Phylogeny and identification of Nocardia species on the basis of multilocus sequence analysis.

Nocardia species identification is difficult due to a complex and rapidly changing taxonomy, the failure of 16S rRNA and cellular fatty acid analysis to discriminate many species, and the unreliability of biochemical testing. Here, Nocardia species identification was achieved through multilocus sequence analysis (MLSA) of gyrase B of the ß subunit of DNA topoisomerase (gyrB), 16S rRNA (16S), subunit A of SecA preprotein translocase (secA1), the 65-kDa heat shock protein (hsp65), and RNA polymerase (rpoB) applied to 190 clinical, 36 type, and 11 reference strains. Phylogenetic analysis resolved 30 sequence clusters with high (>85%) bootstrap support. Since most clusters contained a single type strain and the analysis corroborated current knowledge of Nocardia taxonomy, the sequence clusters were equated with species clusters and MLSA was deemed appropriate for species identification. By comparison, single-locus analysis was inadequate because it failed to resolve species clusters, partly due to the presence of foreign alleles in 22.1% of isolates. While MLSA identified the species of the majority (71.3%) of strains, it also identified clusters that may correspond to new species. The correlation of the identities by MLSA with those determined on the basis of microscopic examination, biochemical testing, and fatty acid analysis was 95%; however, MLSA was more discriminatory. Nocardia cyriacigeorgica (21.58%) and N. farcinica (14.74%) were the most frequently encountered species among clinical isolates. In summary, five-locus MLSA is a reliable method of elucidating taxonomic data to inform Nocardia species identification; however, three-locus (gyrB-16S-secA1) or four-locus (gyrB-16S-secA1-hsp65) MLSA was nearly as reliable, correctly identifying 98.5% and 99.5% of isolates, respectively, and would be more feasible for routine use in a clinical reference microbiology laboratory.

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in pharyngeal and rectal specimens using the BD Probetec ET system, the Gen-Probe Aptima Combo 2 assay and culture.

OBJECTIVES: This study compared the sensitivity and specificity of culture and two nucleic acid amplification tests (NAATs): the BD Probetec ET system (PT) and the Aptima Combo 2 (AC2) in detecting Neisseria gonorrhoeae (GC) and Chlamydia trachomatis (CT) in pharyngeal and rectal specimens. METHODS: Male subjects were prospectively recruited at an MSM clinic in Toronto, Canada. Pharyngeal and rectal specimens were obtained for GC and CT culture, PT and AC2. Urine was also obtained for PT. A true positive was defined as: (1) positive culture, (2) positive PT and AC2 at the same site or (3) a single positive NAAT and detection of the same organism by any method at another site. RESULTS: 248 subjects were recruited. The prevalence of pharyngeal GC was 8.1%, rectal GC 11.7%, pharyngeal CT 2.0% and rectal CT 7.7%. The sensitivity of culture for pharyngeal GC and CT was 0%; 41.4% for rectal GC and 21.1% for rectal CT. The sensitivity of PT for pharyngeal GC, rectal GC, pharyngeal CT and rectal CT was 95.0%, 93.1%, 80.0% and 94.7%, respectively. The sensitivity of AC2 was 95.0% for pharyngeal GC and 100% at all other sites. Specificity was consistently above 98%. CONCLUSIONS: PT and AC2 detected GC and CT with superior sensitivity compared to culture. They detected 73 pharyngeal or rectal GC and CT infections compared to 16 by culture, using a rigorous gold standard. NAATs should be the method of choice for the detection of GC and CT in extragenital sites in men who have sex with men.

Accidental acute clidinium toxicity.

Clidinium bromide (N-methyl-quinuclidinyl-benzylate) is a rarely used antimuscarinic drug that is marketed in combination with chlordiazepoxide as an antispasmodic for use in irritable bowel syndrome. A case is reported of an accidental staggered overdose of clidinium bromide 50 mg in a patient using illicit chlordiazepoxide. The presenting features were mildly dilated pupils and palpitation secondary to sinus tachycardia that persisted for 11 h after the time of first ingestion. Emergency physicians should be aware of the potential for antimuscarinic toxicity in patients using illicit chlordiazepoxide.

Design of a single tube RT-PCR assay for the diagnosis of human infection with highly pathogenic influenza A(H5) viruses.

Concerns about emergence of a pandemic strain of influenza have been increasing. The strains of highly pathogenic influenza A(H5N1) currently circulating are considered among the most plausible candidates for giving rise to a pandemic strain. In this study the design and development of a RT-PCR assay specific for these highly pathogenic influenza A(H5) strains is presented. This is achieved in part by the design of a primer targeting the coding region for the protease cleavage site of the hemagglutinin, and another primer derived from a pan-hemagglutinin RT-PCR assay also presented in this study. It is shown that the HPAI A(H5) specific assay amplifies only the nucleic acids of highly pathogenic A(H5), with a high sensitivity.

The histopathology of the hemolytic uremic syndrome associated with verocytotoxin-producing Escherichia coli infections.

Verocytotoxin-producing Escherichia coli (VTEC) infection was present in three cases of hemolytic uremic syndrome (HUS), two fatal and one non-fatal, in which detailed histopathologic investigations were conducted. Two patients had a prodrome of bloody diarrhea, one of whom required a hemicolectomy for severe bleeding. The renal histopathology was characterized primarily by glomerular thrombotic microangiopathy (TMA) with greater than 95% of glomeruli showing changes of capillary wall thickening, endothelial cell swelling, and narrowing or thrombosis of the capillary lumen. Preglomerular arterioles were frequently thrombosed, and abnormalities of the medium-sized vessels, including endothelial cell damage and thrombosis, were also commonly observed. Gastrointestinal involvement was prominent in all three cases. The colon was most severely involved, with marked mucosal and submucosal edema and hemorrhage, in the absence of significant inflammation or widespread ulceration. Microvascular angiopathy was present in all cases, with changes ranging from endothelial cell damage to overt thrombosis. Similar pathology was seen throughout the small bowel, including the presence of TMA. In one patient, typical morphologic changes of pseudomembranous enterocolitis were found in the absence of infection with Clostridium difficile. The nature of vascular involvement in the kidneys and intestinal tract supports the hypothesis that HUS is mediated by systemic toxemia, and that endothelial cells are the primary target cells for the action of verocytotoxin.

Systemic Candida infection in extremely low birth weight infants: short term morbidity and long term neurodevelopmental outcome.

OBJECTIVE: To describe mortality, morbidity at discharge and neurodevelopmental outcome at 2 years corrected age in extremely low birth weight infants with systemic Candida infection during intensive care stay. METHOD: We identified all extremely low birth weight (birth weight <1000 g) infants diagnosed with Candida sepsis and/or meningoencephalitis between 1988 and mid-1996 in the tertiary neonatal intensive care centers of Toronto. The outcome of the infected infants at discharge and at 2 years corrected age was compared with a cohort of 470 extremely low birth weight infants born between 1990 and 1994. RESULTS: Forty-six extremely low birth weight infants with systemic Candida infection, mean (+/-SD) gestational age of 24.7 +/- 1.6 weeks and birth weight 699 +/- 135 g, were identified. Case fatality rate was 37% (17 of 46), not significantly different from the control group (35%). Data on 27 infected survivors were available at discharge. All had chronic lung disease compared with 33% in the control cases (P = 0.0001), a high incidence of periventricular leukomalacia (26% vs. 12%, P = 0.06) and an increase in severe retinopathy of prematurity (22% vs. 9%, P = 0.04); 60% had adverse neurologic outcomes at 2 years corrected age compared with 35% in the control group, and 41% vs. 12% had severe disabilities (P = 0.005). Cranial ultrasound examination was the only diagnostic modality in 5 of 13 (38%) cases with central nervous system Candida involvement. All infants with brain parenchymal lesions detected by cranial ultrasound had poor outcome. Early diagnosis and commencement of antifungal treatment favorably affected the outcome. CONCLUSIONS: Systemic Candida infection is associated with increased short and long term morbidity in extremely low birth weight infants. Candida infection of the central nervous system has a significant impact on long term neurodevelopmental outcome. Performance of cranial ultrasound examination is recommended as a part of the diagnostic investigation in these infants. Detection of brain parenchymal involvement might provide further information to predict outcome.

Evaluation of the disposable absorption containment trunk for female U-2 and TR-1 pilots.

Female U-2 and TR-1 pilots needed a urine collection device as part of their high flight pressure suit ensembles. The Disposable Absorption Containment Trunk (DACT) had been designed for short term use with the NASA Space Shuttle Program. This study evaluated the DACT for the extended and repetitive use required for integration into the U-2 and TR-1 aircraft. Six female subjects were tested wearing a custom-fit DACT and the 1030 full pressure suit ensemble with torso harness. They were strapped into the U-2 ejection seat with parachute and seat kit for 10 h and 45 min every 3 d for a total of 5 sessions. The DACT was evaluated for absorptive capability and comfort. Subjects were monitored for dermatitis, vaginitis, and urinary tract infections. The DACT reliably absorbed menstrual flow and urinary outputs to 950 cc. Higher urinary outputs resulted in minimal leakage. Dermatitis in the form of mild erythema and chafing was present, but cleared between sessions without medical intervention. Complaints concerning comfort were minor and limited to fit problems. One case of vaginitis developed. There were no cases of urinary tract infection. The DACT was found to be an acceptable method of urine collection for female U-2 and TR-1 pilots. Minor changes in design will enhance the comfort and performance of the DACT.

The Management of Classical Hodgkin's Lymphoma: Past, Present, and Future.

The management of classical Hodgkin's lymphoma (CHL) is a success story of modern multi-agent haemato-oncology. Prior to the middle of the twentieth century CHL was fatal in the majority of cases. Introduction of single agent radiotherapy (RT) demonstrated for the first time that these patients could be cured. Developments in chemotherapy including the mechlorethamine, vincristine, procarbazine and prednisolone (MOPP) and Adriamycin, bleomycin, vinblastine and dacarbazine (ABVD) regimens have resulted in cure rates of over 80%. Even in relapse, CHL patients can be salvaged with high dose chemotherapy and autologous haematopoietic stem cell transplantation (ASCT). Challenges remain, however, in finding new strategies to manage the small number of patients who continue to relapse or progress. In addition, the young age of many Hodgkin's patients forces difficult decisions in balancing the benefit of early disease control against the survival disadvantage of late toxicity. In this article we aim to summarise past trials, define the current standard of care and appraise future developments in the management of CHL.

Evaluation of multiple commercial molecular and conventional diagnostic assays for the detection of respiratory viruses in children.

This study compares the performance of four commercial multiplex PCR assays (Resplex II Panel v2.0, Seeplex RV15, xTAG RVP and xTAG RVP Fast) and direct fluorescent antibody (DFA) staining and viral isolation. Seven hundred and fifty nasopharyngeal swabs were tested for 17 viral agents. In each assay, the sensitivity and specificity for each target were determined against a composite reference standard. Two hundred and eighty-eight out of 750 (38.4%) specimens were positive by DFA or viral isolation, while an additional 214 (28.5%) were positive by multiplex PCR, for a total positivity rate of 66.9%. Of 502 positive specimens, one virus was detected in 420 specimens (83.7%), two in 77 (15.3%), three in four (0.8%) and four in one case (0.2%). Compared with a composite reference standard, the inter-assay accuracy of the multiplex PCR assays varied, but all were superior to conventional diagnostic methods in detecting a broad range of respiratory viral agents in children. In addition, the sensitivity of two commercial assays, Resplex II Plus PRE and Seeplex Influenza A/B Subtyping, was determined relative to the Astra influenza Screen & Type assay for detection of influenza A viruses, including seasonal influenzas and pandemic H1N1 2009 influenza A virus. Using 75 positive and 55 negative nasopharyngeal swabs for influenza A by the Astra assay, the sensitivity of Seeplex and Resplex was 95.9% and 91.8%, respectively, with a specificity of 100% for both.

Changing epidemiology of invasive Haemophilus influenzae in Ontario, Canada: evidence for herd effects and strain replacement due to Hib vaccination.

The epidemiology of invasive Haemophilus influenzae infections was evaluated in Ontario between 1989 and 2007 to assess the impact of the introduction of the conjugate H. influenzae serotype b (Hib) vaccine in the early 1990 s on Hib and non-Hib serotypes in both vaccinated and unvaccinated cohorts as well as the possibility of "strain replacement" with non-vaccine H. influenzae strains. Data were collected by the provincial Public Health Laboratories-Toronto, Ontario Agency for Health Protection and Promotion, which performed almost all serotyping on invasive (blood, CSF, other sterile sites) H. influenzae strains isolated in the province during the study period. Temporal trends for Hib, other typeable strains, and non-typeable H. influenzae were evaluated by Poisson regression, controlling for the specimen submissions. Prior to infant Hib vaccination, the most commonly observed serotype was serotype b (64.9%). Subsequently, 70.3%, 13.6%, and 9.4% of isolates were non-typeable, serotype f, and serotype b, respectively. Infant Hib vaccination resulted in a decrease in Hib incidence in all age groups (pooled IRR 0.432) and marked increases of non-typeable and serotype f H. influenzae in children aged <5 years (IRR 2.4 and 3.0, respectively). Vaccination against Hib has altered the epidemiology of invasive H. influenzae infections in Ontario. Prevention of invasive Hib disease was observed in both vaccinated and unvaccinated age groups. Invasive H. influenzae infection now commonly presents as sepsis due to non-typeable H. influenzae in older individuals. However, strain replacement of Hib with serotype f and non-typeable strains in children under 5 years was documented.

Esophageal temperature after out-of-hospital cardiac arrest: an observational study.

INTRODUCTION: Out-of-hospital cardiac arrest (OHCA) is a significant cause of death and severe neurological disability. The only post-return of spontaneous circulation (ROSC) therapy shown to increase survival is mild therapeutic hypothermia (MTH). The relationship between esophageal temperature post OHCA and outcome is still poorly defined. METHODS: Prospective observational study of all OHCA patients admitted to a single centre for a 14-month period (1/08/2008 to 31/09/2009). Esophageal temperature was measured in the Emergency Department and Intensive Care Unit (ICU). Selected patients had pre-hospital temperature monitoring. Time taken to reach target temperature after ROSC was recorded, together with time to admission to the Emergency Department and ICU. RESULTS: 164 OHCA patients were included in the study. 105 (64.0%) were pronounced dead in the Emergency Department. 59 (36.0%) were admitted to ICU for cooling; 40 (24.4%) died in ICU and 19 (11.6%) survived to hospital discharge. Patients who achieved ROSC and had esophageal temperature measured pre-hospital (n=29) had a mean pre-hospital temperature of 33.9 degrees C (95% CI 33.2-34.5). All patients arriving in the ED post OHCA had a relatively low esophageal temperature (34.3 degrees C, 95% CI 34.1-34.6). Patients surviving to hospital discharge were warmer on admission to ICU than patients who died in hospital (35.7 degrees C vs 34.3 degrees C, p<0.05). Patients surviving to hospital discharge also took longer to reach T(targ) than non-survivors (2h 48min vs 1h 32min, p<0.05). CONCLUSIONS: Following OHCA all patients have esophageal temperatures below normal in the pre-hospital phase and on arrival in the Emergency Department. Patients who achieve ROSC following OHCA and survive to hospital discharge are warmer on arrival in ICU and take longer to reach target MTH temperatures compared to patients who die in hospital. The mechanisms of action underlying esophageal temperature and survival from OHCA remain unclear and further research is warranted to clarify this relationship.

Early diagnosis of SARS: lessons from the Toronto SARS outbreak.

The clinical presentation of SARS is nonspecific and diagnostic tests do not provide accurate results early in the disease course. Initial diagnosis remains reliant on clinical assessment. To identify features of the clinical assessment that are useful in SARS diagnosis, the exposure status and the prevalence and timing of symptoms, signs, laboratory and radiographic findings were determined for all adult patients admitted with suspected SARS during the Toronto SARS outbreak. Findings were compared between patients with laboratory-confirmed SARS and those in whom SARS was excluded by laboratory or public health investigation. Of 364 cases, 273 (75%) had confirmed SARS, 30 (8%) were excluded, and 61 (17%) remained indeterminate. Among confirmed cases, exposure occurred in the healthcare environment (80%) or in the households of affected patients (17%); community or travel-related cases were rare (<3%). Fever occurred in 97% of patients by the time of admission. Respiratory findings including cough, dyspnea and pulmonary infiltrates evolved later and were present in only 59, 37 and 68% of patients, respectively, at admission. Direct exposure, fever on the first day of illness, and elevated temperature, pulmonary infiltrates, lymphopenia and thrombocytopenia at admission were associated with confirmed cases. Rhinorrhea, sore throat, and an elevated neutrophil count at admission were associated with excluded cases. In the absence of fever or significant exposure, SARS is unlikely. Other clinical, laboratory and radiographic findings further raise or lower the likelihood of SARS and provide a rational basis for estimating the likelihood of SARS and directing initial management.

Multicenter comparison of nucleic acid extraction methods for detection of severe acute respiratory syndrome coronavirus RNA in stool specimens.

The emergence of a novel coronavirus (CoV) as the cause of severe acute respiratory syndrome (SARS) catalyzed the development of rapid diagnostic tests. Stool samples have been shown to be appropriate for diagnostic testing for SARS CoV, although it has been recognized to be a heterogeneous and difficult sample that contains amplification inhibitors. Limited information on the efficiency of extraction methods for the purification and concentration of SARS CoV RNA from stool samples is available. Our study objectives were to determine the optimal extraction method for SARS CoV RNA detection and to examine the effect of increased specimen volume for the detection of SARS CoV RNA in stool specimens. We conducted a multicenter evaluation of four automated and four manual extraction methods using dilutions of viral lysate in replicate mock stool samples, followed by quantitation of SARS CoV RNA using real-time reverse transcriptase PCR. The sensitivities of the manual methods ranged from 50% to 100%, with the Cortex Biochem Magazorb method, a magnetic bead isolation method, allowing detection of all 12 positive samples. The sensitivities of the automated methods ranged from 75% to 100%. The bioMérieux NucliSens automated extractor and miniMag extraction methods each had a sensitivity of 100%. Examination of the copy numbers detected and the generation of 10-fold dilutions of the extracted material indicated that a number of extraction methods retained inhibitory substances that prevented optimal amplification. Increasing the volume of sample input did improve detection. This information could be useful for the extraction of other RNA viruses from stool samples and demonstrates the need to evaluate extraction methods for different specimen types.

Fusarium proliferatum as an agent of disseminated infection in an immunosuppressed patient.

Fusarium proliferatum was reported as the agent of a fatal disseminated infection in a child with lymphoblastic leukemia. The fungus has not been reported previously to cause disease in humans, but it is closely related to the known opportunistic pathogen F. verticillioides. It was distinguished by the production of clavate microconidia in chains from proliferating phialides. Resistance to amphotericin B and flucytosine in vitro was shown.

Improved detection of Malassezia species in lipid-supplemented Peds Plus blood culture bottles.

The growth of Malassezia species in BACTEC Peds Plus blood culture bottles was optimized by using various lipid supplements. Palmitic acid (3%, wt/vol) was superior and overcame the inhibitory effect of blood in mock clinical specimens. Palmitic acid (3%) supplementation of Peds Plus bottles may improve recovery of Malassezia species in the BACTEC NR 660.

Detection and identification of Bartonella species pathogenic for humans by PCR amplification targeting the riboflavin synthase gene (ribC).

Several Bartonella species have now been implicated as human pathogens. The recovery of these fastidious organisms in the clinical microbiology laboratory remains difficult, and current methods are still relatively insensitive. Thus, the bartonellae are good candidates for detection by PCR. We have developed a PCR assay which uses a single primer pair targeting the riboflavin synthase gene (ribC) and detected six Bartonella species that have been implicated in human disease, B. henselae, B. quintana, B. bacilliformis, B. clarridgeiae, B. elizabethae, and B. vinsonii subsp. berkhoffii. Species identification is achieved simply by restriction enzyme digestion of the amplicon. This PCR assay appears to be specific for the Bartonella genus because it failed to amplify DNA from several other bacterial species.

Toxicity and immunogenicity of a verotoxin 1 mutant with reduced globotriaosylceramide receptor binding in rabbits.

The verotoxins (VT1 and VT2), produced by strains of enterohemorrhagic Escherichia coli, have been implicated in the pathogenesis of hemorrhagic colitis and the hemolytic uremic syndrome. To better understand the role of globotriaosylceramide (Gb3) receptor binding by the verotoxins in disease production, we examined the clinicopathologic effects of an intravenously (i.v.) administered verotoxin 1 mutant holotoxin (Phe30Ala) in rabbits. The substitution of alanine for phenylalanine 30 in the VT1 B subunit has been shown previously to reduce both Gb3 binding affinity and capacity in vitro. This reduction in receptor binding corresponded to a 10(5)-fold reduction in the toxic activity of VT1 on a Vero cell monolayer. In this study, purified 125I-labeled Phe30Ala was administered i.v. to rabbits to determine its specific distribution in rabbit tissues. In contrast to the rapid elimination of i.v. administered 125I-VT1 from the bloodstream, 125I-Phe30Ala had a 52-fold-longer half-life in serum and failed to localize preferentially in the gastrointestinal tract and central nervous system (CNS). Rabbits challenged with Phe30Ala at a dose equivalent to 10 times the 50% lethal dose (LD50) of VT1 showed no visible clinical symptoms typical of VT effect after 7 days. Administration of Phe30Ala at a dose equivalent to 100 times the LD50 of VT1, however, caused both clinical and histopathologic features indistinguishable from VT1 toxemia in rabbits, although the onset of symptoms was delayed. Rabbits were immunized with Phe30Ala and challenged i.v. with either 125I-VT1 or 125I-VT2. The specific uptake of 125I-VT1 in the gastrointestinal tract and CNS was totally inhibited in Phe30Ala immune rabbits. Only a partial decrease in target organ uptake was observed in Phe30Ala immune rabbits challenged with 125I-VT2. From this study, we conclude that Gb3 binding is responsible for target organ localization of VT1 and disease production in the rabbit. The ability of Phe30Ala to induce both strong antibody and protective responses against VT1 suggests that VT mutants with reduced receptor binding properties may be useful in vaccine strategies. A further reduction in the toxicity of Phe30Ala would be required for its use as a natural toxoid to protect against human verotoxigenic E. coli infections.

Candida infection in pediatric liver transplant recipients.

A retrospective review of 100 liver transplantations in 98 children was performed to determine the incidence of infection caused by Candida organism in these patients and to identify risk factors that may predispose to serious fungal infection. Thirty-one infections caused by Candida organisms developed during the initial 28 days posttransplantation: 19 were definite invasive infections (one deep site or one positive blood culture), 2 were probable invasive infections (three superficial sites), and 10 were urinary tract infections. Eleven of 19 patients had fungemia or a disseminated infection (two noncontiguous deep organs involved and/or positive blood cultures) and 8 of 19 had peritoneal candidiasis. Infection caused by Candida organisms was a contributing factor to mortality in 7 of 21 patients (case fatality rate of 33%) with invasive infection. Risk factors that were predictive for invasive infection by univariate analysis included the following: pretransplantation antibiotic therapy, length of transplant operation, transfusion requirement, number of days in the intensive care unit, number of days intubated, number of concurrent bacterial infections, number of antibiotics administered, number of laparotomies performed posttransplantation, retransplantation, hepatic artery thrombosis, bile leaks, and renal and respiratory failure. By logistic regression analysis, bile leak, hepatic artery thrombosis, preoperative steroid use, transfusion requirement, and the number of days intubated were identified as independent risk factors for invasive infection caused by Candida organisms. The use of prophylactic antifungal agents in high-risk patients may be important in reducing the serious morbidity and mortality associated with sepsis caused by Candida organisms in pediatric liver transplant recipients.