Epidemiology and emm types of invasive group A streptococcal infections in Finland, 2008-2013.

Invasive Streptococcus pyogenes (group A streptococcus, GAS) infections are a major global cause of morbidity and mortality. We analysed the surveillance data on invasive GAS and the microbiological characteristics of corresponding isolates to assess the incidence and emm type distribution of invasive GAS infections in Finland. Cases defined as patients with isolations of blood and cerebrospinal fluid S. pyogenes are mandatorily notified to the National Infectious Disease Registry and sent to the national reference laboratory for emm typing. Antimicrobial data were collected through the network including all clinical microbiology laboratories. Pulsed-field gel electrophoresis (PFGE) analysis was performed to assess clonality. In total, 1165 cases of invasive GAS were reported in Finland during 2008-2013; the median age was 52 years (range, 0-100) and 54% were male. The overall day 7 case fatality rate was 5.1% (59 cases). The average annual incidence was 3.6 cases per 100,000 population. A total of 1122 invasive GAS isolates (96%) were analysed by emm typing; 72 different emm types were identified, of which emm28 (297 isolates, 26%), emm89 (193 isolates, 12%) and emm1 (132 isolates, 12%) were the most common types. During 2008-2013, an increase of erythromycin resistance (1.9% to 8.7%) and clindamycin (0.9% to 9.2%) was observed. This resistance increase was in parallel with the introduction of a novel clone emm33 into Finland. The overall incidence of invasive GAS infections remained stable over the study period in Finland. We identified clonal spread of macrolide-resistant invasive emm33 GAS type, highlighting the importance of molecular surveillance.

Coincidental detection of the first outbreak of carbapenemase-producing Klebsiella pneumoniae colonisation in a primary care hospital, Finland, 2013.

In Finland, occurrence of Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has previously been sporadic and related to travel. We describe the first outbreak of colonisation with KPC-KP strain ST512; it affected nine patients in a 137-bed primary care hospital. The index case was detected by chance when a non-prescribed urine culture was taken from an asymptomatic patient with suprapubic urinary catheter in June 2013. Thereafter, all patients on the 38-bed ward were screened until two screening rounds were negative and extensive control measures were performed. Eight additional KPC-KP-carriers were found, and the highest prevalence of carriers on the ward was nine of 38. All other patients hospitalised on the outbreak ward between 1 May and 10 June and 101 former roommates of KPC-KP carriers since January had negative screening results. Two screening rounds on the hospital's other wards were negative. No link to travel abroad was detected. Compared with non-carriers, but without statistical significance, KPC-KP carriers were older (83 vs 76 years) and had more often received antimicrobial treatment within the three months before screening (9/9 vs 90/133). No clinical infections occurred during the six-month follow-up. Early detection, prompt control measures and repetitive screening were crucial in controlling the outbreak.

Clonality behind the increase of multidrug-resistance among non-invasive pneumococci in Southern Finland.

Multidrug-resistance among Streptococcus pneumoniae isolates, especially of serotype 19A, has increased in several countries recently. Even before the introduction of the pneumococcal conjugate vaccine into the Finnish National Vaccination Programme, the proportion of multidrug-resistant (MDR) pneumococci had doubled from 2007 to 2008, when it reached 3.6% in Southern Finland. Our aim was to look for a possible association between antimicrobial susceptibility and clonality among the MDR isolates. Twelve non-invasive isolates non-susceptible to penicillin, erythromycin, clindamycin, trimethoprim/sulfamethoxazole, and doxycycline from 2008 were available for serotyping, genotyping by multilocus sequence typing (MLST), and detection of genes encoding macrolide resistance and adherence-promoting pili. Two isolates were also resistant to ceftriaxone. Five serotypes, 19F, 19A, 6B, 23F, and 14, and six genotypes from three genetic lineages were found, among which CC320 was the largest. All isolates in this study carried the erm(B) macrolide resistance gene, and the CC320 isolates additionally carried the mef(A/E) macrolide resistance gene. Eleven isolates carried pilus islet 1, while the CC320 isolates also carried the pilus islet 2 genes. The findings emphasize the importance of the careful monitoring of antimicrobial susceptibility and serotype distribution among pneumococci, especially now that antimicrobials and pneumococcal vaccines are in widespread use.

Detection of Streptococcus pneumoniae carriage by the Binax NOW test with nasal and nasopharyngeal swabs in young children.

The rapid detection of carriage of Streptococcus pneumoniae could assist in the management of pneumococcal infection, such as acute otitis media. We evaluated the reliability of the Binax NOW test in the exclusion and detection of pneumococcal carriage by nasal samples from 139 children and using nasopharyngeal samples from 250 children (aged 6-35 months) with respiratory infection with or without acute otitis media. The Binax NOW test results were compared with culture-based detection of carriage of S. pneumoniae. The Binax NOW test from the nasal samples had a sensitivity of 95%, a specificity of 78%, and the positive and negative predictive values were 83 and 93%, respectively; and for the nasopharyngeal samples the corresponding numbers were 88%, 95%, 96%, and 87%. When rapid knowledge of the carriage status of S. pneumoniae is needed, the Binax NOW test is a reliable method for the exclusion of carriage using nasal sampling, and in the detection of carriage using nasopharyngeal sampling.

Incidence, case fatality and genotypes causing Clostridium difficile infections, Finland, 2008.

Since 2000, the epidemiology of C. difficile infections (CDI) has changed in the US and Europe. Few population-based assessments of both incidence and case fatality of CDI have been performed. In this study, the Finnish nationwide laboratory-based surveillance data from the year 2008 were analysed to assess the incidence and case fatality of CDI, and to detect regional differences in relation to molecular epidemiology. A total of 6201 episodes of CDI were identified (118.3/100 000 population; range by regions, 57.2-189.1). The incidence increased by age and was highest in persons aged >84 years (1286.0). Of the CDI episodes, 711 (11.5%; range by regions, 2.2-15.0%) led to death within 30 days. The 30-day case fatality was highest (22.0%) in persons aged >84 years. In total, 334 (5% of all episodes) isolates from 13/21 regions were sent for genotyping: 120 (36%) were of PCR ribotype 027, and it was found in 6/13 regions. Among the rest of the isolates, 53 (16%) were of type 001, and 19 (6%) of 002 and 014. The incidence and case fatality were highest in elderly persons and varied regionally. This may be explained by uneven spread of hypervirulent PCR ribotypes, such as 027, but also differences in diagnostic activity or the patient populations among which the outbreaks are occurring.

Comparison of repetitive extragenic palindromic sequence-based PCR with PCR ribotyping and pulsed-field gel electrophoresis in studying the clonality of Clostridium difficile.

Clostridium difficile infection is most often induced by antibiotic treatment. Recently, morbidity and mortality resulting especially from C. difficile PCR ribotype 027 have increased significantly. In addition, more severe disease has been associated with C. difficile PCR ribotype 078 strains. Thus, reliable typing methods for epidemic control are needed. In the present study, we compared an automated repetitive extragenic palindromic sequence-based PCR (rep-PCR) method (DiversiLab; Bacterial Barcodes, Inc., Athens, GA, USA) to PCR ribotyping and pulsed-field gel electrophoresis (PFGE) typing using 205 isolates of C. difficile (including 24 previously characterized isolates). Among the 181 clinical isolates, a total of 31 different PCR ribotypes, 38 different PFGE types and subtypes and 28 different rep-PCR types were found. Six major rep-PCR groups (DL1-DL6) harboured 86% of the clinical isolates. All isolates belonging to PCR ribotypes 027 and 001 clustered in their own rep-PCR groups, enabling us to screen out the hypervirulent ribotype 027 strain. Within the PCR ribotype 001, four subgroups were found using rep-PCR. Overall, in 75% (135/181) of the isolates, the classification attributed following rep-PCR and PCR ribotyping was comparable. In conclusion, the automated rep-PCR-based typing method represents an option for first-line molecular typing in local clinical microbiology laboratories. The method was easy to use as well as rapid, requiring less hands-on time than PCR ribotyping or PFGE typing. The conventional PCR ribotyping or PFGE, however, are needed for confirmatory molecular epidemiology. In addition, more epidemiology-oriented studies are needed to examine the discriminatory power of automated rep-PCR with isolates collected from a larger geographical area and during a longer period of time.

Emergence of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae during the years 2000 and 2004 in Helsinki, Finland.

The molecular epidemiology of 33 Escherichia coli and 81 Klebsiella pneumoniae extended-spectrum beta-lactamase-producing healthcare-associated and community-acquired isolates collected in the Helsinki district during 2000-2004 was investigated. Clonality studies, antimicrobial susceptibility and genotyping of the isolates were performed. Newly emerging CTX-M-producing E. coli and bla(SHV-12)-producing K. pneumoniae isolates were detected. Clonal clusters of both species persisted throughout the study period.

Epidemiology, outcome and emm types of invasive group A streptococcal infections in Finland.

In 2006, Finnish nationwide surveillance showed an increase of invasive group A streptococcal (iGAS) disease and clinicians were alarmed by severe disease manifestations, prompting the investigation of recent trends and outcome for iGAS. A case of iGAS was defined as Streptococcus pyogenes isolated from blood or cerebrospinal fluid. Cases during 1998-2007 and isolates during 2004-2007 were included. Case-patients' 7-day outcome was available for 2004-2007. Isolates were emm typed. A total of 1,318 cases of iGAS were identified. The average annual incidence was 2.5/100,000 population. The rate was higher in males than females in persons aged 45-64 years, but lower in persons aged 25-34 years. The annual incidence was highest in 2007 (3.9/100,000). Occasional peaks occurred during midwinter and midsummer. The most common emm types were 28 (21%), 1 (16%), 84 (10%), 75 (7%) and 89 (6%). During 2004-2007, emm1 replaced emm28 as the most predominant type. The overall case fatality was 8%. Cases with emm1 were associated with high case fatality (14% vs. 8% in other types; p < 0.02); that of emm28 infections was 2% (p < 0.01). Changes in emm type prevalence influenced incidence and case fatality. Differences in age- and sex-specific incidence and seasonal patterns suggest variations in predisposing factors and underlying conditions.

First isolations of KPC-2-carrying ST258 Klebsiella pneumoniae strains in Finland, June and August 2009.

The first two Klebsiella pneumoniae carbapenemase-producing (KPC) type 2 strains carrying ST258 were detected in Finland in June and early August 2009. They were found colonising two patients transferred from the Mediterranean; one patient referred from a hospital in Greece where isolates were first found in 2007 and another from Italy where the first isolates have been described only very recently.

Induced sputum in the diagnosis of childhood community-acquired pneumonia.

BACKGROUND: The usefulness of induced sputum in searching for causative agents of pneumonia in children has not been studied. METHODS: The study involved 101 children, aged 6 months to 15 years, treated for community-acquired pneumonia at Turku University Hospital (Turku, Finland) from January 2006 to April 2007. Nasopharyngeal aspirate samples were first collected through both nostrils. Sputum production was then induced by inhalation of 5.0% hypertonic saline for 5-10 min and a sputum sample was either aspirated or expectorated. The presence and amount of bacteria and viruses in paired nasopharyngeal aspirate and sputum specimens was analysed and compared using semiquantitative bacterial culture and quantitative PCR techniques. RESULTS: A good quality sputum specimen was obtained from 76 children. The possible causative agent was found in 90% of cases. Streptococcus pneumoniae (46%) and rhinovirus (29%) were the most common microbes detected. Newly discovered viruses human bocavirus and human metapneumovirus were detected in 18% and 13% of the children, respectively. One-quarter of all bacterial findings were only detected in sputum, and the amount of bacteria in the remainder of the sputum specimens compared with nasopharyngeal aspirate was higher in 14% and equal in 70%. The amount of rhinovirus in sputum was higher than in nasopharyngeal aspirate in 82%. CONCLUSIONS: Sputum induction provides good quality sputum specimens with high microbiological yield in children with community-acquired pneumonia. Induced sputum analysis can be useful in the microbiological diagnosis of childhood community-acquired pneumonia.

Pneumolysin polymerase chain reaction for diagnosis of pneumococcal pneumonia and empyema in children.

Streptococcus pneumoniae is the most important cause of childhood pneumonia and empyema, yet the diagnosis of pneumococcal infections by conventional methods is challenging. In this study, the clinical value of the pneumolysin-targeted real-time polymerase chain reaction (PCR) method for the diagnosis of pneumococcal pneumonia and empyema was evaluated with 33 whole blood samples and 12 pleural fluid samples. The analytical sensitivity of the PCR assay was 4 fg of pneumococcal DNA, corresponding to two genome equivalents of pneumococcal DNA per reaction. The PCR assay correctly detected all clinical isolates of S. pneumoniae tested, whereas all nonpneumococcal bacterial organisms tested were negative by PCR. In a clinical trial, S. pneumoniae was detected by PCR in the pleural fluid of 75% of children with empyema, increasing the detection rate of pneumococcus almost tenfold that of pleural fluid culture. However, in whole blood samples, PCR detected S. pneumoniae in only one child with pneumonia and one child with pneumococcal empyema and failed to detect S. pneumoniae in three children with blood cultures positive for S. pneumoniae. The present data indicate that pneumolysin-targeted real-time PCR of pleural fluid is a valuable method for the etiologic diagnosis of pneumococcal empyema in children. The ease and rapidity of the LightCycler technology (Roche Diagnostics, Mannheim, Germany) make real-time PCR an applicable tool for routine diagnostics. In the evaluation of blood samples, blood culture remains the superior method for the diagnosis of bacteremic pneumococcal disease.

Streptococcus pneumoniae isolates resistant to telithromycin.

The telithromycin susceptibility of 210 erythromycin-resistant pneumococci was tested with the agar diffusion method. Twenty-six erm(B)-positive isolates showed heterogeneous resistance to telithromycin, which was manifested by the presence of colonies inside the inhibition zone. When these cells were cultured and tested, they showed stable, homogeneous, and high-level resistance to telithromycin.

Prevalence and molecular genetics of macrolide resistance among Streptococcus pneumoniae isolates collected in Finland in 2002.

The prevalence and mechanisms of macrolide resistance among 1,007 clinical pneumococcal isolates collected in Finland were investigated. Of these, 217 (21.5%) were resistant to erythromycin and 11% to clindamycin. Among the erythromycin-resistant isolates, mef(E) was present in 95 isolates (44%), mef(A) was present in 12 isolates (6%), and erm(B) was present in 90 isolates (41%). A double mechanism, mef(E) and erm(B), was detected in five isolates (2%). Ribosomal mutation was detected in 14 (6%) macrolide-resistant isolates in which no other determinant was found. Based on the telithromycin MICs, two groups of isolates were formed: 83.3% of the isolates belonged to a major group for which the telithromycin MIC range was < or =0.008 to 0.063 microg/ml, and 16.7% belonged to a minor group for which the telithromycin MIC range was 0.125 to 8 microg/ml. All except three isolates in the minor population carried a macrolide resistance gene.

Efficacy of bioabsorbable antibiotic containing bone screw in the prevention of biomaterial-related infection due to Staphylococcus aureus.

Impregnation of antimicrobial agents within biodegradable orthopedic implants provides a possibility for local antimicrobial prophylaxis of biomaterial-related infections. The objective of this study was to evaluate the efficacy of a bioabsorbable ciprofloxacin containing bone screw (Ab-PLGA) in the prevention of biomaterial-related infection due to Staphylococcus aureus in a rabbit model. Animals in Group I (n=8) received a Ab-PLGA screw contaminated with S. aureus, while animals in Group II (n=8) received a stainless steel (SS) screw contaminated with S. aureus. In two negative control groups, the animals received a Ab-PLGA screw (Group III, n=4) or a SS screw (Group IV, n=4) without bacterial contamination. 18F-FDG-PET imaging, performed at 6 weeks, was applied as a novel quantitative in vivo imaging modality of implant-related infection. Infection was verified by swab cultures, direct cultures of the retrieved implant, and quantitative cultures of pulverized bone. The concentrations of ciprofloxacin in serum and local bone tissue were determined by a high performance liquid chromatographic (HPLC) method with fluorescence (FLD) detection. In the group of contaminated Ab-PLGA screws, all cultures were negative. In the group of contaminated SS screws, all cultures of retrieved implants and six cultures out of eight of pulverized bone were positive for inoculated S. aureus. In negative control groups, all cultures were negative except one contaminant (S. cohnii) found in a SS screw culture. Verified infection of contaminated SS screws was collaborated by the increased 18F-FDG-PET uptake (P=0.004 compared with the group of contaminated Ab-PLGA screws). The mean bone tissue concentration of ciprofloxacin varied from 2.54 to 0.83 microg/g bone as a function of distance from the implantation site. The serum concentration of ciprofloxacin remained undetectable and below the resolution of the analytic method (<5.0 ng/ml). This study confirmed the in vivo efficacy of bioabsorbable antibiotic containing bone screw in the prevention of biomaterial-related infection due to S. aureus.

Aetiological diagnosis of brain abscesses and spinal infections: application of broad range bacterial polymerase chain reaction analysis.

OBJECTIVE: To evaluate the usefulness of the broad range bacterial rDNA polymerase chain reaction (PCR) method combined with DNA sequencing in the aetiological diagnosis of intracranial or spinal infections in neurosurgical patients. METHODS: In addition to conventional methods, the broad range bacterial PCR approach was applied to examine pus or tissue specimens from cerebral or spinal lesions in patients treated in a neurosurgical unit for a clinical or neuroradiological suspicion of bacterial brain abscess or spondylitis. RESULTS: Among the 44 patients with intracranial or spinal lesions, the final diagnosis suggested bacterial disease in 25 patients, among whom the aetiological agent was identified in 17. A causative bacterial species was identified only by the rDNA PCR method in six cases, by both the PCR methodology and bacterial culture in six cases, and by bacterial culture alone in five. All samples in which a bacterial aetiology was identified only by the PCR approach were taken during antimicrobial treatment, and in three patients the method yielded the diagnosis even after >/= 12 days of parenteral treatment. One case also identified by the PCR approach alone involved a brain abscess caused by Mycoplasma hominis, which is not readily cultured by routine methods. CONCLUSIONS: In patients with brain abscesses and spinal infections, the broad range bacterial rDNA PCR approach may be the only method to provide an aetiological diagnosis when the patient is receiving antimicrobial treatment, or when the causative agent is fastidious.

Relationships of Scopulariopsis based on LSU rDNA sequences.

Relationships between Scopulariopsis species and allied fungi were studied by sequencing a 350 bp gene region of the large subunit ribosomal RNA gene (LSU rDNA). In addition, a limited morphological dataset of nine characters was included in the cladistic analysis. Nineteen mitosporic strains (nine Scopulariopsis, five Wardomyces, three Doratomyces, one Trichurus and one Scedosporium species) and 21 meiosporic strains (14 Microascus, 4 Kernia and 3 Pithoascus species) were studied. The data were analysed using parsimony methods. Based on the analyses, the studied microascaceous fungi are divided to 12 molecular lineages. Most of the opportunistic human pathogenic Scopulariopsis species are placed in one clade ('Microascus manginii Clade'). Most synnematous anamorphs with Scopulariopsis-like conidia (Doratomyces and Trichurus) are placed in another clade ('Microascus albonigrescens Clade'), together with Wardomyces. Microascus sensu lato can be divided into seven clades which also incorporate all studied Pithoascus, Scopulariopsis, Wardomyces and Trichurus species and most of the Doratomyces species. Most of the Kernia teleomorphs and one Doratomyces species are placed in a different main clade, together with Pseudallescheria and Petriella. Future alternatives in the taxonomy of Microascus include splitting the genus or redefining it to include deviating taxa. More molecular data need to be obtained and considered in either case.

Development of conventional and real-time PCR assays for detection of Legionella DNA in respiratory specimens.

The development and validation of a PCR assay based on the use of new 16S ribosomal DNA (rDNA)-targeted primers to detect Legionella DNA in respiratory specimens are described. The assay was originally developed as conventional PCR followed by electrophoretic detection and was then adapted to Lightcycler format with SYBR Green I detection and melting curve analysis. The 73 Legionella pneumophila strains tested were amplified with both applications. In addition, 21 and 23 out of 27 other Legionella strains were found positive by conventional and real-time PCR assays, respectively, including the clinically important species L. micdadei, L. bozemaniae, and L. dumoffii. Two DNA purification methods were compared using artificially seeded clinical specimens: a standard organic extraction method and a commercial kit based on adsorption of DNA to silica particles. The detection limit of the assay varied from 2 CFU to >200,000 CFU per ml of clinical specimen, depending on the background sample (i.e., pooled sputa or BAL fluids) and the DNA purification method, the silica method achieving lower detection limits. Analysis of 77 clinical samples (66 bronchoalveolar lavage fluid and 11 sputum samples) by conventional PCR yielded results that were consistent with Legionella culture results. The melting curve analysis in the Lightcycler system readily detected the specific amplification products. However, run-to-run variations in the measured melting temperatures required normalization against the standard sample in each run. The results obtained with the clinical specimens were similar to those obtained with conventional PCR, but more samples are required to determine whether the system can be applied to routine screening of samples for the presence of Legionella DNA.

In vitro activities of the novel ketolide telithromycin (HMR 3647) against erythromycin-resistant Streptococcus species.

The in vitro susceptibilities of 184 erythromycin-resistant streptococci to a novel ketolide, telithromycin (HMR 3647), were tested. These clinical isolates included 111 Streptococcus pyogenes, 18 group C streptococcus, 18 group G streptococcus, and 37 Streptococcus pneumoniae strains. The MICs for all but eight S. pyogenes strains were < or =0.5 microg/ml, indicating that telithromycin is active in vitro against erythromycin-resistant Streptococcus strains. All strains for which MICs were > or =1 microg/ml had an erm(B) resistance gene and six strains for which MICs were > or =4 microg/ml had a constitutive erm(B) gene (MIC range, 4 to 64 microg/ml). Interestingly, for S. pneumoniae strains with a constitutive erm(B) gene, MICs were < or =0.25 microg/ml (MIC range, < or =0.008 to 0.25 microg/ml). Our in vitro data show that for S. pyogenes strains which constitutively express the erm(B) methylase gene, MICs are so high that the strains might be clinically resistant to telithromycin.