, 075, 080, 153, 176 and 328), three WEBRIBO types (411, 475, AI-78) and three new profiles (F1-F3) were identified. MLVA revealed six clonal complexes (RTs 016, 027, 176 and F3). MLST showed eleven sequence types (1, 41, 47, 67, 95, 191,192, 223, 229, 264 and new ST). Twenty-two isolates (RTs 016, 080, 176, 328, F1, F2, F3 and WRTAI-78) carried Δ117 in the tcdC gene. Isolates of RTs 016, 027 and 176 were moxifloxacin resistant and harboured Thr82Ile in the GyrA. CONCLUSION: Our results show a high diversity within 28 Finnish RT027-like C. difficile isolates, with twelve CE-ribotyping profiles and eleven STs. MLVA revealed the regional spread of RTs 016, 027, 176 and F3. The presence of Δ117 in the tcdC gene in eight non-027 RTs highlights the importance of careful interpretation of the results from molecular systems targeting this site in the genome of C. difficile and the need of strain typing for epidemiological purposes.

Community- and Healthcare-Associated Clostridium difficile Infections, Finland, 2008-2013

We evaluated incidence, case-fatality rate, and trends of community-associated (CA) and healthcare-associated (HA) Clostridium difficile infections (CDIs) in Finland during 2008-2013. CDIs were identified in the National Infectious Disease Register, deaths in the National Population Information System, hospitalizations to classify infections as CA or HA in the National Hospital Discharge Register, and genotypes in a reference laboratory. A total of 32,991 CDIs were identified: 10,643 (32.3%) were CA (32.9 cases/100,000 population) and 22,348 (67.7%) HA (69.1/100,000). Overall annual incidence decreased from 118.7/100,000 in 2008 to 92.1/100,000 in 2013, which was caused by reduction in HA-CDI rates (average annual decrease 8.1%; p<0.001). The 30-day case-fatality rate was lower for CA-CDIs than for HA-CDIs (3.2% vs. 13.3%; p<0.001). PCR ribotypes 027 and 001 were more common in HA-CDIs than in CA-CDIs. Although the HA-CDI incidence rate decreased, which was probably caused by increased awareness and improved infection control, the CA-CDI rate increased.

Standardised surveillance of Clostridium difficile infection in European acute care hospitals: a pilot study, 2013.

Clostridium difficile infection (CDI) remains poorly controlled in many European countries, of which several have not yet implemented national CDI surveillance. In 2013, experts from the European CDI Surveillance Network project and from the European Centre for Disease Prevention and Control developed a protocol with three options of CDI surveillance for acute care hospitals: a 'minimal' option (aggregated hospital data), a 'light' option (including patient data for CDI cases) and an 'enhanced' option (including microbiological data on the first 10 CDI episodes per hospital). A total of 37 hospitals in 14 European countries tested these options for a three-month period (between 13 May and 1 November 2013). All 37 hospitals successfully completed the minimal surveillance option (for 1,152 patients). Clinical data were submitted for 94% (1,078/1,152) of the patients in the light option; information on CDI origin and outcome was complete for 94% (1,016/1,078) and 98% (294/300) of the patients in the light and enhanced options, respectively. The workload of the options was 1.1, 2.0 and 3.0 person-days per 10,000 hospital discharges, respectively. Enhanced surveillance was tested and was successful in 32 of the hospitals, showing that C. difficile PCR ribotype 027 was predominant (30% (79/267)). This study showed that standardised multicountry surveillance, with the option of integrating clinical and molecular data, is a feasible strategy for monitoring CDI in Europe.

Survey of diagnostic and typing capacity for Clostridium difficile infection in Europe, 2011 and 2014.

Suboptimal laboratory diagnostics for Clostridium difficile infection (CDI) impedes its surveillance and control across Europe. We evaluated changes in local laboratory CDI diagnostics and changes in national diagnostic and typing capacity for CDI during the European C. difficile Infection Surveillance Network (ECDIS-Net) project, through cross-sectional surveys in 33 European countries in 2011 and 2014. In 2011, 126 (61%) of a convenience sample of 206 laboratories in 31 countries completed a survey on local diagnostics. In 2014, 84 (67%) of these 126 laboratories in 26 countries completed a follow-up survey. Among laboratories that participated in both surveys, use of CDI diagnostics deemed 'optimal' or 'acceptable' increased from 19% to 46% and from 10% to 15%, respectively (p < 0.001). The survey of national capacity was completed by national coordinators of 31 and 32 countries in 2011 and 2014, respectively. Capacity for any C. difficile typing method increased from 22/31 countries in 2011 to 26/32 countries in 2014; for PCR ribotyping from 20/31 countries to 23/32 countries, and specifically for capillary PCR ribotyping from 7/31 countries to 16/32 countries. While our study indicates improved diagnostic capability and national capacity for capillary PCR ribotyping across European laboratories between 2011 and 2014, increased use of 'optimal' diagnostics should be promoted.

Differentiating virulent 027 and non-027 Clostridium difficile strains by molecular methods.

OBJECTIVE: Hypervirulent Clostridium difficile clade has been shown to include several lineages of ribotype 027 and also other ribotypes. We present data on additional non-027 strains, identified as presumptive 027 by two commercial molecular C. difficile assays. METHODS: The tested clinical isolates were selected from the national reference laboratory collection on the basis of toxin gene profile similarities with ribotype 027 and tested with XpertC. difficile/Epi and Amplidiag C. difficile+027 assay. RESULT: Xpert misclassified five ribotypes (016, 019, 080, 176 and variant of type 046) as presumptive 027 and Amplidiag two ribotypes (016, 176). The misclassified strains were rare, covering 1.6% of reference laboratory strain collection. CONCLUSION: Our findings confirm the concept that there are closely related outliers to hypervirulent 027 clones that can be misclassified as 027, and that these comprise numerous ribotypes, including previously reported four ribotypes (198, 176, 244, 019), and additional three (016, v046, 080) identified in the present study.

Regional differences in Clostridium difficile infections in relation to fluoroquinolone and proton pump inhibitor use, Finland, 2008-2011.

BACKGROUND: Several antimicrobial agents and proton pump inhibitors (PPIs) have been identified as risk factors for Clostridium difficile infections (CDIs). Nationwide laboratory-based surveillance of CDIs in Finland since 2008 has shown variation in regional CDI rates. We evaluated whether regional differences in CDI rates were associated with antibacterial and PPI use. METHODS: Data on mean annual incidence rates of CDIs during 2008-2011 in 21 healthcare districts (HDs) were obtained from the National Infectious Disease Register, consumption (median annual use) of antimicrobials and PPIs from the Finnish Medical Agency, availability of molecular diagnostics by a laboratory survey and data on ribotypes from the national reference laboratory. The association over the 4 years was measured by incidence rate ratio (IRR) and we performed both bivariate and multivariate analyses. RESULTS: During 2008-2011, PPI use increased 27% but fluoroquinolone use was stable. The level of fluoroquinolone use was strongly associated with the mean annual CDI incidence rate in different HDs over the 4-year period, but PPI use had less effect. The molecular diagnostics methodology and PCR ribotype 027 were not independently associated with CDI rate. The final multivariable model only included fluoroquinolone and PPI use; IRR for fluoroquinolones was 2.20 (95% confidence interval (CI), 1.32-3.67; p = 0.003). CONCLUSIONS: Fluoroquinolone use may play a role in regional differences in CDI rates. Although the use has not recently increased, regionally targeted antimicrobial stewardship campaigns promoting appropriate use of fluoroquinolones should still be encouraged since they may decrease the incidence of CDIs.

Underdiagnosis of Clostridium difficile across Europe: the European, multicentre, prospective, biannual, point-prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID).

BACKGROUND: Variations in testing for Clostridium difficile infection can hinder patients' care, increase the risk of transmission, and skew epidemiological data. We aimed to measure the underdiagnosis of C difficile infection across Europe. METHODS: We did a questionnaire-based study at 482 participating hospitals across 20 European countries. Hospitals were questioned about their methods and testing policy for C difficile infection during the periods September, 2011, to August, 2012, and September, 2012, to August, 2013. On one day in winter, 2012-13 (December, 2012, or January, 2013), and summer, 2013 (July or August), every hospital sent all diarrhoeal samples submitted to their microbiology laboratory to a national coordinating laboratory for standardised testing of C difficile infection. Our primary outcome measures were the rates of testing for and cases of C difficile infection per 10 000 patient bed-days. Results of local and national C difficile infection testing were compared with each other. If the result was positive at the national laboratory but negative at the local hospital, the result was classified as undiagnosed C difficile infection. We compared differences in proportions with the Mann-Whitney test, or McNemar's test if data were matched. FINDINGS: During the study period, participating hospitals reported a mean of 65·8 tests (country range 4·6-223·3) for C difficile infection per 10 000 patient-bed days and a mean of 7·0 cases (country range 0·7-28·7) of C difficile infection per 10 000 patient-bed days. Only two-fifths of hospitals reported using optimum methods for testing of C difficile infection (defined by European guidelines), although the number of participating hospitals using optimum methods increased during the study period, from 152 (32%) of 468 in 2011-12 to 205 (48%) of 428 in 2012-13. Across all 482 European hospitals on the two sampling days, 148 (23%) of 641 samples positive for C difficile infection (as determined by the national laboratory) were not diagnosed by participating hospitals because of an absence of clinical suspicion, equating to about 74 missed diagnoses per day. INTERPRETATION: A wide variety of testing strategies for C difficile infection are used across Europe. Absence of clinical suspicion and suboptimum laboratory diagnostic methods mean that an estimated 40 000 inpatients with C difficile infection are potentially undiagnosed every year in 482 European hospitals. FUNDING: Astellas Pharmaceuticals Europe.

Lactobacillus reuteri DSM 17938 for managing infant colic: protocol for an individual participant data meta-analysis.

INTRODUCTION: Infant colic, or excessive crying of unknown cause in infants less than 3 months old, is common and burdensome. Its aetiology is undetermined, and consensus on its management is still lacking. Recent studies suggest a possible link between infant colic and gut microbiota, indicating probiotics to be a promising treatment. However, only a few strains have been tested, and results from randomised controlled trials are conflicting. It is important to clarify whether probiotics are effective for treating infant colic in general, and to identify whether certain subgroups of infants with colic would benefit from particular strains of probiotics. METHODS AND ANALYSIS: Through an individual participant data meta-analysis (IPDMA), we aim to identify whether the probiotic Lactobacillus reuteri DSM 17938 is effective in the management of infant colic, and to clarify whether its effects differ according to feeding method (breast vs formula vs combined), proton pump inhibitor exposure, and antibiotic exposure. The primary outcomes are infant crying duration and treatment success (at least 50% reduction in crying time from baseline) at 21 days postintervention. Individual participant data from all studies will be modelled simultaneously in multilevel generalised linear mixed-effects regression models to account for the nesting of participants within studies. Subgroup analyses of participant-level and intervention-level characteristics will be undertaken on the primary outcomes to assess if the intervention effect differs between certain groups of infants. ETHICS AND DISSEMINATION: Approved by the Royal Children's Hospital Human Research Ethics Committee (HREC 34081). Results will be reported in a peer-reviewed journal in 2015. TRIAL REGISTRATION NUMBER: PROSPERO CRD42014013210.

Legionella longbeachae infection in a persistent hand-wound after a gardening accident.

INTRODUCTION: Unlike other Legionella species, Legionella longbeachae has been associated with soil and potting composts instead of water systems, and it has caused pneumonia in gardeners. CASE PRESENTATION: We report, to our knowledge, the first case of prolonged localized L. longbeachae infection in an accidental wound on the back of a hand caused by a broken flowerpot. CONCLUSION: Identification of L. longbeachae requires awareness and expertise, since commercial tests are most often specific for L. pneumophila.

Evaluation of a new automated homogeneous PCR assay, GenomEra C. difficile, for rapid detection of Toxigenic Clostridium difficile in fecal specimens.

We evaluated a new automated homogeneous PCR assay to detect toxigenic Clostridium difficile, the GenomEra C. difficile assay (Abacus Diagnostica, Finland), with 310 diarrheal stool specimens and with a collection of 33 known clostridial and nonclostridial isolates. Results were compared with toxigenic culture results, with discrepancies being resolved by the GeneXpert C. difficile PCR assay (Cepheid). Among the 80 toxigenic culture-positive or GeneXpert C. difficile assay-positive fecal specimens, 79 were also positive with the GenomEra C. difficile assay. Additionally, one specimen was positive with the GenomEra assay but negative with the confirmatory methods. Thus, the sensitivity and specificity were 98.8% and 99.6%, respectively. With the culture collection, no false-positive or -negative results were observed. The analytical sensitivity of the GenomEra C. difficile assay was approximately 5 CFU per PCR test. The short hands-on (<5 min for 1 to 4 samples) and total turnaround (<1 h) times, together with the high positive and negative predictive values (98.8% and 99.6%, respectively), make the GenomEra C. difficile assay an excellent option for toxigenic C. difficile detection in fecal specimens.

Two Legionnaires' disease cases associated with industrial waste water treatment plants: a case report.

BACKGROUND: Finnish and Swedish waste water systems used by the forest industry were found to be exceptionally heavily contaminated with legionellae in 2005. CASE PRESENTATION: We report two cases of severe pneumonia in employees working at two separate mills in Finland in 2006. Legionella serological and urinary antigen tests were used to diagnose Legionnaires' disease in the symptomatic employees, who had worked at, or close to, waste water treatment plants. Since the findings indicated a Legionella infection, the waste water and home water systems were studied in more detail. The antibody response and Legionella urinary antigen finding of Case A indicated that the infection had been caused by Legionella pneumophila serogroup 1. Case A had been exposed to legionellae while installing a pump into a post-clarification basin at the waste water treatment plant of mill A. Both the water and sludge in the basin contained high concentrations of Legionella pneumophila serogroup 1, in addition to serogroups 3 and 13. Case B was working 200 meters downwind from a waste water treatment plant, which had an active sludge basin and cooling towers. The antibody response indicated that his disease was due to Legionella pneumophila serogroup 2. The cooling tower was the only site at the waste water treatment plant yielding that serogroup, though water in the active sludge basin yielded abundant growth of Legionella pneumophila serogroup 5 and Legionella rubrilucens. Both workers recovered from the disease. CONCLUSION: These are the first reported cases of Legionnaires' disease in Finland associated with industrial waste water systems.

Detection of virulence genes of Clostridium difficile by multiplex PCR.

Clostridium difficile strains belonging to the PCR ribotype 027, pulse-field gel electrophoresis (PFGE) type NAP1, toxinotype III and restriction endonuclease analysis group BI harbouring mutations in the tcdC gene and possessing binary toxin components A and B have been described to cause epidemics with increased morbidity and mortality. In the present study we developed a conventional multiplex PCR designed to detect selected virulence associated markers of the hypervirulent C. difficile PCR ribotype 027. The multiplex PCR assay detected the major toxins A and B, binary toxin components A and B as well as a possible deletion in the tcdC gene: a characteristic pattern of amplification products for the PCR ribotype 027 strains was detected. This rather simple method was specific for the screening of this hypervirulent C. difficile strain. The correlation between the multiplex PCR and PCR ribotyping methods was excellent. The sensitivity and specificity were 100% in our epidemiological situation. In conclusion, this multiplex PCR was found useful in the preliminary screening for the hypervirulent C. difficile PCR ribotype 027.

Diagnostic trends in Clostridium difficile detection in Finnish microbiology laboratories.

Due to increased interest directed to Clostridium difficile-associated infections, a questionnaire survey of laboratory diagnostics of toxin-producing C. difficile was conducted in Finland in June 2006. Different aspects pertaining to C. difficile diagnosis, such as requests and criteria used for testing, methods used for its detection, yearly changes in diagnostics since 1996, and the total number of investigations positive for C. difficile in 2005, were asked in the questionnaire, which was sent to 32 clinical microbiology laboratories, including all hospital-affiliated and the relevant private clinical microbiology laboratories in Finland. The situation was updated by phone and email correspondence in September 2008. In June 2006, 28 (88%) laboratories responded to the questionnaire survey; 24 of them reported routinely testing requested stool specimens for C. difficile. Main laboratory methods included toxin detection (21/24; 88%) and/or anaerobic culture (19/24; 79%). In June 2006, 18 (86%) of the 21 laboratories detecting toxins directly from feces, from the isolate, or both used methods for both toxin A (TcdA) and B (TcdB), whereas only one laboratory did so in 1996. By September 2008, all of the 23 laboratories performing diagnostics for C. difficile used methods for both TcdA and TcdB. In 2006, the number of specimens processed per 100,000 population varied remarkably between different hospital districts. In conclusion, culturing C. difficile is common and there has been a favorable shift in toxin detection practice in Finnish clinical microbiology laboratories. However, the variability in diagnostic activity reported in 2006 creates a challenge for national monitoring of the epidemiology of C. difficile and related diseases.