Comparison of AP-PCR typing and PCR-ribotyping for estimation of nosocomial transmission of Clostridium difficile.

We recently attempted to clarify an increased incidence of Clostridium difficile-associated diarrhoea (CDAD) in our hospital by arbitrarily primed polymerase chain reaction (AP-PCR) typing of isolates from 147 consecutive patients collected during a 12 month period (Wullt et al. J Hosp Infect 1999;43:265-273). In the present study we compared the results based on previous AP-PCR data with those based on recent PCR ribotyping of the same isolates and re-analysis of a subset of isolates by AP-PCR typing. The pattern of PCR ribotypes was similar among inpatients and outpatients. A cluster of three closely related PCR ribotypes, related to those of the serogroup H and A8 type strains, dominated and comprised 31% of inpatient and 28% of outpatient C. difficile isolates. The apparent nosocomial transmission rate among inpatients with CDAD was only 9% by AP-PCR typing compared with 18 or 36% by PCR ribotyping depending on the definition used (proportion of patients sharing C. difficile type and ward within two or 12 months). Corresponding rates for all CDAD patients were 5% by AP-PCR and 11 or 21% by PCR ribotyping. Thus, most CDAD patients apparently became ill due to their endogenous strain of C. difficile. Because of the low concordance between the two typing methods the proportion of patients fulfilling the criteria for nosocomial transmission by both methods was only 1%. Re-examination of isolates from patients with recurrences revealed a reproducibility problem with AP-PCR typing. We conclude, that of these two PCR-based options for typing of C. difficile PCR ribotyping offers a superior experimental robustness compared with AP-PCR typing.

Carriage of penicillin-resistant Streptococcus pneumoniae by children in day-care centers during an intervention program in Malmo, Sweden.

BACKGROUND: An increasing incidence of penicillin-resistant Streptococcus pneumoniae (PRP) was detected in Malmo in 1994. OBJECTIVE: To evaluate clonality and factors facilitating the spread of PRP among children in day-care centers (DCCs). METHODS: We used phenotypic and DNA-fingerprinting methods in conjunction with epidemiologic data from the South Swedish Pneumococcal Intervention Project's investigation of 63 DCCs during a 3-year period (1995 to 1997) in the Malmo region. RESULTS: A questionnaire about building and hygiene standards disclosed no statistically significant risk factor for carriage of pneumococci. However, age younger than the mean age at the DCC or in the child group was positively associated with carriage. Contrary to expectations no association with the number of children, either at the DCC or in the individual groups, was found. Of 2912 investigated children 1224 (42%) were carriers of S. pneumoniae, and 373 (12.8%) were PRP carriers (MIC > or = 0.1 microg/ml). Among isolates with MIC > or = 0.5 microg/ml 9 serogroups and 30 genetic types were found. Two clones in serogroups 9 (33%) and 19 (24%) were dominant in most municipality districts, and dominance was sustained during the whole study period. The previously internationally recognized serotype 9V clone seemed to be very stable, with a single DNA type and resistance pattern during the study period. In contrast the serogroup 19 isolates and other serogroups had diverse DNA types and resistance patterns, supporting the hypothesis that DCCs have a unique microenvironment facilitating the recombination of penicillin-binding protein genes among streptococci. In five DCCs we found PRP isolates with two different serogroups but an identical genetic type, indicating that serotype shift may be a common phenomenon in DCCs. CONCLUSION: Multivariate logistic regression of risk factors disclosed that young age of the children in the child groups was a significant risk factor for carriage of S. pneumoniae.

Several different clones present during the penetration phase of resistant Streptococcus pneumoniae in the city of Malmö, Sweden.

A rapid increase in the prevalence of Streptococcus pneumoniae with reduced sensitivity to penicillin (MIC > or = 0.12 microgram/mL) was noted among clinical isolates during a 15-month period in 1994-1995 in the city of Malmö, Sweden. All first-time clinical isolates (n = 178) were consecutively collected and investigated for genetic relatedness with BOX-A PCR and arbitrarily primed (AP) PCR. An improved method for chromosomal DNA extraction and the use of three reliable discriminatory primers for AP-PCR of S. pneumoniae are described. Using molecular fingerprinting, 30 different genotypes were discerned among the 178 isolates. The majority (87%) of isolates belonged to serogroups 6, 9, 15, 19, and 23. Resistance patterns and serogrouping indicated the presence of at least three major phenotypic clones. DNA fingerprinting in conjunction with minimum inhibitory concentration (MIC) values, resistance patterns, and epidemiological data confirmed the existence of two clones belonging to serogroups 6 and 9. Of the 178 isolates, 82% were from children of preschool age, most of whom attended day-care centers. We conclude that abundant S. pneumoniae strains with MICs > or = 0.12 microgram/mL for penicillin were present in the city of Malmö during the study period. At least two genetically discrete clones causing clinical illness were identified, and attendance at day-care centers may be a major factor in the spread of these strains.

Low prevalence of nosocomial Clostridium difficile transmission, as determined by comparison of arbitrarily primed PCR and epidemiological data.

An increased prevalence of patients with C. difficile-associated diarrhoea in a hospital setting suggested the possible existence of an endemic occurrence. A study was therefore designed to determine clonal relatedness among 173 isolates of C. difficile, collected consecutively during 1995 from 147 patients (89 inpatients and 58 outpatients) and to estimate the probability of nosocomial transmission. Arbitrarily primed PCR (AP-PCR) with three different primers, AP1, AP2 and CLD1, was used for fingerprinting and identified 21, 92 and 70 types, respectively. Overall DNA analysis of the combined AP-PCR data yielded 140 types, of which 130 were unique, whereas 10 types occurred repeatedly in 36 isolates from 33 patients; seven isolates were non-typeable by one of the primers. Epidemiological data confirmed that in eight of the 33 patients there was a high probability of nosocomial transmission. Despite a high prevalence of C. difficile among hospitalized patients, a low frequency of nosocomial transmission was suggested by high resolution molecular typing of bacterial isolates in conjunction with traditional epidemiological methods.