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.

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.

Prevalence of pilus-encoding islets and clonality of pneumococcal isolates from children with acute otitis media.

We characterized the prevalence of pilus islets 1 (PI-1) and 2 (PI-2) and the clonality of Streptococcus pneumoniae isolates taken from children with acute otitis media (AOM) to study the association between pilus existence and AOM disease potential prior to pneumococcal conjugate vaccine and increased antimicrobial resistance. The study material consisted of 75 pneumococcal isolates cultured from the middle ear fluid and/or nasopharyngeal aspirate of 56 children with AOM in Finland during the period 1990-1992. Isolates were studied for antimicrobial susceptibility and were serotyped, genotyped by multilocus sequence typing (MLST), and tested for the presence of pneumococcal PI-1 and PI-2 genes. All isolates were susceptible to penicillin, 14 different serotypes were found, and 20% of the isolates were positive for PI-1 genes. PI-2 genes were not found. MLST showed high heterogeneity: 52 AOM isolates belonged to 18 known clonal complexes (CC). PI-1 was associated with serotypes 6A, 6B, and 9V, and genotype CC490. In the time prior to 7-valent pneumococcal conjugate vaccine (PCV7) and increased antimicrobial resistance, pneumococcal AOM isolates carried PI-1 genes at a rather low prevalence. PI-2 genes were not detected. PI-1 was related to serotype rather than genotype. The importance of PI-1 in AOM infections and its association with the spread of antimicrobial resistance requires further research.

Adapting spa typing for national laboratory-based surveillance of methicillin-resistant Staphylococcus aureus.

Laboratory-based surveillance of methicillin-resistant Staphylococcus aureus (MRSA) monitors the baseline occurrence of different genotypes and identifies strains and transmission chains responsible for outbreaks. The consequences of substituting pulsed-field gel electrophoresis (PFGE) with spa typing as a first-line typing method were analyzed by typing 589 strains isolated between 1997 and 2006, with a focus on both short- and long-term correspondence between the PFGE and spa typing results. The study, covering these ten years, included all Finnish MRSA blood isolates and representatives of the two most prevalent MRSA strains (PFGE types FIN-4 and FIN-16) in Finland. In addition, all sporadic isolates from 2006 were included. spa typing was more expensive but approximately four times faster to perform than PFGE. Nearly 90% of FIN-4 and FIN-16 isolates showed consistent spa types, t172 and t067, respectively. spa typing predicted the PFGE result of the blood isolates by a Wallace coefficient of 0.9009, recognized internationally successful strains (t041, t067) to be common also in Finland, and identified a separate cluster of isolates, also related in time and place among the FIN-4 strains. Additional typing by another method was needed to provide adequate discrimination or to characterize isolates with a newly recognized spa type in Finland.

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.

A report of Streptococcus pneumoniae serotype 6D in Europe.

Serotype 6D of Streptococcus pneumoniae has been reported in Asia and the Fijian islands among nasopharyngeal carriage isolates. We now report a 6D isolate from a Finnish adult with invasive pneumococcal disease. Interestingly, the Finnish isolate and Asian isolate capsule gene loci are almost identical.

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.

Low incidence of antibiotic resistance among invasive and nasopharyngeal isolates of Streptococcus pneumoniae from children in rural Philippines between 1994 and 2000.

The purpose of this study was to determine the prevalence of acquired antimicrobial resistance in Streptococcus pneumoniae isolated from nasopharyngeal swabs and blood and cerebrospinal fluid (CSF) specimens of 3,028 children hospitalized with signs or symptoms of pneumonia, sepsis, or meningitis in rural Philippines between 1994 and 2000. Pneumococci were identified using standard methods, serotyped, and their susceptibility to oxacillin, erythromycin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole was determined using the disk diffusion method. Penicillin minimum inhibitory concentrations (MICs) of the oxacillin-resistant isolates were further tested. The clonality of the penicillin-nonsusceptible (PNSP) isolates was analyzed using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Altogether 1,048 isolates were analyzed, of which 35 were invasive and 1,013 nasopharyngeal isolates. None was resistant, but 22 (2.1%) were intermediately resistant to penicillin, 4 (0.2%) were resistant to chloramphenicol, 3 (0.2%) to erythromycin, 39 (3.7%) to tetracycline, and 4 (0.2%) to trimethoprim/sulfamethoxazole. Twelve of the 22 PNSP isolates were of serotype 14 and of sequence type 63. These included the two invasive PNSP isolates. PFGE profiling further identified three separate clusters among the sequence of type 63, serotype 14 (ST63(14)) isolates. Antimicrobial resistance in both invasive and nasopharyngeal pneumococcal pediatric isolates in rural Philippines is rare. In spite of this remote setting, the PNSP isolates of the serotype 14 clusters were of ST63 type, which has been described previously on other continents.

Clonality of epidemic methicillin-resistant Staphylococcus aureus strains in Finland as defined by several molecular methods.

In Finland, the incidence of methicillin-resistant Staphylococcus aureus (MRSA) strains has increased ten fold within the last decade. In order to follow the changing epidemiology of MRSA, accurate typing of S. aureus strains is important. The purpose of this study was to reanalyse 44 previously recognised Finnish epidemic MRSA strains (EMRSA) by several molecular typing methods and to revise their nomenclature. The 44 EMRSA strains were grouped into 26 pulsed-field gel electrophoresis (PFGE) clusters, 20 multi locus sequence typing (MLST) sequence types (ST) belonging to 12 clonal complexes (CC) of which CC8 was the most prevalent, and 27 spa types belonging to four clonal complexes. The staphylococcal cassette chromosome mec (SCCmec) type IV was predominant, and 48% of the strains were nonmultiresistant to antibiotics. The discriminatory power of PFGE clusters, MLST, and spa typing was high. The overall concordance values of typing methods differed when assessed by two different methods. Adjusted Rand coefficient provided fairly low correlations for all comparisons. However, spa type was able to efficiently predict types and clonal complexes of most of the other methods with high probability (> or =80%).

Panton-Valentine leukocidin genes and staphylococcal chromosomal cassette mec types amongst Finnish community-acquired methicillin-resistant Staphylococcus aureus strains, 1997-1999.

Methicillin-resistant Staphylococcus aureus (MRSA) strains from Finland covering years 1997-1999 were studied for the presence of Panton-Valentine leukocidin (PVL) gene loci, and the clinically well-defined community-acquired MRSA (CA-MRSA) strains (n = 108) also for staphylococcal chromosomal cassette mec (SCCmec) and multilocus sequence types (MLST). Only a minority (12%) of the CA-MRSA strains contained the PVL gene loci and possessed genotypes formerly described as typical to CA-MRSA strains. The majority of these strains were heterogenous by MLST and pulsed-field gel electrophoresis (PFGE) analysis but, however, harboured the SCCmec cassette type IV. In conclusion, it seems doubtful to consider only molecular characteristics such as the presence of PVL genes as definite markers for CA-MRSA strains.

Clinical outcome of acute pneumococcal otitis media and serum antibody responses to pneumococcal pneumolysin and polysaccharides in children.

Serum antibody responses to pneumococcal antigens and their relationship to the clinical outcome were determined in a prospective study of 121 children with acute otitis media (AOM). Pneumococcus positive children with a pneumolysin response more often had a recurrence and middle ear effusion (MEE) after 1 month than did the non-responders (p = 0.005 and p = 0.04, respectively). All the children who responded to pneumolysin also had clinically strong symptoms and signs of AOM. Children who responded to pneumococcal polysaccharides developed otitis media with effusion within a 6-month follow-up period more often than did the non-responders (p = 0.005). The results of this study suggest that children with pneumococcal AOM and an antibody response to the intracellular pneumococcal protein pneumolysin behave clinically differently from children with an antibody response to polysaccharides.

Human antibodies to pneumococcal surface protein A in health and disease.

BACKGROUND: Diseases caused by Streptococcus pneumoniae have a high impact in young children whose ability to mount antibodies to capsular polysaccharides is impaired. Pneumococcal surface protein A (PspA) is a potential vaccine candidate for this age group. METHODS: We used Western blot analysis and enzyme immunoassay to study human sera of healthy adults from Alabama (n = 20) and from Finland (n = 21), healthy children from Finland (n = 20) and ill children from Finland, those with pneumococcal invasive infection (n = 26) and those with nonpneumococcal invasive infection (n = 26). RESULTS: Human antibodies to PspA exhibited strong cross-reactivity among different pneumococcal strains. The geometric mean titer of IgG antibody to PspA in sera from 21 healthy adults was 4,040, from ten 3-year-old healthy children 1,080 and from ten 2-month-old healthy children 1,650. The geometric mean titer of PspA antibody of acute phase sera of children with invasive pneumococcal disease was 140, significantly (P < 0.001) lower than the respective value, 1,020, for children with infection caused by other bacteria. CONCLUSIONS: We demonstrate for the first time the existence of antibodies to PspA in human sera in health and disease. The findings in ill children suggest that antibodies to PspA might play a role in protection against pneumococcal disease.

Intranasal immunization of mice with a mixture of the pneumococcal proteins PsaA and PspA is highly protective against nasopharyngeal carriage of Streptococcus pneumoniae.

Acquisition of pneumococci is generally from carriers rather than from infected individuals. Therefore, to induce herd immunity against Streptococcus pneumoniae it will be necessary to elicit protection against carriage. Capsular polysaccharide-protein conjugates, PspA, and PsaA are known to elicit some protection against nasopharyngeal carriage of pneumococci but do not always completely eliminate carriage. In this study, we observed that PsaA elicited better protection than did PspA against carriage. Pneumolysin elicited no protection against carriage. Immunization with a mixture of PsaA and PspA elicited the best protection against carriage. These results indicate that PspA and PsaA may be useful for the elicitation of herd immunity in humans. As PspA and pneumolysin are known to elicit immunity to bacteremia and pneumonia, their inclusion in a mucosal vaccine may enable such a vaccine to prevent invasive disease as well as carriage.

Polymerase chain reaction-based detection of rhinovirus, respiratory syncytial virus, and coronavirus in otitis media with effusion.

OBJECTIVES: To study the association of human rhinovirus (HRV), respiratory syncytial virus (RSV), and human coronavirus infections in children aged 6 months to 12 years with otitis media with effusion (OME). To determine how long HRV RNA can be detected after HRV infection. METHODS: Middle ear effusion (MEE) samples collected at the time of tympanostomy tube placement from 100 children with OME were examined. Viral RNA was detected by reverse-transcriptase polymerase chain reaction. For HRV the results were compared with virus isolation in cell culture. In vitro studies of the persistence of HRV infectivity and RNA were conducted by combining approximately 10(5) median cell culture infectious doses of HRV with pooled MEE at 37 degrees C and assaying serial samples for 12 weeks. RESULTS: Virus RNA was detected in 30 children. HRV was detected by reverse-transcriptase polymerase chain reaction in 19 children with OME and by virus isolation in 5 children. RSV RNA was found in 8 and HCV in 3 children with OME. No dual viral infection was found. Bacterial pathogens were isolated from 35 MEE samples and were associated with viral RNA in 11 cases, most often with HRV (9 cases). Under in vitro conditions, HRV culture positivity declined rapidly (<2 days), but RNA was detectable for up to 8 weeks. CONCLUSIONS: These results suggest that virus infection, particularly HRV infection, either alone or concurrent with bacteria, is present in a larger percentage of children with OME than previously suspected. It remains to be determined how often the presence of viral RNA in MEE represents persistent RNA, ongoing viral replication, or recurrent infection.

Detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction.

OBJECTIVE: To determine the frequencies of human rhinovirus (HRV), respiratory syncytial virus (RSV), and coronavirus (HCV) infection in children with acute otitis media (AOM). METHODS: Middle ear fluids (MEF) collected by tympanocentesis and nasopharyngeal aspirates (NPA) at the time of the AOM diagnosis were examined by reverse transcriptase polymerase chain reaction for HRV, RSV, and HCV RNA. PATIENTS: Ninety-two children aged 3 months to 7 years during a 1-year period. RESULTS: Virus RNA was detected in a total of 69 children (75%) and in 44 MEF samples (48%) and 57 NPA samples (62%) at the time of AOM diagnosis. HRV RNA was detected in both MEF and NPA in 18 (20%), in MEF alone in 4 (4%), and in NPA alone in 10 (11%). RSV was detected in both MEF and NPA in 12 (13%), in MEF alone in 5 (5%), and in NPA alone in 9 (10%). HCV RNA was detected in both MEF and NPA in 5 (5%), in MEF alone in 2 (2%), and in NPA alone in 9 (10%). Dual viral infections were detected in 5% of children. HRV and RSV were detected simultaneously in 2 MEF samples and in 2 NPA samples; RSV and HCV were detected in 1 NPA sample. Bacterial pathogens were detected in 56 (62%) MEF from 91 children. Viral RNA was detected in 20 (57%) MEF of 35 bacteria-negative and in 25 (45%) of 56 bacteria-positive MEF samples. No important differences in the risk of treatment failure, relapse, or occurrence of late secretory otitis media were noted between children with virus-positive and virus-negative MEF aspirates. CONCLUSION: These findings highlight the importance of common respiratory viruses, particularly HRV and RSV, in predisposing to and causing AOM in young children.

Circulating antibody secreting cell response to parenteral pneumococcal vaccines as an indicator of a salivary IgA antibody response.

This study assessed the mucosal immune response in healthy adult volunteers immunized parenterally with either pneumococcal polysaccharide (N = 8) or pneumococcal polysaccharide-protein conjugate (N = 10) vaccine with an aim to evaluate the relevance of antibody secreting cell (ASC) response after parenteral vaccination. An ASC response to the four types of capsular polysaccharide tested was observed in all vaccinees 7-9 days after immunization. IgA was the predominant class in the ASC response, and IgG the next common, with very few IgM ASCs. The IgA/IgG ratio in the ASC response was higher after immunization with the polysaccharide than the conjugate vaccine. Antibodies of the IgA class were frequently seen in the saliva already before immunization; especially to serotypes 14 and 19F. A twofold increase of the type specific secretory IgA antibodies in saliva was found in eight of the 16 instances in which the specific IgA ASC response was > 100 ASC per 10(6) cells and in only one of the 52 instances with fewer ASCs. We conclude that the ASC response in the peripheral blood is a useful parameter of the antibody response to pneumococcal vaccines and a good indicator of a secretory IgA response in the saliva.

Establishment of a Streptococcus pneumoniae nasopharyngeal colonization model in adult mice.

Human nasopharyngeal carriage of Streptococcus pneumoniae constitutes the major natural reservoir of pneumococci and is thought to be the prelude to virtually all pneumococcal disease. If carriage could be greatly reduced, pneumococcal transmission and disease could be largely eliminated. To facilitate the studies of mechanisms important in carriage and to identify immunogens that can elicit protection against carriage, we characterized an adult mouse model of nasopharyngeal carriage. Non-anaesthetized mice were inoculated intranasally with pneumococci in 10 microl of fluid. Nasopharyngeal carriage was observed with strains of capsular types 3, 4, 6A, 6B, 14, 19, and 23. Carriage was stable over time, and the numbers of pneumococci carried were relatively independent of inoculation dose; findings which indicate that the recovery of pneumococci from 1 day to 2 weeks post inoculation was dependent on colonization, rather than just temporary contamination. To ensure carriage in the largest percentage of mice, without causing sepsis or death, inoculations of 10(7) colony forming units (cfu) should be used. In this model, carriage was generally observed without concomitant bacteremia or sepsis and carriage was observed even with strains that were avirulent when injected i.v. The model should be useful for the identification of protection-eliciting antigens, since intranasal immunization with heat-killed pneumococci or lysates of pneumococci protected against carriage.

Prognosis of acute otitis media: factors associated with poor outcome.

Factors associated with poor outcome of acute otitis media (AOM) were analysed in 131 children aged 1/4 to 7 1/2 (median 2 1/2) years. After AOM, altogether 37 (28%) of the children had poor outcome: 15 children (12%) clinical failure (unimprovement or worsening of pre-treatment signs and symptoms within 2 weeks of onset of therapy) and 31 (24%) persistent middle ear effusion (MEE) > or = 1 month post-treatment. Of the different variables studied in multivariate analysis, age < 2 years (p < 0.01), history of allergic skin or respiratory symptoms (p = 0.02), > or = 6 h duration of pre-treatment earache (p = 0.01) and B. catarrhalis in MEE (p = 0.05) were associated with clinical failure. Children with previous adenotomy or unilateral AOM had no failures. Persistence of MEE at 1 month was associated with age < 2 years (p = 0.05), otitis proneness (p = 0.03), bilaterality of AOM (p < 0.01) and S. pneumoniae in MEE (p = 0.01) in univariate but not in multivariate analysis.