Ghada N. Al-Rawahi

Community-Associated CMRSA-10 (USA-300) is the Predominant Strain Among Methicillin-Resistant Staphylococcus aureus Strains Causing Skin and Soft Tissue Infections in Patients Presenting to the Emergency Department of a Canadian Tertiary Care Hospital

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging pathogen first described among individuals with no contact with health care facilities. The purpose of this study was to determine the proportion of CA-MRSA, defined by pulsed field gel electrophoresis (PFGE), in MRSA skin and soft tissue infections presenting to the Emergency Department (ED). We also aimed to describe the laboratory and clinical characteristics of CA-MRSA infections. From June 1, 2001 to May 30, 2005, MRSA isolates from skin and soft tissue infections presenting to the ED were reviewed. They were characterized by antibiotic susceptibilities and PFGE, and the presence of staphylococcal cassette chromosome (SCC) mec type IVa and Panton-Valentine leukocidin (PVL) genes was assessed on representative isolates. The medical records were reviewed to define risk factors. There were 95 isolates available for analysis, of which 58 (61%) were CMRSA-10 (USA-300), the predominant clone from 2003 onward. All representative isolates (24%) tested in this group had PVL genes and SCCmec type IVa. Their antibiogram showed 100% susceptibility to trimethoprim-sulfamethoxazole, rifampin, and fusidic acid, and 79% to clindamycin. Clinical comparison of CMRSA-10 vs. hospital PFGE type strains showed 22% vs. 60%, respectively, for recent antibiotic use (p < 0.0001), 26% vs. 6%, respectively, for intravenous drug use (p < 0.05), and 57% vs. 6%, respectively, for soft tissue abscess (p < 0.001). CMRSA-10 is a major pathogen in skin and soft tissue abscesses in our ED. It has a characteristic susceptibility, and was associated with intravenous drug use, but not with recent antibiotic usage.

Carriage of methicillin-resistant Staphylococcus aureus by wild urban Norway rats (Rattus norvegicus).

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of multi-drug-resistant infections in people, particularly indigent populations. MRSA can be transmitted between people and domestic animals, but the potential for transmission between people and commensal pests, particularly rodents, had not been investigated. The objective of this study was to identify the presence and characterize the ecology of MRSA in rats (Rattus spp.) from in an impoverished, inner-city neighborhood. Oropharyngeal swabs were collected from rats trapped in 33 city blocks and one location within the adjacent port. Bacterial culture was performed and MRSA isolates were characterized using a variety of methods, including whole-genome sequencing (WGS). The ecology of MRSA in rats was described using phylogenetic analysis, geospatial analysis, and generalized linear mixed models. MRSA was identified 22 of 637 (3.5%) rats tested, although prevalence varied from 0 – 50% among blocks. Isolates belonged to 4 clusters according to WGS, with the largest cluster (n = 10) containing isolates that were genetically indistinguishable from community-acquired USA300 MRSA strains isolated from people within the study area. MRSA strains demonstrated both geographic clustering and dispersion. The odds of an individual rat carrying MRSA increased with increased body fat (OR = 2.53, 95% CI = 1.33 – 4.82), and in the winter (OR = 5.29, 95% CI = 1.04 – 26.85) and spring (OR = 5.50, 95% CI = 1.10 – 27.58) compared to the fall. The results show that urban rats carried the same MRSA lineages occurring in local human and/or animal populations, supporting recent transmission from external sources. MRSA carriage was influenced by season, most likely as a result of temporal variation in rat behavior and rat-human interactions.

First Report of Isolation and Characterization of Aurantimonas altamirensis from Clinical Samples

ABSTRACT The genus Aurantimonas, proposed in 2003, encompasses four species from environmental sources, including Aurantimonas altamirensis, isolated from a cave wall in Spain. Here, we report what we believe are the first cases of the recovery of A. altamirensis from human clinical materials.

Short-Term Stability of Pathogen-Specific Nucleic Acid Targets in Clinical Samples

ABSTRACT The stability of pathogen-specific DNA or RNA amplification targets in clinical samples following short-term storage at room temperature, 4°C, and −80°C was assessed by real-time PCR. In purified nucleic acid extracts, both DNA and RNA targets were stable for up to 30 days, irrespective of storage temperature. In unextracted samples, temperature-dependent loss of targets (P < 0.05) was observed in serum and cerebrospinal fluid specimens, while no changes were observed for EDTA blood samples.

Evaluation of Amplification Targets for the Specific Detection of Bordetella pertussis Using Real-Time Polymerase Chain Reaction

BACKGROUND: Bordetella pertussis infections continue to be a major public health challenge in Canada. Polymerase chain reaction (PCR) assays to detect B pertussis are typically based on the multicopy insertion sequence IS481, which offers high sensitivity but lacks species specificity. METHODS: A novel B pertussis real-time PCR assay based on the porin gene was tested in parallel with several previously published assays that target genes such as IS481, ptx-promoter, pertactin and a putative thialase. The assays were evaluated using a reference panel of common respiratory bacteria including different Bordetella species and 107 clinical nasopharyngeal specimens. Discrepant results were confirmed by sequencing the PCR products. RESULTS: Analytical sensitivity was highest for the assay targeting the IS481 element; however, the assay lacked specificity for B pertussis in the reference panel and in the clinical samples. False-positive results were also observed with assays targeting the ptx-promoter and pertactin genes. A PCR assay based on the thialase gene was highly specific but failed to detect all reference strains of B pertussis. However, a novel assay targeting the porin gene demonstrated high specificity for B pertussis both in the reference panel and in clinical samples and, based on sequence-confirmed results, correctly predicted all B pertussis-positive cases in clinical samples. According to Probit regression analysis, the 95% detection limit of the new assay was 4 colony forming units/reaction. CONCLUSION: A novel porin assay for B pertussis demonstrated superior performance and may be useful for improved molecular detection of B pertussis in clinical specimens.

Granulomatous Herpes Simplex Encephalitis in an Infant With Multicystic Encephalopathy: A Distinct Clinicopathologic Entity?

BACKGROUND Herpes simplex virus encephalitis can manifest as a range of clinical presentations including classic adult, neonatal, and biphasic chronic-granulomatous herpes encephalitis. METHOD We report an infant with granulomatous herpes simplex virus type 2 encephalitis with a subacute course and multicystic encephalopathy. CASE A 2-month-old girl presented with lethargy and hypothermia. Computed tomography scan of the head showed multicystic encephalopathy and calcifications. Cerebrospinal fluid analysis by polymerase chain reaction testing for herpes simplex virus 1 and 2, enterovirus, and cytomegalovirus was negative. Normal cerebrospinal fluid interferon-α levels argued against Aicardi-Goutières syndrome. The patient died 2 weeks after presentation. At autopsy, multicystic encephalopathy was confirmed with bilateral gliosis, granulomatous inflammation with multinucleated giant cells, and calcifications. Bilateral healing necrotizing retinitis suggested a viral etiology, but retina and brain were free of viral inclusions and immunohistochemically negative for herpes simplex virus-2 and cytomegalovirus. However, polymerase chain reaction analysis showed herpes simplex virus-2 DNA in four cerebral paraffin blocks. Subsequent repeat testing of the initial cerebrospinal fluid sample using a different polymerase chain reaction assay was weakly positive for herpes simplex virus-2 DNA. CONCLUSION Granulomatous herpes simplex virus encephalitis in infants can present with subacute course and result in multicystic encephalopathy with mineralization and minimal cerebrospinal fluid herpes simplex virus DNA load. Infectious etiologies should be carefully investigated in the differential diagnosis of multicystic encephalopathy with mineralization, in particular if multinucleated giant cells are present.

Measles Lymphadenopathy in a Child With PFAPA Syndrome

Periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome is a common cause of periodic fever in children. The pathogenesis of PFAPA is unknown but likely involves immune system dysregulation and may be initiated by an environmental trigger. Tonsillectomy resolves or improves symptoms in some patients, but the reason for this is unknown; moreover, specific abnormalities in tonsillectomy specimens from PFAPA patients have not been described. Here, we report measles virus in tonsil from a child with PFAPA. Measles-type viral cytopathic effect was discovered on histological examination of tonsillar tissue after therapeutic tonsillectomy for PFAPA. Molecular testing showed the left tonsil was positive for measles RNA by reverse transcription polymerase chain reaction (RT-PCR) while the right tonsil was inconclusive (weakly positive). Real-time RT-PCR specific for measles vaccine strain RNA (genotype A) was weakly reactive in the left tonsil tissue when tested in 3 independent replicates, but this result could not be confirmed with conventional genotyping by sequencing. The relationship and clinical significance between measles virus and PFAPA in this case is unclear but may be related to PFAPA-associated immune dysregulation. Additional investigation of measles virus in PFAPA patients would be helpful in further exploring this potential association.

Comparative evaluation of laboratory developed real-time PCR assays and RealStar(®) BKV PCR Kit for quantitative detection of BK polyomavirus.

BACKGROUND Quantitative, viral load monitoring for BK virus (BKV) by real-time PCR is an important tool in the management of polyomavirus associated nephropathy in renal transplant patients. However, variability in PCR results has been reported because of polymorphisms in viral genes among different subtypes of BKV, and lack of standardization of the PCR assays among different laboratories. In this study we have compared the performance of several laboratory developed PCR assays that target highly conserved regions of BKV genome with a commercially available, RealStar(®) BKV PCR Kit. METHOD Three real-time PCR assays (i) VP1 assay: selected from the literature that targets the major capsid protein (VP1) gene (ii) VP1MOD assay: VP1 assay with a modified probe, and (iii) BKLTA assay: newly designed assay that targets the large T antigen gene were assessed in parallel, using controls and clinical specimens that were previously tested using RealStar(®) BKV PCR Kit (Altona Diagnostics GmbH, Hamburg, Germany). Nucleic acid from all samples were extracted using the QIA symphony virus/bacteria kit on an automated DNA extraction platform QIA symphony SP (Qiagen). Primer and probe concentration, and reaction conditions for laboratory developed assays were optimized and the limit of detection of different assays was determined. Positive control for laboratory developed BK assays was prepared through construction of a plasmid carrying respective amplicon sequences. RESULTS The 95% detection limit of VP1, VP1MOD and BKLTA assays were 1.8×10(2), 3×10(3) and 3.5×10(2) genomic copies/ml, respectively, as determined by Probit regression analysis of data obtained by testing a dilution series of a titered patient specimen, using RealStar(®) BKV PCR Kit. The inter-assay and intra-assay, coefficient of variations of these assays using calibrated, plasmid standards were <1%. All assays, including the RealStar(®) BKV PCR assay, were highly specific when tested against a panel of external proficiency specimens containing both BK and JC viruses. All assays, except the VP1MOD assay determined BK viral load in proficiency specimens within the same log values. With reference to results obtained by RealStar(®) BKV PCR assay, the sensitivity and specificity of different assays tested in 116 serum specimens submitted for BK viral load assay were 91% and 97% for VP1 assay, 88% and 97% for VP1MOD assay, and 97% and 98% for BKLTA assay, respectively. BK Viral load in positive specimens determined by various assays was highly correlated (R(2)>0.97), based on linear regression analysis. CONCLUSIONS The performance characteristics of the newly designed, BKLTA assay were highly comparable to RealStar(®) BKV PCR assay, and can be used for routine detection and viral load monitoring of BKV in a cost-effective manner.