Race has no impact on prostate cancer-specific mortality, when comparing patients with similar risk of other-cause mortality: An analysis of a population-based cohort.

BACKGROUND: Other-cause mortality (OCM) can serve as a surrogate for access-to-care. The authors sought to compare prostate cancer-specific mortality (PCSM) in Black versus White men matched based on their calculated OCM risk. METHODS: The Surveillance, Epidemiology, and End Results (SEER) database was queried for Black and White men diagnosed with prostate cancer between 2004 to 2009, to collect long-term follow-up. A Cox regression was used to calculate the OCM risk using all available covariates. This calculated OCM risk was used to construct a 1:1 propensity score matched (PSM) cohort. Then, a competing-risks multivariable tested the impact of race on PCSM. RESULTS: A total of 94,363 patients were identified, with 19,398 Black men and 74,965 White men. The median (IQR) follow-up was 11.3 years (9.8-12.8). In the unmatched-cohort at 10-years, PCSM and OCM were 5.5% versus 3.5% and 13.8% versus 8.4% in non-Hispanic Black (NHB) versus non-Hispanic White (NHW) patients (all p < .0001). The standardized mean difference was <0.15 for all covariates, indicating a good match. In the matched cohort at 10-years, OCM was 13.6% and 10.0% in NHB versus NHW (p < .0001), whereas the PCSM was 5.3% versus 4.7% (p < .01). On competing-risks multivariable analysis on PCSM, Black men had a hazard ratio of 1.08 (95% confidence interval, 0.98-1.20) compared to White men with a p = .13. CONCLUSIONS: The results of this study showed similar PCSM in Black and White patients, when matched with their calculated OCM risk. This report is the first to indicate at a population-based level that race has no impact on PCSM. PLAIN LANGUAGE SUMMARY: Prostate cancer is a very common cancer among men and it is associated with health disparities that disproportionately impact Black men compared to White men. There is an on-going discussion of whether disparities between these two groups stem from genetic or environmental factors. This study sought to examine if matching based on overall health status, a proxy for the impact of social determinants of health, mitigated significant differences in outcomes. When matched using risk of death from any cause other than prostate cancer, Black and White men had no significant differences in prostate cancer death.

Impact of lymphovascular invasion on survival in surgically treated upper tract urothelial carcinoma: a nationwide analysis.

OBJECTIVES: To assess the prognostic ability of lymphovascular invasion (LVI) in upper tract urothelial carcinoma (UTUC) as a predictor of overall survival (OS) using a large North American cohort. PATIENTS AND METHODS: Our cohort included 5940 patients with clinical M0 UTUC who underwent a radical nephroureterectomy (RNU), between 2010 and 2016, within the National Cancer Database. The main variable of interest was LVI status, and its interaction with pathological nodal (pN) status. Kaplan-Meier curves were used to depict the OS also stratifying patients on LVI status. Cox regression analysis tested the impact of LVI status on OS after accounting for the available covariates. RESULTS: The median (interquartile range [IQR]) age at diagnosis was 71 (63-78) years and most patients had pathological T1 stage disease (48.6%). Nodal status was pN0, pN1 and pNx in 45.8%, 6.3% and 47.9%, respectively. Overall, 22.1% had LVI. The median (IQR) follow-up time was 32.6 (16.0-53.3) months. At the 5-year postoperative follow-up, the estimated OS rate was 28% in patients with LVI vs 66% in those without LVI (P < 0.001). When patients were stratified based on nodal status those rates were 32% vs 68% in pN0 patients (P < 0.001), 23% vs 30% in pN1 patients (P = 0.8), and 28% vs 65% in pNx patients (P < 0.001). On multivariable analysis, the presence of LVI was associated with less favourable OS (hazard ratio 1.79, 95% confidence interval 1.60-1.99; P < 0.001). CONCLUSION: Our study assessed the impact of LVI on OS in patients with UTUC in a large North American nationwide cohort. Our series, as the largest to date, indicate that LVI is associated with less favourable survival outcomes in patients with UTUC after RNU, and this variable could be used in counselling patients about their prognosis and might be a useful tool for future trials to risk-stratify patients.

Comparison of patient background between a real-world North American cohort and the Göteborg-2 trial.

OBJECTIVES: To analyze the generalizability of the Göteborg-2 findings to a North American cohort. METHODS: We replicated the Göteborg-2 inclusion criteria in our Henry Ford Health (HFH) cohort, by identifying all patients 50-60 years old who had a PSA test from 2013 to 2018. The first PSA within the study period was considered PSA at entry, and included in the analysis. Chi-square test was used to compare categorical variables between the Göteborg-2 and HFH cohort, with a particular focus on Black men, who were also analyzed separately. RESULTS: The HFH patients included in the cohort were 49 456, of which 8562 were Black. In patients within the entire HFH cohort, HFH Black cohort, Göteborg Reference cohort, and Göteborg Experimental cohort, the rate of PSA ≥3 ng/mL was, respectively, 6.8%, 10.2%, 6.8%, and 6.6%. The rate of biopsy performed was, respectively, 1.8%, 4.1%, 5.8%, and 2.5%. PCa was found in, respectively, 1.4%, 3.0%, 2.3%, and 1.5%; Gleason score 3 + 3 in, respectively, 0.5%, 0.8%, 1.2%, and 0.6%; Gleason score > 3 + 3 in, respectively, 0.9%, 2.2%, 1.1%, and 0.9%. CONCLUSIONS: Our cohort had a lower biopsy rate and a lower incidence of non-csPCa diagnosis than both Göteborg cohorts, while still maintaining the same incidence of csPCa. This implies that the benefits of reducing non-csPCa diagnosis, as observed in the Experimental Göteborg cohort, are not necessarily replicable in U.S. "real-world practice" patients. Also noteworthy, we had a significantly higher percentage of Black men, who showed more aggressive disease.

Time to second biochemical recurrence as a prognostic indicator in postprostatectomy patients who undergo salvage radiation therapy: An RTOG 9601 based post hoc analysis.

INTRODUCTION AND OBJECTIVE: The prognostic significance of a "second" biochemical recurrence (sBCR) after salvage radiation therapy (sRT) with/without hormonal therapy following primary radical prostatectomy in men with prostate cancer has not been examined. We hypothesized that a shorter time to sBCR will be associated with worse cancer control outcomes. METHODS: The RTOG 9601 study included 760 patients with tumor stage pT2/T3, pN0, who had either persistently elevated prostate-specific antigen (PSA) postradical prostatectomy or developed subsequent biochemical recurrence with PSA levels between 0.2 and 4.0 ng/ml. All patients received sRT (with or without 2 years of Bicalutamide) from 1998 to 2015. For our study, we focused on 421 patients who had sBCR after sRT-which was defined as a PSA increase of at least 0.3 ng/ml over the first nadir. Patients were divided into two categories: early sBCR (n = 210) and late sBCR (n = 211) using median time to sBCR (3.51 years). All patients who experienced sBCR received salvage hormonal therapy. Competing-risk analysis was used to examine the impact of early versus late sBCR on prostate cancer specific mortality (CSM), after accounting for available covariates. RESULTS: The majority of patients were age 60 years or older (75.8%), had pT3 disease (74.8%), and Gleason score 7 (75.2%). Overall, 13.8% had persistent PSA initially after surgery. At 10 years, starting at the time of sBCR, CSM rate was 31.3% in the early sBCR group versus 20.0% in the late sBCR group. In competing-risk analysis, time to sBCR was an independent predictor of CSM, where patients with early sBCR had 1.7-fold higher CSM risk (p = 0.026) than their counterparts with late sBCR. CONCLUSIONS: Time to sBCR after sRT (with or without concomitant Bicalutamide) is a significant predictor of CSM following initial radical prostatectomy. This information can be used to guide subsequent treatments, and to counsel patients.

A state-wide information campaign during a pertussis epidemic in New South Wales, 2010.

Pertussis notifications increased dramatically in New South Wales in 2008, exceeding the rates in previous epidemic years. A state-wide, multi-faceted campaign was launched in March 2009 to provide information about pertussis prevention. A population-based survey was conducted using a Computer Assisted Telephone Interviewing facility to assess the effectiveness of sending letters to households with young infants. A representative sample of 1,200 adults across all 8 area health services was interviewed between July 2009 and September 2010, with responses weighted against the state population. Many respondents (39.7%) reported receiving the letter, while fewer (29.6%) reported receiving an adult pertussis booster in the last year, mostly in response to General Practitioner advice (40.4%). Letter receipt was associated with the uptake of an adult pertussis booster in the past 12 months by respondents (OR 5.8; 95%CI 4.1, 8.2) and other adults in the household (OR 5.1; 95%CI 3.5, 7.5), as well as knowledge about pertussis prevention. Health providers remain crucial for vaccination decision making; however letters may have contributed to an increased uptake of pertussis booster vaccination and knowledge. Health authorities may consider mailing households in future pertussis epidemics as a component of a wider communication strategy.

Assessment of the gender gap in urology industry payments: An Open Payments Program data analysis.

PURPOSE: The Open Payments Program (OPP), established in 2013 under the Sunshine Act, mandated medical device and pharmaceutical manufacturers to submit records of financial incentives given to physicians for public availability. The study aims to characterize the gap in real general and real research payments between man and woman urologists. MATERIALS AND METHODS: The study sample included all urologists in the United States who received at least one general or research payment in the OPP database from 2015 to 2021. Recipients were identified using the National Provider Identifier and National Downloadable File datasets. Payments were analyzed by geography, year, payment type, and years since graduation. Multivariable analysis on odds of being in above the median in terms of money received was done with gender as a covariate. This analysis was also completed for all academic urologists. RESULTS: There was a total of 15,980 urologists; 13.6% were woman, and 86.4% were man. Compared to man urologists, woman urologists were less likely to be in the top half of total payments received (odds ratio [OR] 0.62) when adjusted for other variables. When looking at academic urologists, 18.1% were woman and 81.9% were man. However, woman academic urologists were even less likely to be in the top 50% of payments received (OR 0.55). CONCLUSIONS: This study is the first to characterize the difference in industry payments between man and woman urologists. The results should be utilized to educate physicians and industry, in order to achieve equitable engagement and funding for woman urologists.

Adequacy of prostate cancer prevention and screening recommendations provided by an artificial intelligence-powered large language model.

PURPOSE: We aimed to assess the appropriateness of ChatGPT in providing answers related to prostate cancer (PCa) screening, comparing GPT-3.5 and GPT-4. METHODS: A committee of five reviewers designed 30 questions related to PCa screening, categorized into three difficulty levels. The questions were formulated identically for both GPTs three times, varying the prompts. Each reviewer assigned a score for accuracy, clarity, and conciseness. The readability was assessed by the Flesch Kincaid Grade (FKG) and Flesch Reading Ease (FRE). The mean scores were extracted and compared using the Wilcoxon test. We compared the readability across the three different prompts by ANOVA. RESULTS: In GPT-3.5 the mean score (SD) for accuracy, clarity, and conciseness was 1.5 (0.59), 1.7 (0.45), 1.7 (0.49), respectively for easy questions; 1.3 (0.67), 1.6 (0.69), 1.3 (0.65) for medium; 1.3 (0.62), 1.6 (0.56), 1.4 (0.56) for hard. In GPT-4 was 2.0 (0), 2.0 (0), 2.0 (0.14), respectively for easy questions; 1.7 (0.66), 1.8 (0.61), 1.7 (0.64) for medium; 2.0 (0.24), 1.8 (0.37), 1.9 (0.27) for hard. GPT-4 performed better for all three qualities and difficulty levels than GPT-3.5. The FKG mean for GPT-3.5 and GPT-4 answers were 12.8 (1.75) and 10.8 (1.72), respectively; the FRE for GPT-3.5 and GPT-4 was 37.3 (9.65) and 47.6 (9.88), respectively. The 2nd prompt has achieved better results in terms of clarity (all p < 0.05). CONCLUSIONS: GPT-4 displayed superior accuracy, clarity, conciseness, and readability than GPT-3.5. Though prompts influenced the quality response in both GPTs, their impact was significant only for clarity.

Testing the external validity of the POUT III trial (adjuvant platnium-based chemotherapy in upper tract urothelial carcinoma) in a North American cohort.

OBJECTIVE: The European POUT III randomized controlled trial provided level-one evidence that adjuvant platinum-based chemotherapy is the standard of care following nephroureterectomy (RNU) for locally invasive or node-positive upper tract urothelial carcinoma. We aim to assess this European randomized controlled trial's generalizability (external validity) to a North American cohort, using a nationwide database. MATERIALS AND METHODS: To compare trial patients with those seen in real-world practice, we simulated the trial inclusion criteria using data from the National Cancer Database (NCDB). We identified patients with histologically confirmed transitional cell carcinoma who underwent RNU. The available demographic characteristics of the NCDB cohort were compared with the POUT III trial cohort using Chi-squared test. RESULTS: The NCDB cohort (n = 3,380) had a significantly higher proportion of older patients (age ≥ 80: 23.5% vs. 5%), and more males (68% vs. 56.2%) than the POUT cohort (Table 1, both p < 0.001). Additionally, the rate of advanced nodal disease was higher in the NCDB (N1 9.6%, N2 9.3%) than in the POUT (N1 6%, N2 3%) cohort (p < 0.001). A more extensive lymph node dissection was performed in NCDB vs. POUT patients (node≥10 10.9% vs. 3%, p < 0.001). Sensitivity analysis removing all subjects with a Charlson Comorbidity Index > 0 did not change the significance of any results. CONCLUSIONS: While the primary disease stage was similar, the rate of advanced nodal disease was significantly higher in NCDB, which might be explained partially by the more extensive lymph node dissection performed in the latter. These differences warrant caution when applying the POUT III findings to North American patients.

Demonstration of the Managing for Daily Improvement Quality Improvement Methodology in an Alcohol Withdrawal Protocol Audit.

Background: The Clinical Institute Withdrawal Assessment for Alcohol, Revised (CIWA-Ar) is an assessment tool designed to standardize care and minimize the risk of complications in patients experiencing alcohol withdrawal. After discovering an increase in medication errors and late assessments under this protocol, pharmacists at a 218-bed community hospital performed an audit of protocol compliance using a performance improvement methodology known as Managing for Daily Improvement (MDI). Methods: A daily audit of CIWA-Ar protocol compliance was performed across all hospital units, followed by discussions with frontline nurses regarding barriers to compliance. The daily audit included assessments of appropriate monitoring frequency, medication administration, and medication coverage. Nurses caring for CIWA-Ar patients were interviewed to identify perceived barriers to protocol compliance. The MDI methodology provided a framework and tools to visualize audit results. The visual management tools used in the methodology include daily tracking of 1 or more discrete process measures, daily identification of barriers to perfect process performance at the patient and process level, and collaborative action plan tracking to resolve barriers. Results: Forty-one audits were collected for 21 unique patients over 8 days. After conversations with multiple nurses across different units, the most commonly reported barrier to compliance was a lack of communication at shift handoff. The results of this audit were discussed with nurse educators, patient safety and quality leaders, and frontline nurses. Process improvement opportunities identified from this data included improved widespread nursing education, development of protocol auto-discontinuation criteria based on scores, and determination of downtime processes for the protocol. Conclusion: The MDI quality tool successfully assisted in identifying end-user barriers to and focused areas of improvement of compliance with a nurse-driven CIWA-Ar protocol. This tool is elegant in its simplicity and ease of use. It can be customized to cover any timeframe or monitoring frequency while providing visualization of progress over time.

Assessing the impact of lymphovascular invasion on overall survival in surgically treated renal cell carcinoma patients: A nationwide cohort analysis.

INTRODUCTION: Lymph-vascular invasion (LVI) is recognized as an adverse pathological feature in patients with renal cell carcinoma (RCC). However, its impact on overall survival (OS) is not clear and scarcely addressed in the literature. We aimed to assess the prognostic ability of LVI as a predictor of OS in RCC patients using a large, North American cohort. METHODS: We included 95,783 cM0 RCC patients, diagnosed between 2010 and 2015, who underwent partial or radical nephrectomy within the National Cancer Database. Kaplan-Meier curves and log-rank tests were used to depict and compare survival curves. Cox regression analysis tested the impact of LVI on OS, after adjusting for all available confounders. RESULTS: Mean age (SD) was 59 (12), and most patients had pT1 stage (72.2%). Nodal status was pN0, pN1, and pNx, in 14.5%, 2.3%, and 83.3%, respectively. Overall, 9.0% of patients had LVI. The mean (SD) follow-up of the cohort was 39 months (24). At 5 years, OS was 65% in patients with LVI vs. 86% in patients without LVI (p<.0001). When patients were stratified based on nodal stage, these rates were 64% vs. 78% in pN0 patients, 31% vs. 41% in pN1 patients, and 69% vs. 87% in pNx patients (all P < 0.001). On multivariable analysis, and in comparison to patients without LVI, those with LVI had 1.37- (P < 0.001), 1.18- (P = 0.068), and 1.53-fold (P < 0.001) greater risk of death, when also harboring pN0, pN1, and pNx disease, respectively. CONCLUSIONS: Our findings are the first, to our best knowledge, to illustrate the clear detrimental impact of LVI on OS in surgically treated RCC patients. These findings might be useful in postoperative patient counseling and need to be accounted for when designing future clinical trials.

Trends in Prostate Cancer Screening in the Pre- and Peri-COVID-19 Pandemic Period.

INTRODUCTION: This study sought to examine PSA testing rates before, early in, and later in the COVID-19 pandemic. METHODS: Our cohort included test results from men >45 years who received PSA testing at least once at our institution from November 2018 to September 2021 and were alive at the end of that period. Monthly trends were evaluated for 3 periods: pre-COVID (November 2018-February 2020), early-COVID (March-May 2020), and late-COVID (June 2020-September 2021). Univariable and multivariable analysis tested the impact of these periods on PSA testing rate, after accounting for available confounders. All analyses were stratified by prostate cancer diagnosis status. RESULTS: A total of 141,777 PSA tests met inclusion criteria. The monthly number of tests in men without prostate cancer declined from 3,669 pre-COVID to 1,760 early-COVID (52% decrease; P = .0086) before increasing to 4,171 (14% increase from pre-COVID; P < .0001) late-COVID. The monthly average of first-time tests declined from 805 pre-COVID to 315 early-COVID (61% decrease; P = .008) before rebounding to 795 (1% decrease from pre-COVID; P = .7) late-COVID. The monthly number of tests in prostate cancer patients declined from 343 pre-COVID to 195 early-COVID (43% decrease; P = .008) before partially rebounding to 313 (9% decrease; P = .03) late-COVID. These differences remained within multivariable models. CONCLUSIONS: A number of men have forgone first-time PSA testing opportunities following the COVID-19 outbreak; thus, early cancer diagnoses in some individuals might have been missed. Likewise, many prostate cancer patients have forgone follow-up in the late-COVID period, which might compromise their oncologic outcomes.

Near-monodisperse poly(2-(methacryloyloxy)ethyl phosphorylcholine)-based macromonomers prepared by atom transfer radical polymerization and thiol-ene click chemistry: novel reactive steric stabilizers for aqueous emulsion polymerization.

Poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC) macromonomers have been prepared by the atom transfer radical polymerization (ATRP) of 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) using a bifunctional disulfide-based initiator. To attach a terminal polymerizable methacrylate group, the central disulfide bond was cleaved and the resulting thiols were conjugated to 3-(acryloyloxy)-2-hydroxypropyl methacrylate using tris(2-carboxyethyl)phosphine (TCEP) in water. Here TCEP serves as both the disulfide cleavage agent and also the catalyst for the subsequent Michael addition, which is highly selective for the acrylate group. The resulting methacrylate-terminated macromonomers were used as a reactive steric stabilizer for the aqueous emulsion polymerization of styrene, yielding near-monodisperse PMPC-stabilized polystyrene (PS) latexes of around 100-200 nm in diameter. As a comparison, the disulfide-containing PMPC homopolymer precursor and the intermediate thiol-functional PMPC homopolymer (PMPC-SH) were also evaluated as potential steric stabilizers. Interestingly, near-monodisperse latexes were also obtained in each case. These three sterically-stabilized latexes, prepared using either PMPC macromonomer, disulfide-based PMPC homopolymer, or PMPC-SH homopolymer as a reactive steric stabilizer, remained colloidally stable after both freeze-thaw experiments and the addition of an electrolyte, indicating that a coronal layer of PMPC chains prevented flocculation in each case. In contrast, both a charge-stabilized PS latex prepared in the absence of any steric stabilizer and a PS latex prepared in the presence of a nonfunctional PMPC homopolymer exhibited very poor colloidal stability when subjected to a freeze-thaw cycle or the addition of an electrolyte, as expected.

Phylogenetically distinct Staphylococcus aureus lineage prevalent among indigenous communities in northern Australia.

The aim was to determine the evolutionary position of the Staphylococcus aureus clonal complex 75 (CC75) that is prevalent in tropical northern Australia. Sequencing of gap, rpoB, sodA, tuf, and hsp60 and the multilocus sequence typing loci revealed a clear separation between conventional S. aureus and CC75 and significant diversity within CC75.

Assignment of Streptococcus agalactiae isolates to clonal complexes using a small set of single nucleotide polymorphisms.

BACKGROUND: Streptococcus agalactiae (Group B Streptococcus (GBS)) is an important human pathogen, particularly of newborns. Emerging evidence for a relationship between genotype and virulence has accentuated the need for efficient and well-defined typing methods. The objective of this study was to develop a single nucleotide polymorphism (SNP) based method for assigning GBS isolates to multilocus sequence typing (MLST)-defined clonal complexes. RESULTS: It was found that a SNP set derived from the MLST database on the basis of maximization of Simpsons Index of Diversity provided poor resolution and did not define groups concordant with the population structure as defined by eBURST analysis of the MLST database. This was interpreted as being a consequence of low diversity and high frequency horizontal gene transfer. Accordingly, a different approach to SNP identification was developed. This entailed use of the "Not-N" bioinformatic algorithm that identifies SNPs diagnostic for groups of known sequence variants, together with an empirical process of SNP testing. This yielded a four member SNP set that divides GBS into 10 groups that are concordant with the population structure. A fifth SNP was identified that increased the sensitivity for the clinically significant clonal complex 17 to 100%. Kinetic PCR methods for the interrogation of these SNPs were developed, and used to genotype 116 well characterized isolates. CONCLUSION: A five SNP method for dividing GBS into biologically valid groups has been developed. These SNPs are ideal for high throughput surveillance activities, and combining with more rapidly evolving loci when additional resolution is required.

Methicillin-resistant Staphylococcus aureus genotyping using a small set of polymorphisms.

The aim of this study was to identify a set of genetic polymorphisms that efficiently divides methicillin-resistant Staphylococcus aureus (MRSA) strains into groups consistent with the population structure. The rationale was that such polymorphisms could underpin rapid real-time PCR or low-density array-based methods for monitoring MRSA dissemination in a cost-effective manner. Previously, the authors devised a computerized method for identifying sets of single nucleotide polymorphisms (SNPs) with high resolving power that are defined by multilocus sequence typing (MLST) databases, and also developed a real-time PCR method for interrogating a seven-member SNP set for genotyping S. aureus. Here, it is shown that these seven SNPs efficiently resolve the major MRSA lineages and define 27 genotypes. The SNP-based genotypes are consistent with the MRSA population structure as defined by eBURST analysis. The capacity of binary markers to improve resolution was tested using 107 diverse MRSA isolates of Australian origin that encompass nine SNP-based genotypes. The addition of the virulence-associated genes cna, pvl and bbp/sdrE, and the integrated plasmids pT181, pI258 and pUB110, resolved the nine SNP-based genotypes into 21 combinatorial genotypes. Subtyping of the SCCmec locus revealed new SCCmec types and increased the number of combinatorial genotypes to 24. It was concluded that these polymorphisms provide a facile means of assigning MRSA isolates into well-recognized lineages.

An exploration into the relationships people with dementia have with physical objects: an ethnographic study.

In recent years there has been increasing interest in how different aspects of object relations theory might apply to dementia. While attachment theory in dementia has been well studied, there have been no systematic investigations of the way in which transitional objects are used by people with dementia. This study explores the relationship people with dementia have with physical objects using a focussed ethnographic method. Twenty-one residents and the staff of a care home for people with dementia were observed over a two-month period. Observations were recorded and analysed in light of Winnicott's criteria for transitional objects and incorporated the work of other key theorists. The ethnography found evidence that people with dementia have varied relationships with objects and can employ objects in a transitional way. The paper then explores the implications of this research for understanding the function of transitional objects for people with dementia. The findings suggest that that Winnicott's theory of transitional objects can provide a framework for understanding some of the processes of dementia.

High-resolution melting analysis of the spa repeat region of Staphylococcus aureus.

BACKGROUND: The staphylococcal protein A (spa) locus of Staphylococcus aureus contains a complex repeat structure and is commonly used for single-locus sequence-based genotyping. The real-time PCR platform supports genotyping methods that are single step and closed tube and potentially can be carried out simultaneously with diagnosis. We describe here a method for genotyping S. aureus using high-resolution melting (HRM) analysis of the spa polymorphic region X. METHODS: The conventional PCR spa assay was modified and optimized for the Rotor-Gene 6000 instrument (Corbett Life Science). HRM analysis on the Corbett Rotor-Gene 6000 instrument was used to test 22 known spa sequences obtained from 44 diverse methicillin-resistant S. aureus (MRSA) isolates. Criteria for calling pairs of melting curves "same" or "different" were developed empirically by converting the data to difference graph format with one curve defined as the control. HRM curve comparison between runs was done to determine the portability of the method. The assay performance was assessed by genotyping uncharacterized isolates, carrying out blind trials, and comparing HRM profiles from different runs. RESULTS: HRM analysis of 44 diverse MRSA isolates generated 20 profiles from 22 spa sequence types. The 2 unresolved HRM spa types differed by only 1 bp. Two blind trials demonstrated complete reproducibility with respect to calling the different spa types. Interrun comparisons of HRM curves were successfully developed, indicating the robustness of the method. CONCLUSION: Analysis of the spa locus by HRM resolves spa sequence variants. This single- and closed-tube single-step method for S. aureus genotyping can be easily combined with the interrogation of other genetic markers.

Systematic derivation of marker sets for staphylococcal cassette chromosome mec typing.

The aim of this study was to identify optimized sets of genotyping targets for the staphylococcal cassette chromosome mec (SCCmec). We analyzed the gene contents of 46 SCCmec variants in order to identify minimal subsets of targets that provide useful resolution. This was achieved by firstly identifying and characterizing each available SCCmec element based on the presence or absence of 34 binary targets. This information was used as input for the software "Minimum SNPs," which identifies the minimum number of targets required to differentiate a set of genotypes up to a predefined Simpson's index of diversity (D) value. It was determined that 22 of the 34 targets were required to genotype the 46 SCCmec variants to a D of 1. The first 6, 9, 12, and 15 targets were found to define 21, 29, 35, and 39 SCCmec variants, respectively. The genotypes defined by these marker subsets were largely consistent with the relationships between SCCmec variants and the accepted nomenclature. Consistency was made virtually complete by forcing the computer program to include ccr1 and ccr5 in the target set. An alternative target set biased towards discriminating abundant SCCmec variants was derived by analyzing an input file in which common SCCmec variants were repeated, thus ensuring that markers that discriminate abundant variants had a large effect on D. Finally, it was determined that mecA single nucleotide polymorphisms (SNPs) can increase the overall genotyping resolution, as different mecA alleles were found in otherwise identical SCCmec variants.

Use of a single-nucleotide polymorphism genotyping system to demonstrate the unique epidemiology of methicillin-resistant Staphylococcus aureus in remote aboriginal communities.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has emerged as a major public health problem in Australia, as in many other parts of the world. High rates of CA-MRSA skin and soft tissue infection have been reported from Aboriginal communities. We used a single-nucleotide polymorphism (SNP) genotyping typing system based on the multilocus sequence type (MLST) database to investigate the epidemiology of CA-MRSA and methicillin-sensitive S. aureus (MSSA) over a 12-month period in three remote Aboriginal communities of Northern Australia. This was supplemented by real-time PCR for Panton-Valentine leukocidin (PVL) genes, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial susceptibility testing. S. aureus was recovered from pyoderma lesions on 221 occasions and throat swabs on 44 occasions. The median monthly recovery rate of S. aureus from skin sores was 58% (interquartile range, 62 to 78%), and there was no seasonal variation. Twenty-three percent of isolates were CA-MRSA; the proportion was similar across the communities and did not vary over the study period. Erythromycin resistance was found in 47% of CA-MRSA and 21% of MSSA. SNP-based typing identified 14 different clonal complexes (cc); however, cc75 was predominant, accounting for 71% of CA-MRSA isolates. These were confirmed as ST75-like by using an additional SNP and MLST of selected isolates. All but one of the cc75 isolates had SSCmec type IV (one had type V), and all were PVL negative. Monthly tracking of SNP-based cc types showed a highly dynamic process. ST75-MRSA-IV appears to be unique to the region and probably evolved de novo in remote Aboriginal communities.