The Dissemination and Molecular Characterization of Clonal Complex 361 (CC361) Methicillin-Resistant Staphylococcus aureus (MRSA) in Kuwait Hospitals.

Methicillin-resistant Staphylococcus aureus (MRSA) belonging to clonal complex 361 (CC361-MRSA) is rare among patients' populations globally. However, CC361-MRSA has been isolated with an increasing trend among patients in Kuwait hospitals since 2010. This study investigated the molecular characteristics of CC361-MRSA isolated from patients in Kuwait hospitals in 2016-2018 to understand their genetic relatedness and virulence determinants. Of 5,223 MRSA isolates investigated by DNA microarray, 182 (3.4%) isolates obtained in 2016 (N = 55), 2017 (N = 56), and 2018 (N = 71) were identified as CC361-MRSA. The CC361-MRSA isolates were analyzed further using antibiogram, spa typing and multi locus sequence typing (MLST). Most of the isolates were resistant to fusidic acid (64.8%), kanamycin (43.4%), erythromycin (36.3%), and clindamycin (14.3%) encoded by fusC, aphA3, and erm(B)/erm(C) respectively. Nine isolates (4.9%) were resistant to linezolid mediated by cfr. The isolates belonged to 22 spa types with t3841 (N = 113), t315 (N = 16), t1309 (N = 14), and t3175 (N = 5) constituting 81.3% of the spa types, four genotypes (strain types), CC361-MRSA-[V/VT + fus] (N = 112), CC361-MRSA-IV, WA MRSA-29 (N = 36), CC361-MRSA-V, WA MRSA-70/110 (N = 33) and CC361-MRSA-[V + fus] variant (N = 1). MLST conducted on 69 representative isolates yielded two sequence types: ST361 (11/69) and ST672 (58/69). All CC361-MRSA isolates were positive for cap8, agr1, and the enterotoxin egc gene cluster (seg, sei, selm, seln, selo, and selu). The tst1 was detected in 19 isolates. The immune evasion cluster (IEC) genes type B (scn, chp, and sak) and type E (scn and sak) were detected in 20 and 152 isolates, respectively. The CC361-MRSA circulating in Kuwait hospitals consisted of two closely related sequence types, ST361 and ST672 with ST672-MRSA [V/VT + fus] as the dominant genotype. The dissemination of these newly emerged clones and the emergence of linezolid resistance limits therapeutic options, as well as present significant challenges for the control of MRSA infections in Kuwait hospitals.

Development of a miniaturized protein microarray as a new serological IgG screening test for zoonotic agents and production diseases in pigs.

In order to monitor the occurrence of zoonotic agents in pig herds as well as to improve herd health management, the development of new cost-effective diagnostic methods for pigs is necessary. In this study, a protein microarray-based assay for the simultaneous detection of immunoglobulin G (IgG) antibodies against different zoonotic agents and pathogens causing production diseases in pigs was developed. Therefore, antigens of ten different important swine pathogens (Toxoplasma gondii, Yersinia enterocolitica, Salmonella spp., Trichinella spp., Mycobacterium avium, Hepatitis E virus, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, the porcine reproductive and respiratory syndrome virus, Influenza A virus) were spotted and covalently immobilized as 'antigen-spots' on microarray chips in order to test pig serum for the occurrence of antibodies. Pig serum was sampled at three German abattoirs and ELISA tests for the different pathogens were conducted with the purpose of creating a panel of reference samples for microarray analysis. To evaluate the accuracy of the antigens on the microarray, receiver operating characteristic (ROC) curve analysis using the ELISA test results as reference was performed for the different antigens. High area under curve values were achieved for the antigens of two zoonotic agents: Toxoplasma gondii (0.91), Yersinia enterocolitica (0.97) and for three production diseases: Actinobacillus pleuropneumoniae (0.77), Mycoplasma hyopneumoniae (0.94) and the porcine reproductive and respiratory syndrome virus (0.87). With the help of the newly developed microarray assay, collecting data on the occurrence of antibodies against zoonotic agents and production diseases in pig herds could be minimized to one measurement, resulting in an efficient screening test.

Fast, economic and simultaneous identification of clinically relevant Gram-negative species with multiplex real-time PCR.

AIM: A newly designed multiplex real-time PCR (rt-PCR) was validated to detect four clinically relevant Gram-negative bacteria (Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa). MATERIALS & METHODS: Serial dilutions of genomic DNA were used to determine the limit of detection. Colony PCR was performed with isolates of the four selected species and other species as negative controls. Isolates were characterized genotypically and phenotypically to evaluate the assay. RESULTS: Specific signals of all target genes were detected with diluted templates comprising ten genomic equivalents. Using colony rt-PCR, all isolates of the target species were identified correctly. All negative control isolates were negative. CONCLUSION: The genes gad, basC, khe and ecfX can reliably identify these four species via multiplex colony rt-PCR.

The impact of Skp2 overexpression on recurrence-free survival following radical prostatectomy.

BACKGROUND: In several human cancers, overexpression of Skp2 (S-phase kinase associated protein 2), which targets p27 for degradation, portends a poorer prognosis. We examined whether Skp2 overexpression is associated with recurrence following radical prostatectomy (RP) for prostate cancer. METHODS: Immunohistochemical staining for Skp2, p27, and MIB-1 was performed on 109 men with node-negative prostate cancer surgically managed from 1985-1996. Associations between the stains were tested and Cox regression was used to determine the association between Skp2 expression and time to biochemical recurrence following RP. RESULTS: The 12 tumors (11%) with Skp2 overexpression all had correspondingly low p27 expression (P=0.006), and a similar inverse Skp2/p27 relationship was seen in vitro in LNCap cells. Skp2 overexpression in tissue was associated with higher Gleason score (P=0.002), more advanced pathological stage (P=0.01), and higher MIB-1 index (P=0.03), but a more favorable PSA profile (P=0.04). Five men received a TURP. Among 104 who received RP, median follow-up was 67 months (range: 0.2-218). After adjusting for PSA, pathologic stage, and Gleason score, Skp2 overexpression remained significantly associated with a shorter time to biochemical recurrence (adjusted hazard ratio 4.8 (95% C.I. 1.6-14, P=0.004)). The median time to recurrence with high vs. low Skp2 was 4 vs. 54 months. CONCLUSIONS: Skp2 overexpression was seen in a significant minority of surgically-managed men and was independently associated with a higher risk of recurrence, raising the possibility that Skp2 could be useful as a prognostic biomarker and as a potential molecular target for novel systemic agents in prostate cancer.

Endothelial NOS, estrogen receptor beta, and HIFs cooperate in the activation of a prognostic transcriptional pattern in aggressive human prostate cancer.

The identification of biomarkers that distinguish between aggressive and indolent forms of prostate cancer (PCa) is crucial for diagnosis and treatment. In this study, we used cultured cells derived from prostate tissue from patients with PCa to define a molecular mechanism underlying the most aggressive form of PCa that involves the functional activation of eNOS and HIFs in association with estrogen receptor beta (ERbeta). Cells from patients with poor prognosis exhibited a constitutively hypoxic phenotype and increased NO production. Upon estrogen treatment, formation of ERbeta/eNOS, ERbeta/HIF-1alpha, or ERbeta/HIF-2alpha combinatorial complexes led to chromatin remodeling and transcriptional induction of prognostic genes. Tissue microarray analysis, using an independent cohort of patients, established a hierarchical predictive power for these proteins, with expression of eNOS plus ERbeta and nuclear eNOS plus HIF-2alpha being the most relevant indicators of adverse clinical outcome. Genetic or pharmacologic modulation of eNOS expression and activity resulted in reciprocal conversion of the transcriptional signature in cells from patients with bad or good outcome, respectively, highlighting the relevance of eNOS in PCa progression. Our work has considerable clinical relevance, since it may enable the earlier diagnosis of aggressive PCa through routine biopsy assessment of eNOS, ERbeta, and HIF-2alpha expression. Furthermore, proposing eNOS as a therapeutic target fosters innovative therapies for PCa with NO inhibitors, which are employed in preclinical trials in non-oncological diseases.

p27Kip1 in stage III colon cancer: implications for outcome following adjuvant chemotherapy in cancer and leukemia group B protocol 89803.

BACKGROUND: In retrospective studies, loss of p27(Kip1) (p27), a cyclin-dependent kinase inhibitor, has been associated with poor prognosis following colorectal cancer treatment. In a prospective study, we validated this relationship in patients enrolled on a trial of adjuvant chemotherapy for stage III colon cancer. METHODS: Cancer and Leukemia Group B protocol 89803 randomized 1,264 stage III colon cancer patients to receive weekly bolus 5-fluorouracil/leucovorin or weekly bolus irinotecan, 5-fluorouracil, and leucovorin (IFL). The primary endpoint was overall survival (OS); disease-free survival was a secondary endpoint. Expression of p27 and DNA mismatch repair proteins were determined by immunohistochemistry in primary tumor and normal tissue from paraffin blocks. Data were analyzed using log-rank test. RESULTS: Of 601 tumors analyzed, 207 (34.4%) showed p27 loss, 377 (62.8%) retained p27, and 17 (2.8%) were indeterminate. Patients with p27-negative tumors showed reduced OS [5-year OS 66%: 95% confidence interval (95% CI), 0.59-0.72 versus 75%: 95% CI, 0.70-0.79; log-rank P = 0.021]. This relationship was not influenced by treatment arm. Combination of p27 status with mismatch repair status, however, identified a small subset of patients that may benefit from IFL (n = 36; 5-year disease-free survival 81%: 95% CI, 0.64-0.98 versus 47%: 95% CI, 0.21-0.72; log-rank P = 0.042; 5-year OS 81%: 95% CI, 0.64-0.98 versus 60%: 95% CI, 0.35-0.85; log-rank P = 0.128). CONCLUSIONS: Loss of p27 is associated with reduced survival in stage III colon cancer but by itself does not indicate a significant difference in outcome between patients treated IFL or 5-fluorouracil/leucovorin.

Detection of procalcitonin (PCT) in healthy controls and patients with local infection by a sensitive ILMA.

BACKGROUND: Procalcitonin (PCT) is an established marker for severe systemic bacterial infection and sepsis. So far the relevance of PCT in healthy individuals or patients with local infections is unclear due to the lack of highly sensitive assays. The aim of our study was the characterization of a new sensitive PCT assay, the establishment of reference values and the assessment of diagnostic accuracy. METHODS: We assessed PCT values in 522 patients with different infectious and non infectious conditions and 410 healthy controls by a new coated tube sandwich chemiluminescence assay B.R.A.H.M.S ProCa-S (2 step assay, time to result 2.5 hours). RESULTS: The lower detection limit was 6.0 ng/L, with a functional assay sensitivity below 7 ng/L. Samples above 250 ng/L gave excellent correlation to the LUMItest PCT (r = 0.98, p < 0.0001). There was no high dose hook effect up to a concentration of 21,300 ng/L. The 410 healthy controls had a median concentration of 12.7 ng/L (95% CI: 12.6-14.7 ng/L). 65 controls had non-detectable PCT values (defined as 5 ng/L). The 2.5th percentile of the normal population was 5 ng/L and the 97.5th percentile was 46.7 ng/L. ROC plot analysis resulted in an area under the curve (AUC) of 0.90. The optimal decision threshold was at 50 ng/L, with a sensitivity for infection of 77.8% and a specificity of 98.5% (positive predictive value 97.7%, negative predictive value 84.9%). There was a highly significant (p < 0.0001) difference in the PCT median between healthy individuals and patients with infections (e.g. pneumonia, peritonitis) but not non-infectious controls (e.g. pregnancy, autoimmune disease). CONCLUSIONS: The new PCT assay is 30 times more sensitive than the established routine assay LUMItest PCT, thus allowing for the first time PCT detection in healthy individuals. First results indicate that the assay is suitable to differentiate local bacterial infections from other non-infectious diseases.