Nosocomial Staphylococcal scalded skin syndrome caused by intra-articular injection.

BACKGROUND: The pathogenic role of nasal carriage as a source for cutaneous and soft-tissue Staphylococcus aureus (SA) infections, and Staphylococcal scalded skin syndrome (SSSS) in particular, is unclear. OBSERVATION: We herein describe a nosocomial outbreak of SSSS in three orthopaedic patients who received intra-articular injections by a single orthopaedic surgeon. Bacteriological samples from the index patients and medical personnel involved in their care were assessed by phage typing, polymerase chain reaction for exfoliative toxin genes, SmaI macro-restriction analysis and molecular spa-typing. These studies first revealed SA cultural growth in synovial fluid of all three patients as well as nasal mucosa of one medical assistant. Moreover, all SA isolates had the same phage typing and antibiotic susceptibilities and were positive for exfoliative toxin ETa by polymerase chain reaction. SmaI macro-restriction and spa-typing further confirmed all proband isolates to be identical. CONCLUSION: These findings provide evidence that SA nasal colonization of otherwise healthy carriers is a risk factor for SA infections, including SSSS, in predisposed individuals.

Introducing a fast and simple PCR-RFLP analysis for the detection of mutant thiopurine S-methyltransferase alleles TPMT*3A and TPMT*3C.

BACKGROUND: Azathioprine, in combination with corticosteroids, is the first-line therapy of severe forms of pemphigus vulgaris. Patients with an impaired thiopurine S-methyltransferase (TPMT) activity are at risk of developing severe myelo-suppression upon treatment with thiopurines such as azathioprine. Analysis of the TPMT status prior to drug administration is therefore highly recommended. However, because of the limited availability of TPMT testing outside of specialized centres, pre-emptive TPMT testing is not widespread. To avoid laborious biochemical and sequencing assays, we evaluated a new restriction fragment length polymorphism (RFLP) analysis. METHODS: We designed a rapid genetic polymerase chain reaction (PCR)-RFLP screen for the most prevalent mutant TPMT*3A and TPMT*3C alleles that are known to result in reduced TPMT enzyme activity. RESULTS: Validating our fast system on 871 Caucasian DNA samples, we observed that 8.61% of our probands carried the TPMT*3A allele and 0.23% were heterozygous for the TPMT*3C allele, which is in concordance with previously reported allele frequencies. CONCLUSION: This simple and low-cost PCR-RFLP TPMT polymorphism testing approach can be performed in a standard laboratory. It should be applied to all patients prior to receiving thiopurine drug therapy to avoid the severe, but predictable, haematopoietic side-effects of thiopurine drug administration.

Relative quantitation of protein-protein interaction strength within the yeast two-hybrid system via fluorescence beta-galactosidase activity detection in a high-throughput and low-cost manner.

The yeast two-hybrid (Y2H) method is capable of delivering vast amounts of interacting positive yeast colonies from a single library screen, particularly if a multifunctional protein is used as bait. However, the selection of definitive colonies for further molecular analysis is limited by both technical practicality and high costs. Here we demonstrate a cost-effective and simple method for the rapid selection and ranking of those Y2H-positive interaction clones that are suitable for further analysis. We performed a Y2H screen for the identification of human transforming growth factor beta2- interacting proteins in a human skin keratinocyte library. The identified clones were ranked by the amount of beta-galactosidase enzyme produced, as well as by the interaction strength of the positive colonies. The combination of high-throughput microplate fluorescence readers and specific fluorescence assays can be utilized for relative quantitation of protein-protein interaction strength of Y2H-positive colonies in crude yeast-cell lysates. We demonstrate here that the high sensitivity of the fluorescence approach can bypass cumbersome conventional methods of cell lysis used in beta-galactosidase assays, and still deliver accurate values for analysis of protein interaction data. Finally, we also achieved a better understanding of general aspects of beta-galactosidase measurements in the Y2H system, such as protein normalization, the influence of yeast culture incubation time on optimal beta-galactosidase detection, and the linearity of beta-galactosidase detection in crude cell lysates.