It is well known that some isolates of Staphylococcus aureus produce pathogenic toxin, Panton-Valentine leukocidin (PVL), and that the toxin has been reported to be highly associated with community acquired methicillin resistant S. aureus (CA-MRSA). Currently, the PCR method using specific primers for the PVL gene (LukS-PV-lukF-PV) have been widely used to detect PVL. In this study, we evaluated the PVL-RPLA "Seiken", diagnostic reagent based on a reserved passive latex agglutination reaction with a specific monoclonal antibody for detecting PVL. A total of 630 clinical isolates were used. PCR method detected 34 PVL-positive (28 MRSA and 6 MSSA), and, of these, PVL-RPLA "Seiken" read positive for 32 isolates (27 MRSA and 5 MSSA), the result indicating two false negative occurrences. The concordance rate was 99.7%. In addition the recommended BHI broth, CCY medium, Dolman broth and Todd-Hewitt broth were applied for toxin preparation media. Toxin concentration produced in CCY medium was significantly higher than those in the remaining culture medium (p < 0.05). PVL-RPLA "Seiken" is a method for detecting the PVL in the culture broth by antigen antibody reaction after an overnight shaking culture. This method does not require any expensive equipments or facilities. Thus this reagent provides us with rapid, easy-to-perform, less expensive test method to detect PVL in clinical microbiology laboratories.
Heteromorphic sex-determining regions or mating-type loci can contain large regions of non-recombining sequence where selection operates under different constraints than in freely recombining autosomal regions. Detailed studies of these non-recombining regions can provide insights into how genes are gained and lost, and how genetic isolation is maintained between mating haplotypes or sex chromosomes. The Chlamydomonas reinhardtii mating-type locus (MT) is a complex polygenic region characterized by sequence rearrangements and suppressed recombination between its two haplotypes, MT+ and MT-. We used new sequence information to redefine the genetic contents of MT and found repeated translocations from autosomes as well as sexually controlled expression patterns for several newly identified genes. We examined sequence diversity of MT genes from wild isolates of C. reinhardtii to investigate the impacts of recombination suppression. Our population data revealed two previously unreported types of genetic exchange in Chlamydomonas MT--gene conversion in the rearranged domains, and crossover exchanges in flanking domains--both of which contribute to maintenance of genetic homogeneity between haplotypes. To investigate the cause of blocked recombination in MT we assessed recombination rates in crosses where the parents were homozygous at MT. While normal recombination was restored in MT+ ×MT+ crosses, it was still suppressed in MT- ×MT- crosses. These data revealed an underlying asymmetry in the two MT haplotypes and suggest that sequence rearrangements are insufficient to fully account for recombination suppression. Together our findings reveal new evolutionary dynamics for mating loci and have implications for the evolution of heteromorphic sex chromosomes and other non-recombining genomic regions.
We experienced hospital-acquired infection in March 2008 that three nurses became infected with Panton-Valentine leukocidin (PVL)-positive methicillin-resistant Staphylococcus aureus (MRSA). Accordingly, we performed the retrospective study to determine the prevalence of PVL-positive S. aureus in Okinawa. A total of 731 clinical isolates, consisting of 600 MRSA and 131 methicillin-susceptible isolates in Okinawa, were included. Of the isolates, 16 were positive for PVL gene (lukS-PV-lukF-PV). All the PVL-positive isolates were MRSA, and the first appeared in March 2008. The isolates from the University Hospital were characterized as staphylococcal chromosomal cassette mec type IVa. Through the analysis of pulsed-field gel electrophoresis (PFGE), 16 PVL-positive MRSA isolates were divided in three groups. One isolate (the first group) from the other hospital was less similar (< 40% similarity) when compared with the remaining 15 isolates from the University Hospital. The second group consisted of two respective paired isolates from the same department wards, and those were very similar with each other, indicating possible patient-to-patient transmission. The 11 isolates were characterized as the third group with >80% similarity. The DiversiLab system (bioMérieux) based on repetitive-sequence-based PCR typing demonstrated that the isolates of the third group were similar and indistinguishable with the strains of USA300 clone. However, the first and second groups were not determinable which USA clone was the origin. With these, we could conclude that the PVL-positive MRSA close to USA300 clone first appeared in Okinawa in 2008 and is now becoming prevalent multi-focally. Also, person-to-person transmission is already likely in a hospital setting.
In response to the revision of social medical insurance policy, in which hospital clinics can additionally charge for laboratory testing when the test results are presented to an outpatient in a print-out form on a visiting day, we evaluated laboratory-spending times, so-called turnaround times (TATs). A total of 14,802 outpatients during the period from October 2010 to May 2011 were enrolled. TATs from venipuncture accession to completing blood collection revealed a log-normal distribution with 5 to 6 min of mode and 10(0.95 +/- 0.26) (4.90 to 16.2) min of mean +/- standard deviation. Order waiting time figured a half-normal distribution, 50% tile and 90%-tile being 4 and 16 min, respectively. TATs of blood collection and order waiting time were significantly influenced by days of the week and accession time. Through analysis of TATs from specimen receipt to reporting test results, it became apparent that the tests determined by immunoassay and erythrocyte sedimentation rate (ESR) required more minutes when compared to the remaining tests. Total TATs from venipuncture accession to reporting test results ranged 28 to 29 min (50%-tile) for complete blood count and hemoglobin A1c, whereas those of endocrinology and tumor markers were 65 to 73 min. In conclusion, the tests determined by immunoassay are rate-limiting for rapid reporting efforts in clinical laboratories. Secondly, TATs of blood collection are mostly influenced by order waiting time depending on days of the week and accession time. At present, there is no target value for TATs, however it is important to recognize the necessity to shorten laboratory-spending TATs.
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