Laura Lindholm

Nasopharyngeal bacterial colonization and gene polymorphisms of mannose-binding lectin and toll-like receptors 2 and 4 in infants.

Background Human nasopharynx is often colonized by potentially pathogenic bacteria. Gene polymorphisms in mannose-binding lectin (MBL), toll-like receptor (TLR) 2 and TLR4 have been reported. The present study aimed to investigate possible association between nasopharyngeal bacterial colonization and gene polymorphisms of MBL, TLR2 and TLR4 in healthy infants. Methodology/Principal Findings From August 2008 to June 2010, 489 nasopharyngeal swabs and 412 blood samples were taken from 3-month-old healthy Finnish infants. Semi-quantitative culture was performed and pyrosequencing was used for detection of polymorphisms in MBL structural gene at codons 52, 54, and 57, TLR2 Arg753Gln and TLR4 Asp299Gly. Fifty-nine percent of subjects were culture positive for at least one of the four species: 11% for Streptococcus pneumoniae, 23% for Moraxella catarrhalis, 1% for Haemophilus influenzae and 25% for Staphylococcus aureus. Thirty-two percent of subjects had variant types in MBL, 5% had polymorphism of TLR2, and 18% had polymorphism of TLR4. Colonization rates of S. pneumoniae and S. aureus were significantly higher in infants with variant types of MBL than those with wild type (p = .011 and p = .024). Colonization rates of S. aureus and M. catarrhalis were significantly higher in infants with polymorphisms of TLR2 and of TLR4 than those without (p = .027 and p = .002). Conclusions Our study suggests that there is an association between nasopharyngeal bacterial colonization and genetic variation of MBL, TLR2 and TLR4 in young infants. This finding supports a role for these genetic variations in susceptibility of children to respiratory infections.

Occurrence and characterization of methicillin- resistant staphylococci from bovine mastitis milk samples in Finland

BackgroundMethicillin-resistant staphylococci (MRS) are increasingly being isolated in bovine mastitis. The aim of our study was to evaluate the occurrence of MRS in Finnish mastitis milk samples and characterize the MRS isolates using molecular methods.ResultsMethicillin-resistant S. aureus (MRSA) was a rare finding in bovine mastitis in Finland. Only two out of 135 (1.5%) S. aureus isolates were positive for mec genes. One of these carried mec A and was of spa type t172, SCCmec type IV and ST375, and the other harboured mec C, being spa type t3256, and ST130. MRSA ST375 is common among human MRSA isolates in Finland, but this is the first report in the country of bovine mec C MRSA. In coagulase-negative staphylococci (CoNS) originating from bovine mastitis, methicillin resistance was more common. In the two CoNS collections studied, 5.2% (17/324) and 1.8% (2/110) of the isolates were mecA positive. Eighteen of these were methicillin-resistant S. epidermidis (MRSE), which were divided into 6 separate PFGE clusters. One pulsotype was detected in different parts of the country, indicating clonal spread. Most MRSE (13/18) were of SCCmec type IV, one was of type V and four were non-typeable. Comparison with a human staphylococcal database indicated that bovine MRSE strains were not closely related to human MRSE isolates.ConclusionsThe occurrence of MRS, especially MRSA, in bovine mastitis in Finland was low. Most methicillin-resistant bovine CoNS are MRSE, and we found evidence of a bovine MRSE strain that may spread clonally. This is the first report of a Finnish bovine isolate of MRSAmecC ST130. The study provides a baseline for further MRS monitoring.

Invasive Group A Streptococcal Infections in Children: A Nationwide Survey in Finland.

Background: The incidence of invasive group A streptococcus (iGAS) infections varies in time and geographically for unknown reasons. We performed a nationwide survey to assess the population-based incidence rates and outcomes of children with iGAS infections. Methods: We collected data on patients from hospital discharge registries and the electronic databases of microbiological laboratories in Finland for the period 1996–2010. We then recorded the emm types or serotypes of the strains. The study physician visited all university clinics and collected the clinical data using the same data entry sheet. Results: We identified 151 children with iGAS infection. Varicella preceded iGAS infection in 20% of cases and fasciitis infection in 83% of cases. The annual incidence rate of iGAS infection was 0.93 per 100,000 in 1996–2000, 1.80 in 2001–2005 and 2.50 in 2006–2010. The proportion of emm 1.0 or T1M1 strains peaked in 1996–2000 and again in 2006–2010, to 44% and 37% of all typed isolates. The main clinical diagnoses of the patients were severe soft-tissue infection (46%), sepsis (28%), empyema (10%), osteoarticular infection (9%) and primary peritonitis (5%). Severe pain was the most typical symptom for soft-tissue infections. More than half of the patients underwent surgery and received clindamycin. The readmission rate was 7%, and the case fatality rate was 2%. Conclusions: The incidence rate of pediatric iGAS infections tripled during our study. The increase was not, however, the result of a change in the strain types causing iGAS. Varicella immunization would likely have prevented a significant number of the cases.

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.

Evaluation of two commercial carbapenemase gene assays, the Rapidec Carba NP test and the in-house Rapid Carba NP test, on bacterial cultures

Sir, Carbapenemase-producing organisms (CPO) are recognized as one of the biggest problems in infectious diseases today: how do you treat a patient infected with an organism that might no longer be eliminated by antibiotics? Not only do you have to produce susceptibility results, but knowing the mechanism behind the resistance is also crucial, e.g. many of the new antibiotic/inhibitor combinations are effective only against class A enzymes. With a long list of enzymes and a broad spectrum of susceptibility phenotypes, this is a big challenge for clinical laboratories. We need methods that are simple to set up and perform, have a low rate of false negatives and are reasonably specific. We compared two commercial molecular assays [Check-Direct CPE (Check-Points Health, Wageningen, the Netherlands) and Xpert Carba-R (Cepheid, Maurens-Scopont, France)] and two phenotypic tests [the in-house Rapid Carba NP test and the commercially available version, i.e. Rapidec Carba NP (bioMérieux, Marcy-l’Étoile, France)] using a panel of bacteria with known carbapenemase status, selected among strains sent to our reference laboratory during 2008 to March 2014 from all Finnish clinical laboratories. Of the 94 isolates tested, 57 were CPO (Table 1): 45 Enterobacteriaceae (11 KPC, 15 NDM, 9 OXA-48, 5 OXA-181 and 5 VIM), 9 Pseudomonas aeruginosa (6 VIM and 3 IMP) and 3 Acinetobacter spp. (2 NDM and 1 VIM). They had been confirmed with our in-house PCR (testing for KPC, NDM, OXA-48-like, VIM, IMP, GES and SPM) and UV-spectrophotometric imipenem hydrolysis assay. Of the non-CPO, all had been referred to us because of non-WT susceptibility to carbapenems (screening cut-off for Enterobacteriaceae, zone diameters: meropenem ,22 mm, imipenem ,21 mm, ertapenem ,25 mm; and screening cut-off for Pseudomonas, zone diameters: meropenem ,24 mm and imipenem ,20 mm); 11 Enterobacteriaceae isolates had ambiguous (borderline) results in the hydrolysis assay, but no carbapenemase genes were found with the in-house PCR; the borderline result was assumed to be caused by increased chromosomal AmpC enzyme activity. Whole-genome sequences of two similar strains have since been studied and only chromosomal genes found. Nineteen of the non-CPO Enterobacteriaceae had ESBL or transferable ampC genes (tested for CTX-M, SHV, TEM, CMY, DHA, ACC, FOX, MOX and EBC), seven P. aeruginosa and one Klebsiella pneumoniae strains had reduced susceptibility to carbapenems but no carbapenemase activity or transferable b-lactamase genes. Check-Direct CPE was performed as outlined in the manufacturer’s manual and run on a LightCycler 480 II (Roche, Basel, Switzerland). Xpert Carba-R was performed using the manufacturer’s standard protocol and run on a GeneXpert IV. False negative results were retested once. In-house Carba NP was conducted using the Rapid protocol, as taught to the participants of the Capacity-Building Workshop of the ECDC EuSCAPE project. The Rapidec Carba NP test was done according to the kit instructions, without modifications. For both phenotypic tests, strains were grown on Mueller–Hinton-II agar overnight and results were read after 2 h. The performers and interpreters of all the assays were blinded to the properties of the isolates and each Carba NP assay was independently interpreted by two technicians. No false positives were seen with the first three assays (Table 1), but the Rapidec Carba NP test surprisingly produced 14 false positives. On the other hand, there were only 4 false negatives among the OXA-48-like positive isolates, compared with 12 with the in-house protocol. Others have also experienced difficulties with OXA-48 and seen false positives with the Rapidec Carba NP test. The reason we observed this many false positives is probably due to our negative panel, which was not random, but hand-picked to be extra challenging (most had some kind of b-lactamase activity, not defined as carbapenemase). The species and mechanisms of the false positives were more or less a cross-section of the negative panel: eight ESBL positive (1 ESBL-SHV Escherichia coli, 7 CTX-M: 2 K. pneumoniae, 5 E. coli), one CMY-positive Proteus mirabilis, two Enterobacter cloacae and one Klebsiella oxytoca with borderline results in the hydrolysis assay, and the K. pneumoniae without transferable genes and negative hydrolysis (ertapenem disc 23 mm). Interpretation was also more difficult with the commercial kit, but interpreting orange-red wells as negative, to get fewer false positives, would have given more false negatives. pH might be the fundamental problem with OXA-48-like enzymes: Studentova et al. showed that bicarbonate added to test media significantly enhanced enzyme activity, i.e. a drop in pH, such as happens in a positive Carba NP test, can act to inhibit OXA-48. The two technicians had the same Carba NP results for all but one OXA-181-carrying isolate; this was interpreted in favour of the positive result. In-house Carba NP surprisingly did not detect one KPC isolate and we found no explanation for this: the strain was a non-mucous ST258 K. pneumoniae of Greek origin that was fully resistant (meropenem MIC, 8 mg/L; imipenem MIC, 16 mg/L). It was also weakly positive with the Rapidec Carba NP test. Hands-on time with Xpert Carba-R was much less than with the other methods and it allowed testing directly from a transport swab and also produced the fastest results (,1 h). Check-Direct CPE required isolated DNA and took 2.5 h. The Rapidec Carba NP test took up to 2 h and 40 min from colony to result; the in-house version took slightly longer, depending on how far in advance the reagents had been prepared.

Combined interventions are effective in MRSA control.

Abstract Background: A large healthcare-associated epidemic mainly caused by one methicillin-resistant Staphylococcus aureus (MRSA) strain broke out in Pirkanmaa County, Finland, in 2001. This study describes the impact of infection control and screening practices on the epidemic. Methods: The number of hospital-acquired (HA)-MRSA findings obtained from clinical and screening samples during the epidemic was calculated. Strains were typed by pulsed-field electrophoresis (PFGE) or spa typing. Strain type distribution was studied in relation to sample type, year of the epidemic and site of transmission. Several infection control interventions were launched stepwise and screening protocols were expanded. Results: A total of 4118 cases were identified during 2001–2014, of which 3527 were classified as HA. One strain (spa t067) dominated in the epidemic. HA-MRSA cases decreased constantly from the year 2011. The number of new HA-MRSA cases was 57% less in the year 2014 (n = 171) as compared with the year 2011 (n = 399). The proportion of the epidemic strain declined significantly over the years. Screening samples comprised 71% (2439/3527) and clinical samples 29% (1034/3527) of HA-MRSA findings. The number of HA-MRSA cases found from clinical samples started to decrease when screening was expanded. An increase in hand-rub consumption was associated with a decrease in transmissions in Tampere University Hospital (TAUH). Conclusion: Implementation of universal screening together with several other interventions is effective in containing an MRSA epidemic. The proportion of other than Pirkanmaa epidemic (PE)-MRSA strain findings increased throughout the period, indicating the changing epidemiology of MRSA.

Impact of antimicrobial treatment for acute otitis media on carriage dynamics of penicillin-susceptible and penicillin non-susceptible Streptococcus pneumoniae: secondary analysis of a randomized, double-blind, placebo-controlled trial

Background: Concerns that antimicrobial treatment for acute otitis media (AOM) may select for resistant bacterial lineages have led to conflicting guidelines for clinical management of AOM. Methods: We measured effects of amoxicillin-clavulanate on carriage of penicillin-susceptible Streptococcus pneumoniae (PSSP) strains and on carriage of strains with reduced penicillin susceptibility (RSSP) at end-of-treatment and 15d, 30d, and 60d after treatment in a previously-conducted randomized, double-blind, placebo-controlled trial in Finland. The trial enrolled 322 children 6-35 months of age with stringently-defined AOM. Children who did not show clinical improvement received open-label antimicrobial rescue treatment, irrespective of the blinded treatment assignment. The intention-to-treat populations of the trial arms thus received care resembling immediate antimicrobial therapy and watchful waiting. Results: We analyzed 1358 nasopharyngeal specimens. Immediate amoxicillin-clavulanate reduced PSSP carriage prevalence by 88% (95%CI: 76 to 96%) at end-of-treatment and 27% (-3 to 49%) after 60d, but did not measurably alter RSSP carriage prevalence. By end-of-treatment, 7% of children who carried PSSP at enrollment remained colonized in the amoxicillin-clavulanate arm, compared to 61% of PSSP carriers who received placebo; efficacy persisted 60d after treatment among children who carried PSSP at enrollment. Among children not carrying pneumococci, amoxicillin-clavulanate reduced PSSP acquisition by >80% over 15d. No effect was detected among children who carried RSSP at enrollment. Thus, although amoxicillin-clavulanate had a selective impact favoring continued carriage of RSSP, absolute risk of carrying non-susceptible lineages was unaffected by treatment. Conclusions: Antimicrobial treatment for AOM reduced penicillin-susceptible pneumococcal colonization but did not alter children9s risk of carrying resistant pneumococcal lineages.Background Concerns that antimicrobial treatment may foster selection and transmission of resistant bacterial lineages have led to conflicting guidelines for clinical management of common non-severe infections. However, the impact of antimicrobial treatment on colonization dynamics is poorly understood. We used data from a previously-conducted trial of amoxicillin-clavulanate therapy for acute otitis media (AOM) to understand how antimicrobial treatment impacts the acquisition and clearance of Streptococcus pneumoniae lineages with varying susceptibility to penicillin. Methods and findings We measured impacts of antimicrobial treatment on nasopharyngeal carriage of penicillin-susceptible S. pneumoniae (PSSP) and penicillin–non-susceptible S. pneumoniae (PNSP) lineages at end-of-treatment and 15d, 30d, and 60d after treatment in a previously-conducted randomized, double-blind, placebo-controlled trial. Analyses were not specified in the original protocol. Among children 6-35 months of age with stringently-defined AOM, 162 were assigned amoxicillin-clavulanate, and 160 were assigned placebo. Children who did not show clinical improvement received open-label antimicrobial rescue treatment with amoxicillin-clavulanate irrespective of the randomized treatment assignment, to which both patients and physicians were blinded. The intention-to-treat populations of the intervention and placebo arms thus received care resembling immediate antimicrobial therapy and watchful waiting, respectively. Immediate amoxicillin-clavulanate reduced PSSP carriage prevalence by 88% (95%CI: 76-96%) at end-of-treatment and by 27% (–3-49%) after 60d, but did not measurably alter PNSP carriage prevalence throughout follow-up. By end-of-treatment, 7% of children who carried PSSP at enrollment remained colonized in the amoxicillin-clavulanate arm, compared to 61% of PSSP carriers who received placebo; differences in carriage prevalence persisted at least 60d after treatment among children who carried PSSP at enrollment. Among children not carrying pneumococci at enrollment, amoxicillin-clavulanate reduced PSSP acquisition by >80% over 15d. Among children who carried PNSP at enrollment, no differences in carriage prevalence of S. pneumoniae, PSSP, or PNSP were detected at follow-up visits. Conclusions In a setting with low PNSP prevalence, antimicrobial therapy for AOM conferred a selective impact on colonizing S. pneumoniae by accelerating clearance, and delaying acquisition, of penicillin-susceptible lineages. Absolute risk of carrying PNSP was unaffected by treatment (ClinicalTrials.gov: NCT00299455; Funding: NIH/NIGMS).