Major challenges in diagnostic molecular microbiology are to develop a simple assay to distinguish Staphylococcus aureus from the less virulent but clinically important coagulase-negative staphylococci (CoNS) and to simultaneously determine their antibiotic resistance profiles. Multiplex PCR assays have been developed for the detection of methicillin-and mupirocin-resistant S. aureus and CoNS but not for the simultaneous discrimination of S. aureus from CoNS. We designed a new set of Staphylococcus genus-specific primers and developed a novel quadriplex PCR assay targeting the 16S rRNA (Staphylococcus genus specific), nuc (S. aureus species specific), mecA (a determinant of methicillin resistance), and mupA (a determinant of mupirocin resistance) genes to identify most staphylococci, to discriminate S. aureus from CoNS and other bacteria, and to simultaneously detect methicillin and mupirocin resistance. Validation of the assay with 96 ATCC control strains and 323 previously characterized clinical isolates, including methicillin-and mupirocin-sensitive and -resistant S. aureus and CoNS isolates and other bacteria, demonstrated 100% sensitivity, specificity, and accuracy. This assay represents a simple, rapid, accurate, and reliable approach for the detection of methicillin-and mupirocin-resistant staphylococci and offers the hope of preventing their widespread dissemination through early and reliable detection.

Clostridium difficile-associated diarrhea (CDAD) is the most commonly identified cause of nosocomial diarrhea (10,12,17). The pathogenicity of C. difficile is due to the production of two exotoxins: toxin A, an enterotoxin, and toxin B. Both toxins A and B contribute to human disease (19).TechLab Inc. (Blacksburg, Va.) has recently marketed an enzyme immunoassay, C. DIFF CHEK-60, which detects glutamate dehydrogenase (GDH), a common C. difficile antigen (the assay is hereinafter referred to as TL-GDH), and also markets an enzyme immunoassay, C. difficile Tox A/B II (Tox-A/B), for the detection of toxins A and B. The aim of this study was to evaluate and compare these two assays with another immunoassay, the Triage C. difficile Panel assay (Biosite Diagnostics, San Diego, Calif.), which detects both GDH (TR-GDH) and toxin A (TR-Tox-A); an in-house cytotoxin assay (C-Tox); and stool cultures for C. difficile.All nonformed stool specimens from inpatients suspected of having CDAD were included in the study. Except for stool cultures, all tests were performed within 48 h of the arrival of specimens in the laboratory. Specimens were kept at 4°C if not processed immediately. A portion of stool was stored at Ϫ70°C for subsequent culture. Specimens of insufficient quantity were excluded from all tests.The results of TL-GDH and Tox-A/B were read by using a dual-wavelength spectrometer (450 and 620 nm). TR-GDH, TR-Tox-A, TL-GDH, and Tox-A/B were carried out according to the manufacturer's instruction. Details of the Triage panels and Tox-A/B and C-Tox have been described previously (16).After thawing, fecal samples were planted onto prereduced cycloserine-cefoxitin-fructose agar (cefoxitin, 8 mg/liter; cycloserine, 250 mg/liter; fructose agar; Oxoid, Ottawa, Canada) and incubated for 96 h at 35°C in an anaerobic glove box. Suspected colonies were identified based on their growth on selective media, their colonial morphology, and a positive reaction by the MicroScreen C. difficile latex slide agglutination test (Microgen Bioproducts Ltd., Surrey, United Kingdom) (2, 11).All C. difficile isolates were tested by PCR for the presence of toxin A and B genes by use of primers NK9 and NK11, derived from the repeating portion of toxin A, and NK104 and NK105, derived from the nonrepeating portion of toxin B. These primers have been described by Kato and coworkers (13). The composition of our master mix and the amplification conditions were the same as used by those investigators.For the purpose of this study, a true positive sample was defined as a sample that was positive for toxin A and/or B and from which a toxigenic C. difficile strain (positive by PCR for a toxin gene) was isolated. If a positive toxin detection test result was seen for a sample which was negative for C. difficile by culture or yielded a nontoxigenic strain, the reaction was considered to be falsely positive. Sensitivity, specificity, and positive and negative predictive values for each test were calculated on this basis.C. difficile was isolated from 93 of 49...

MRSASelect agar was compared to CHROMagar, mannitol-salt agar with oxacillin, and mannitol-salt agar with cefoxitin (MSA-CFOX) for the isolation of methicillin-resistant Staphylococcus aureus (MRSA). The sensitivities and specificities were 97.3% and 99.8%, 82.9% and 99.1%, 80.2% and 79%, and 99.1% and 84.8%, respectively. MSA-CFOX and MRSASelect had a high sensitivity. MRSASelect, however, was more specific and proved to be a more reliable and rapid medium for the detection of MRSA.

BackgroundPeritonsillar abscesses (PTA) are a common complication of tonsillitis. Recent global epidemiological data regarding PTAs have demonstrated increasing antimicrobial resistance patterns. No similar studies have been conducted in Canada and no Canadian study has examined the post-discharge course of treated patients.MethodsA prospective observational study of the epidemiology, antibiotic resistance and post-discharge course of patients presenting with a peritonsillar abscess to the Emergency Department in London, Ontario over one year. A follow-up telephone survey was conducted 2–3 weeks after abscess drainage.Results60 patients were diagnosed with an abscess, giving an incidence of 12/100,000. 46 patients were enrolled in the study; the average duration of symptoms prior to presentation was 6 days, with 51% treated with antibiotics prior to presentation. Streptococcus pyogenes and Streptococcus anginosus were present in 56% of isolates and of those, 7/23 (32%) of specimens demonstrated resistance to clindamycin. Eight patients were treated with clindamycin and had a culture that was resistant, yet only one had recurrence. Telephone follow-up was possible for 38 patients: 51% of patients reported a return to solid food within 2 days, and 75% reported no pain by 5 days. Resolution of trismus took a week or longer for 51%.InterpretationClindamycin resistance was identified in a third of Streptococcus isolates, which should be taken into account when prescribing antibiotics. Routine culture appears unnecessary as patients recover quickly from outpatient drainage and empiric therapy, with less pain than expected, but trismus takes time to resolve.

The pattern of calculous disease changed from a predominantly lower tract site in the mid 1980s to the upper tract in the mid 1990s. Stone composition, urinary risk factors and dietary analysis suggest that diet, dehydration and poor nutrition are the main causative factors of stone disease.

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We retrospectively analyzed the results of 75 living-related pediatric renal transplants performed at our center between January 1986 and December 1999. The major causes of end-stage renal disease (ESRD) were glomerulonephritis (26%) and nephrolithiasis (16%), while the etiology was unknown in 50%. The mean age of the recipients was 12 yr (range 6-17 yr) and that of the donors was 39 yr (range 20-65 yr). The majority (73%) of donors were parents. Eighty five per cent of donors were one-haplotype matched and the rest identical. Immunosuppression was based on a triple drug regimen. Thirty per cent of recipients were rapid metabolizers of cyclosporin A (CsA) (area under the curve [AUC]: < 6,000 ng/mL/h), while 16% were slow metabolizers (AUC: > 8,000 ng/mL/h). Forty three (57%) children encountered 59 rejection episodes, the majority of which (59%) were recorded in the first month post-transplant. Seventy-four per cent of the rejection episodes were steroid sensitive and the rest, except two, were resolved by therapy with antithymocyte globulin (ATG) or orthoclone thymocyte 3 (OKT3). After a mean follow-up of 37 months, 17 (22%) grafts had chronic rejection and 76% of these recipients had previously experienced acute rejection episodes. The overall infection rate was high, necessitating two hospital admissions/patient/year. The majority (53%) of the infections were bacterial. Urinary tract infections (UTIs) were seen in 17 (23%) recipients. Twelve of these had ESRD as a result of stone disease and eight grafts were lost because of UTIs. Eight per cent of recipients developed tuberculosis (TB), and extra-pulmonary lesions were seen in 50%. Surgical complications were encountered in eight patients. Free medication to all recipients and parental support ensured a compliance rate of 93%. Baseline growth deficit was seen in children of the two groups studied (the 6-12 yr and 13-17 yr age-groups), with Z-scores of - 2.39 and - 2.12, respectively. No growth catch-up was observed at 12 and 24 months in either group. Post-donation complications were seen most commonly in donors > 50 yr of age and included: proteinuria (> 300 mg/24 h, four patients), hypertension (three patients), and diabetes (one patient). Twenty-four grafts were lost, 54% as a result of immunological and the rest as a result of non-immunological causes, and 17 recipients died during the follow-up period. Infections were the main cause of patient and graft loss. Overall 1- and 5-yr graft and patient survival rates were 88% and 65%, and 90% and 75%, respectively.

The estimated incidence of end-stage renal disease (ESRD) in Pakistan is 100 per million population. Paucity and high costs of renal replacement therapy allows only 10% to get dialysis and 4-5% transplants. Our center, a government organization, started a dialysis and transplant program in 1980s where all services were provided free of charge to all patients. It was based on the concept of community government partnership funded by both partners. The guiding principles were equity, transparency, accountability and development of all facilities under one roof. This partnership has sustained itself for 30 years with an annual budget of $25 million in 2009. Daily 600 patients are dialyzed and weekly 10-12 receive transplants. Oneand 5-year graft survival of 3000 transplants is 92% and 85%, respectively. The institute became a focus of transplantation in Pakistan and played a vital role in the campaign against transplant tourism and in promulgation of transplant law of 2007, and also helped to increase altruistic transplants in the country. This model emphasizes that in developing countries specialized centers in government sector are necessary for transplantation to progress and community support can make it available to the common man.