Detection of an outbreak of methicillin-resistant Staphylococcus aureus with reduced susceptibility to glycopeptides in a French hospital.

Staphylococcus aureus isolates were screened for reduced susceptibility to glycopeptides with an initial glycopeptide agar screening test, followed by confirmation of the strains thus identified by two Etest strip techniques and population analysis. This procedure detected 48 methicillin-resistant S. aureus (MRSA) isolates with reduced susceptibility to glycopeptides from 24 patients among 883 MRSA isolates tested. The dissemination of a single clone was confirmed by pulsed-field gel electrophoresis.

[Value of an antibiotic lock for the prevention of bacteremia recurrence from central catheters in chronic hemodialysis]. / Intérêt d'un verrou antibiotique pour la prévention des récurrences de bactériémies à point de départ des cathéters centraux en hémodialyse chronique.

Prospectively since 11/1997, all central venous catheter related bacteremias in our dialysis center (n = 60) was recorded. We systematically tested antibiotic lock technique using pure heparin (1 ml = 5000 Ul) mixed with antibiotic matched to isolated micro-organism after 15 days of systemic antibiotherapy. During 39 months of study, 27 bacteremias were documented from 23 patients. Seventeen locks in 15 patients were performed after each dialysis sessions during one month. Associated tunnel infection did not allow to stop the lock in 3 cases. In the 12 remaining patients, we observed 4 recurrences for 3 patients after the stop of the lock with the same micro-organism in 3 times/4 without modifications of antibiotics sensibility. No septic metastases were notified and the patency of all catheters were respected. The incidence of bacteremias was 4.6 per 1000 catheters days before the lock and 0.88 after, during a mean observation period of 15 months per patients. Sterilisation of infected catheters seems possible and the incidence of bacteremias is reduce by the lock technique without coming out of septic complications or selected micro-organisms.

Detection of Pneumocystis carinii DNA in blood specimens from human immunodeficiency virus-infected patients by nested PCR.

The detection of Pneumocystis carinii DNA in blood by PCR could be useful for studying the natural history of pneumocystosis and could also be a noninvasive diagnostic method. The results of previous studies are nevertheless conflicting. In our study, we compared three commercially available DNA extraction kits (GeneReleaser, QIAamp Tissue Kit, and ReadyAmp Genomic DNA Purification System) and proteinase K and proteinase K-phenol-chloroform treatments for the extraction of P. carinii DNA from dilutions of a P. carinii f. sp. hominis cyst suspension mixed with human whole blood. A rapid and simple nested PCR protocol which amplifies a portion of the mitochondrial large-subunit rRNA gene was applied to all the extraction products. The QIAmp Tissue Kit was the most effective kit for the isolation of amplification-ready P. carinii DNA and was used with nested PCR for the testing of whole-blood specimens from 35 immunocompetent control patients and 84 human immunodeficiency virus (HIV)-infected patients investigated for pulmonary disease and/or fever. In HIV-infected patients, P. carinii DNA was detected by nested PCR in blood samples from 3 of 14 patients with microscopically proven P. carinii pneumonia, 7 of 22 patients who were considered to be colonized with P. carinii, and 9 of 48 patients who were neither infected nor colonized with P. carinii. P. carinii DNA was not detected in blood specimens from the 35 immunocompetent patients. P. carinii DNA in blood might represent viable P. carinii organisms or DNA complexes released from pulmonary phagocytes. In conclusion, P. carinii DNA may be detected in whole blood from HIV-infected patients, but the nature and the meaning of the circulating form of P. carinii remain to be established.

Rapid detection of Pneumocystis carinii in bronchoalveolar lavage specimens from human immunodeficiency virus-infected patients: use of a simple DNA extraction procedure and nested PCR.

We report on the development of a rapid nested PCR protocol for the detection of Pneumocystis carinii DNA in bronchoalveolar lavage (BAL) specimens in which the protocol included the use of a commercially available DNA extraction kit (GeneReleaser). GeneReleaser enabled us to obtain amplification-ready DNA within 20 min without requiring the purification of the DNA. The nested PCR was performed with the primers pAZ102-E, pAZ102-H, and pAZ102-L2 (A. E. Wakefield, F. J. Pixley, S. Banerji, K. Sinclair, R. F. Miller, E. R. Moxon, and J. M. Hopkin, Lancet 336:451-453, 1990.). Results were obtained in about 4 h with the adoption of denaturation, annealing, and extension steps shortened to 20 seconds. The sensitivity of the nested PCR was tested with a P. carinii cyst suspension and was found to be less than one cyst (one to eight nuclei). The detection limit was the same with the use of GeneReleaser or proteinase K-phenol chloroform for DNA extraction. The nested PCR assay was prospectively compared with staining with Giemsa and methenamine silver stains for the detection of P. carinii in 127 BAL samples from 105 human immunodeficiency virus-infected patients investigated for acute respiratory illness. Twenty-five BAL specimens (20%) were positive by staining and the nested PCR and 25 (20%) were negative by staining and positive by the nested PCR. These 25 BAL specimens with conflicting results were obtained from 23 patients, 82% of whom were receiving prophylactic therapy against P. carinii pneumonia (PCP). Only two patients were diagnosed with possible PCP. The final diagnosis was not PCP for 20 patients who were considered to be colonized or to have a low level of infection. This colonization is not of clinical importance but is of epidemiological importance. Our rapid, simple, and sensitive amplification protocol may be performed in clinical laboratories for the routine diagnosis of PCP with BAL specimens.