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1.  Development and evaluation of a novel fast broad-range 16S ribosomal DNA PCR and sequencing assay for diagnosis of bacterial infective endocarditis: multi-year experience in a large Canadian healthcare zone and a literature review 
BMC Infectious Diseases  2016;16:146.
The study aimed to explore the sensitivity and specificity of a novel fast 16S rDNA PCR and sequencing assay for the improved diagnosis of infective endocarditis (IE) in patients with suspected native or prosthetic heart valve (HV) infection over a multi-year period at our cardiovascular center.
Sixty-eight patients were prospectively enrolled who underwent HV replacement for suspected or confirmed IE between February 1, 2009 and September 1, 2014. Patient demographics, medical co-morbidities, Duke’s criteria, culture results, and antibiotic therapy were collected by detailed chart reviews. Dual-priming oligonucleotide primers targeted to 500 bps of the V1-V3 region of the 16S rRNA gene were used to perform fast broad-range 16S rDNA PCR and Sanger sequencing on ribosomal DNA extracted from HV tissues. The performance/diagnostic efficiency of the molecular test was evaluated against blood cultures and Gram stain and culture of HV tissue in patients’ with definite IE according to Duke’s criteria.
Fifty patients (73.5 %) had definite IE and another 8 (11.8 %) had possible IE according to Duke’s criteria. Cardiac surgery was delayed an average of 15.4 days from the time of the patient’s last positive blood culture, and appropriate antibiotic therapy was given in the pre-operative period. While 44/50 (88 %) patients had a positive blood culture, HV tissue culture was only positive in 23 (46 %) of them. Molecular testing of all HV tissues had sensitivity, specificity, NPV and PPV of 92, 77.8, 77.8 and 92 % compared to 44, 100, 39.1 and 100 % respectively for culture for diagnosis of definite IE. For prosthetic HV tissue, 16S rDNA PCR had sensitivity of 93 % and specificity of 83 % compared to 35 and 100 % respectively for culture. A literature review showed that the diagnostic accuracy of our novel fast broad-range 16S rDNA PCR assay was similar or better than that of previously published studies.
This novel fast broad-range 16S rDNA PCR/sequencing test had superior sensitivity compared to tissue Gram stain and culture for identifying underlying bacterial pathogen in both native and prosthetic valve endocarditis.
PMCID: PMC4828839  PMID: 27066823
Endocarditis; Broad-range 16S rDNA PCR; Bacterial pathogen; Molecular diagnosis
2.  The Validation of a Novel Surveillance System for Monitoring Bloodstream Infections in the Calgary Zone 
Background. Electronic surveillance systems (ESSs) that utilize existing information in databases are more efficient than conventional infection surveillance methods. The objective was to assess an ESS for bloodstream infections (BSIs) in the Calgary Zone for its agreement with traditional medical record review. Methods. The ESS was developed by linking related data from regional laboratory and hospital administrative databases and using set definitions for excluding contaminants and duplicate isolates. Infections were classified as hospital-acquired (HA), healthcare-associated community-onset (HCA), or community-acquired (CA). A random sample of patients from the ESS was then compared with independent medical record review. Results. Among the 308 patients selected for comparative review, the ESS identified 318 episodes of BSI of which 130 (40.9%) were CA, 98 (30.8%) were HCA, and 90 (28.3%) were HA. Medical record review identified 313 episodes of which 136 (43.4%) were CA, 97 (30.9%) were HCA, and 80 (25.6%) were HA. Episodes of BSI were concordant in 304 (97%) cases. Overall, there was 85.5% agreement between ESS and medical record review for the classification of where BSIs were acquired (kappa = 0.78, 95% Confidence Interval: 0.75–0.80). Conclusion. This novel ESS identified and classified BSIs with a high degree of accuracy. This system requires additional linkages with other related databases.
PMCID: PMC4914721  PMID: 27375749
3.  Population-Based Epidemiology and Microbiology of Community-Onset Bloodstream Infections 
Clinical Microbiology Reviews  2014;27(4):647-664.
Bloodstream infection (BSI) is a major cause of infectious disease morbidity and mortality worldwide. While a positive blood culture is mandatory for establishment of the presence of a BSI, there are a number of determinants that must be considered for establishment of this entity. Community-onset BSIs are those that occur in outpatients or are first identified <48 h after admission to hospital, and they may be subclassified further as health care associated, when they occur in patients with significant prior health care exposure, or community associated, in other cases. The most common causes of community-onset BSI include Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae. Antimicrobial-resistant organisms, including methicillin-resistant Staphylococcus aureus and extended-spectrum β-lactamase/metallo-β-lactamase/carbapenemase-producing Enterobacteriaceae, have emerged as important etiologies of community-onset BSI.
PMCID: PMC4187633  PMID: 25278570
4.  Development and validation of a Pneumocystis jirovecii real-time polymerase chain reaction assay for diagnosis of Pneumocystis pneumonia 
Pneumocystis pneumonia is caused by Pneumocystis jirovecii, an opportunistic fungal pathogen. Presently, many clinical microbiology laboratories rely on direct microscopic detection of P jirovecii. The validation, and clinical and laboratory development of a qualitative P jirovecii real-time polymerase chain reaction assay for the rapid detection of Pneumocystis pneumonia is discussed by the authors. In addition, this new technique is compared with the existing gold-standard immunofluorescence assay.
Pneumocystis jirovecii (PJ), a pathogenic fungus, causes severe interstitial Pneumocystis pneumonia (PCP) among immunocompromised patients. A laboratory-developed real-time polyermase chain reaction (PCR) assay was validated for PJ detection to improve diagnosis of PCP.
Forty stored bronchoalveolar lavage (BAL) samples (20 known PJ positive [PJ+] and 20 known PJ negative [PJ−]) were initially tested using the molecular assay. Ninety-two sequentially collected BAL samples were then analyzed using an immunofluorescence assay (IFA) and secondarily tested using the PJ real-time PCR assay. Discrepant results were resolved by retesting BAL samples using another real-time PCR assay with a different target. PJ real-time PCR assay performance was compared with the existing gold standard (ie, IFA) and a modified gold standard, in which a true positive was defined as a sample that tested positive in two of three methods in a patient suspected to have PCP.
Ninety of 132 (68%) BAL fluid samples were collected from immunocompromised patients. Thirteen of 92 (14%) BALs collected were PJ+ when tested using IFA. A total of 40 BAL samples were PJ+ in the present study including: all IFA positive samples (n=13); all referred PJ+ BAL samples (n=20); and seven additional BAL samples that were IFA negative, but positive using the modified gold standard. Compared with IFA, the PJ real-time PCR had sensitivity, specificity, and positive and negative predictive values of 100%, 91%, 65% and 100%, respectively. Compared with the modified gold standard, PJ real-time PCR had a sensitivity, specificity, and positive and negative predictive values of 100%.
PJ real-time PCR improved detection of PJ in immunocompromised patients.
PMCID: PMC4644010  PMID: 26600815
Diagnosis; Immunocompromised; P jirovecii; Pneumocystis pneumonia; Real-time PCR
5.  Validity of calendar day-based definitions for community-onset bloodstream infections 
BMC Research Notes  2015;8:123.
Community-onset (CO) bloodstream infections (BSI) are those BSI where the blood culture is drawn <48 hours from hospital admission. However, exact times of culture draw or hospital admission are not always available. We evaluated the validity of using 2- or 3- calendar day based definitions for CO-BSI by comparing to a “gold standard” 48-hour definition.
Among the population-based cohort of 14,106 episodes of BSI studied, 10,543 were classified as CO based on “gold standard” 48-hour criteria. When 2-day and 3-day definitions were applied, 10,396 and 10,707 CO-BSI episodes were ascertained, respectively. All but 147 (1.4%) true CO-BSI cases were included by using the 2-day definition. When the 3-day definition was applied, all cases of CO-BSI were identified but and additional 164 (1.5%) cases of hospital-onset HO-BSI were also included. Thus the sensitivity and specificity of the 2-day definition was 98.6% and 100% and for the 3-day definition was 100% and 98.5%, respectively. Overall, only 311 (2.2%) cases were potentially miss-classifiable using either the 2- or 3-calendar day based definitions.
Use of either a 2- or 3-day definition is highly accurate for classifying CO-BSI.
PMCID: PMC4389998  PMID: 25889421
Nosocomial; Bacteremia; Community-acquired; Incidence; Epidemiology
6.  Invasive amoebiasis: A review of Entamoeba infections highlighted with case reports 
Entamoeba infections primarily involve the gastrointestinal tract and, although rare in North America, are common in the developing world. Infections can range from asymptomatic to severe or fatal invasions of multiple organ systems. Most cases in North America involve first-generation immigrant populations and returning international travellers. It can be difficult to differentiate Entamoeba histolytica-associated colitis from inflammatory bowel disease and invasive bacterial dysentery. Moreover, specific tests for E histolytica infection are not readily available in many centres, and stool studies and sigmoidoscopy can miss cases. Following two case presentations, this review discusses several aspects of these types of infection and stresses the importance of keeping E histolytica-associated colitis in differential diagnoses.
Entamoeba histolytica infections of the gastrointestinal tract are common in the developing world but rare in North America. The authors present two cases: one involving an individual who had not travelled to an endemic area and another involving an individual who was born in Bulgaria. Both presented with severe abdominal pain and diarrhea. Endoscopic assessment revealed scattered colonic ulcerations and one patient was found to have a liver abscess on imaging. Stool ova and parasite studies were negative in both cases and both were diagnosed on review of colonic biopsies. On review of all Entamoeba cases in the Calgary Health Zone (Alberta), ova and parasite analysis found an average of 63.7 Entamoeba cases per year and a pathology database review revealed a total of seven cases of invasive E histolytica (2001 to 2011). Both patients responded well to antibiotic therapy. E histolytica should be considered in new-onset colitis, especially in individuals from endemic areas.
PMCID: PMC4144452  PMID: 25157525
Amoebic colitis; Entamoeba histolytica; Extraintestinal abscesses
7.  International Multicenter Evaluation of the DiversiLab Bacterial Typing System for Escherichia coli and Klebsiella spp. 
Journal of Clinical Microbiology  2013;51(12):3944-3949.
Successful multidrug-resistant clones are increasing in prevalence globally, which makes the ability to identify these clones urgent. However, adequate, easy-to-perform, and reproducible typing methods are lacking. We investigated whether DiversiLab (DL), an automated repetitive-sequence-based PCR bacterial typing system (bioMérieux), is suitable for comparing isolates analyzed at different geographic centers. A total of 39 Escherichia coli and 39 Klebsiella species isolates previously typed by the coordinating center were analyzed. Pulsed-field gel electrophoresis (PFGE) confirmed the presence of one cluster of 6 isolates, three clusters of 3 isolates, and three clusters of 2 isolates for each set of isolates. DL analysis was performed in 11 centers in six different countries using the same protocol. The DL profiles of 425 E. coli and 422 Klebsiella spp. were obtained. The DL system showed a lower discriminatory power for E. coli than did PFGE. The local DL data showed a low concordance, as indicated by the adjusted Rand and Wallace coefficients (0.132 to 0.740 and 0.070 to 1.0 [E. coli] and 0.091 to 0.864 and 0.056 to 1.0 [Klebsiella spp.], respectively). The central analysis showed a significantly improved concordance (0.473 to 1.0 and 0.290 to 1.0 [E. coli] and 0.513 to 0.965 and 0.425 to 1.0 [Klebsiella spp.], respectively). The misclassifications of profiles for individual isolates were mainly due to inconsistent amplification, which was most likely due to variations in the quality and amounts of the isolated DNA used for amplification. Despite local variations, the DL system has the potential to indicate the occurrence of clonal outbreaks in an international setting, provided there is strict adherence to standardized, reproducible DNA isolation methods and analysis protocols, all supported by a central database for profile comparisons.
PMCID: PMC3838019  PMID: 24025914
8.  Community-associated methicillin-resistant Staphylococcus aureus necrotizing pneumonia without evidence of antecedent viral upper respiratory infection 
Pneumonia caused by community-associated methicillin-resistant Staphylococcus aureus is frequently complicated and severe, with a high mortality rate. This case series describes a group of patients with necrotizing pneumonia caused by community-associated methicillin-resistant Staphylococcus aureus who were treated at three hospitals in Calgary, Alberta. Laboratory results and clinical outcomes are described.
USA300 community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) strains causing necrotizing pneumonia have been reported in association with antecedent viral upper respiratory tract infections (URI).
A case series of necrotizing pneumonia presenting as a primary or coprimary infection, secondary to CA-MRSA without evidence of antecedent viral URI, is presented. Cases were identified through the infectious diseases consultation service records. Clinical and radiographic data were collected by chart review and electronic records. MRSA strains were isolated from sputum, bronchoalveolar lavage, pleural fluid or blood cultures and confirmed using standard laboratory procedures. MRSA strains were characterized by susceptibility testing, pulsed-field gel electrophoresis, spa typing, agr typing and multilocus sequence typing. Testing for respiratory viruses was performed by appropriate serological testing of banked sera, or nucleic acid testing of nasopharyngeal or bronchoalveloar lavage specimens.
Ten patients who presented or copresented with CA necrotizing pneumonia secondary to CA-MRSA from April 2004 to October 2011 were identified. The median length of stay was 22.5 days. Mortality was 20.0%. Classical risk factors for CA-MRSA were identified in seven of 10 (70.0%) cases. Chest tube placement occurred in seven of 10 patients with empyema. None of the patients had historical evidence of antecedent URI. In eight of 10 patients, serological or nucleic acid testing testing revealed no evidence of acute viral coinfection. Eight strains were CMRSA-10 (USA300). The remaining two strains were a USA300 genetically related strain and a USA1100 strain.
Pneumonia secondary to CA-MRSA can occur in the absence of an antecedent URI. Infections due to CA-MRSA are associated with significant morbidity and mortality. Clinicians need to have an awareness of this clinical entity, particularly in patients who are in risk groups that predispose to exposure to this bacterium.
PMCID: PMC4173983  PMID: 25285117
Community-associated methicillin-resistant; Staphylococcus aureus; Necrosis; Pneumonia; Viral infection
9.  Gastrointestinal Viral Load and Enteroendocrine Cell Number Are Associated with Altered Survival in HIV-1 Infected Individuals 
PLoS ONE  2013;8(10):e75967.
Human immunodeficiency virus type 1 (HIV-1) infects and destroys cells of the immune system leading to an overt immune deficiency known as HIV acquired immunodeficiency syndrome (HIV/AIDS). The gut associated lymphoid tissue is one of the major lymphoid tissues targeted by HIV-1, and is considered a reservoir for HIV-1 replication and of major importance in CD4+ T-cell depletion. In addition to immunodeficiency, HIV-1 infection also directly causes gastrointestinal (GI) dysfunction, also known as HIV enteropathy. This enteropathy can manifest itself as many pathological changes in the GI tract. The objective of this study was to determine the association of gut HIV-1 infection markers with long-term survival in a cohort of men who have sex with men (MSM) enrolled pre-HAART (Highly Active Antiretroviral Therapy). We examined survival over 15-years in a cohort of 42 HIV-infected cases: In addition to CD4+ T cell counts and HIV-1 plasma viral load, multiple gut compartment (duodenum and colon) biopsies were taken by endoscopy every 6 months during the initial 3-year period. HIV-1 was cultured from tissues and phenotyped and viral loads in the gut tissues were determined. Moreover, the tissues were subjected to an extensive assessment of enteroendocrine cell distribution and pathology. The collected data was used for survival analyses, which showed that patients with higher gut tissue viral load levels had a significantly worse survival prognosis. Moreover, lower numbers of serotonin (duodenum) and somatostatin (duodenum and colon) immunoreactive cell counts in the gut tissues of patients was associated with significant lower survival prognosis. Our study, suggested that HIV-1 pathogenesis and survival prognosis is associated with altered enteroendocrine cell numbers, which could point to a potential role for enteroendocrine function in HIV infection and pathogenesis.
PMCID: PMC3797816  PMID: 24146801
10.  Point prevalence study of antibiotic susceptibility of genital group B streptococcus isolated from near-term pregnant women in Calgary, Alberta 
Genital group B streptococcus (GBS) may be transmitted from a colonized mother to her infant if appropriate intrapartum antibiotic prophylaxis is not given. A recent case of GBS neonatal sepsis occurred due to an erythromycin-intermediate strain after empirical use of this drug as intrapartum prophylaxis.
To determine the regional antibiotic resistance rates of genital GBS isolates to penicillin, erythromycin and clindamycin.
A total of 309 genital GBS strains cultured from vaginal/rectal swabs were prospectively isolated and randomly selected between March and May 2011. Etest strips (bioMèrieux, France) were used to determine the minimum inhibitory concentrations to penicillin, erythromycin and clindamycin according to standard methods. All isolates that either demonstrated intermediate or full resistance to erythromycin had a D-test performed to detect inducible resistance to clindamycin. The resistance mechanism for each isolate was inferred from its antibiogram phenotype.
All genital GBS isolates were susceptible to penicillin, but high rates of resistance were found to both erythromycin (25%) and clindamycin (22%), mainly due to acquisition of erythromycin ribosomal methylation genes (erm) that result in the MLSB resistance phenotype. Most often the MLSB resistance phenotype was constitutive (MLSB-C; 14.2%) rather than inducible (MLSB-I; 8.1%), and an efflux mechanism (msrA; 3%) was much less common.
The present article is the first point prevalence study of genital GBS antibiogram profile that has been reported from a Canadian health care region. The high rates of resistance of genital GBS to both erythromycin and clindamycin is mainly due to the acquisition and spread of erm genes conveying the MSLB phenotype.
Changes to clinical and laboratory practice in the Calgary, Alberta, region were made to prevent additional cases of neonatal GBS sepsis due to inappropriate intrapartum antibiotic prescription.
PMCID: PMC3476555  PMID: 23997778
Antibiotic resistance; Group B streptococcus; Neonatal sepsis
11.  Laboratory Detection of Enterobacteriaceae That Produce Carbapenemases 
Journal of Clinical Microbiology  2012;50(12):3877-3880.
A study was designed to evaluate the modified Hodge test (MHT), Mastdiscs ID inhibitor combination disks (MDI), Rosco Diagnostica Neo-Sensitabs (RDS), metallo-β-lactamase (MBL) Etest, and in-house multiplex PCR for the detection of well-characterized carbapenemase-producing Enterobacteriaceae. One hundred forty-two nonrepeat clinical isolates of carbapenemase-producing Enterobacteriaceae (including Klebsiella spp., Escherichia coli, Citrobacter freundii, and Enterobacter spp.) obtained from the SMART worldwide surveillance program during 2008 to 2009 were included. These included 49 KPC-, 27 NDM-, 19 VIM-, 14 OXA-48-like enzyme-, and 5 IMP-producing isolates and 28 carbapenem-resistant, carbapenemase-negative isolates. The manufacturer's instructions were followed for MDI, RDS, and MBL Etest and CLSI guidelines for MHT. A multiplex PCR was designed to detect KPC, NDM, VIM, IMP, and OXA-48-like carbapenemases. Overall, the sensitivity and specificity were 78% and 93% for MDI, 80% and 93% for RDS, 58% and 93% for MHT, and 55% and 100% for MBL Etest, respectively. The PCR had 100% sensitivity and specificity. MDI and RDS performed well for the detection of KPCs and NDMs but poorly for VIMs, IMPs, and OXA-48-like enzymes. MHT performed well for KPCs and OXA-48-like enzymes but poorly for NDMs, VIMs, and IMPs. MDI and RDS were easy to perform and interpret but lacked sensitivity for OXA-48-like enzymes, VIMs, and IMPs. MHT and MBL Etest were often difficult to interpret. We recommend using molecular tests for the optimal detection of carbapenemase-producing Enterobacteriaceae.
PMCID: PMC3503014  PMID: 22993175
12.  Population-Based Laboratory Surveillance of Imported Malaria in Metropolitan Calgary, 2000–2011 
PLoS ONE  2013;8(4):e60751.
Increased travel leads to a heightened risk of imported infectious diseases. Patterns of immigration to countries like Canada have changed such that countries of malaria endemicity are frequented in larger numbers. In keeping with the changes in travel patterns and immigration, the major metropolitan city of Calgary has seen a dramatic rise in malaria incidence over the last decade. Fuelling this rise in Calgary has been the apparent complacence with prophylaxis in individuals visiting friends and relatives and potentially inadequate public health intervention in areas of the city with increased immigration and lower socioeconomic status.
PMCID: PMC3626683  PMID: 23613742
13.  The distinct category of healthcare associated bloodstream infections 
Bloodstream infections (BSI) have been traditionally classified as either community acquired (CA) or hospital acquired (HA) in origin. However, a third category of healthcare-associated (HCA) community onset disease has been increasingly recognized. The objective of this study was to compare and contrast characteristics of HCA-BSI with CA-BSI and HA-BSI.
All first episodes of BSI occurring among adults admitted to hospitals in a large health region in Canada during 2000-2007 were identified from regional databases. Cases were classified using a series of validated algorithms into one of HA-BSI, HCA-BSI, or CA-BSI and compared on a number of epidemiologic, microbiologic, and outcome characteristics.
A total of 7,712 patients were included; 2,132 (28%) had HA-BSI, 2,492 (32%) HCA-BSI, and 3,088 (40%) had CA-BSI. Patients with CA-BSI were significantly younger and less likely to have co-morbid medical illnesses than patients with HCA-BSI or HA-BSI (p < 0.001). The proportion of cases in males was higher for HA-BSI (60%; p < 0.001 vs. others) as compared to HCA-BSI or CA-BSI (52% and 54%; p = 0.13). The proportion of cases that had a poly-microbial etiology was significantly lower for CA-BSI (5.5%; p < 0.001) compared to both HA and HCA (8.6 vs. 8.3%). The median length of stay following BSI diagnosis 15 days for HA, 9 days for HCA, and 8 days for CA (p < 0.001). Overall the most common species causing bloodstream infection were Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae. The distribution and relative rank of importance of these species varied according to classification of acquisition. Twenty eight day all cause case-fatality rates were 26%, 19%, and 10% for HA-BSI, HCA-BSI, and CA-BSI, respectively (p < 0.001).
Healthcare-associated community onset infections are distinctly different from CA and HA infections based on a number of epidemiologic, microbiologic, and outcome characteristics. This study adds further support for the classification of community onset BSI into separate CA and HCA categories.
PMCID: PMC3364909  PMID: 22487002
14.  A case of acute cholecystitis caused by methicillin-resistant Staphylococcus aureus in an immunocompromised patient 
Although infections with Staphylococcus aureus can implicate multiple organ systems, involvement of the biliary tract is rare. A case of acute cholecystitis and bacteremia with methicillin-resistant S aureus (MRSA) in a patient with HIV infection is presented. The MRSA isolate was found to be a community-associated strain. The present case highlights the invasive nature of staphylococcal infections and the emerging importance of community-associated MRSA strains.
PMCID: PMC3076155  PMID: 22379489
Cholecystitis; Hematogenous seeding; HIV; Methicillin-resistant Staphylococcus aureus
15.  Community-Based Outbreaks in Vulnerable Populations of Invasive Infections Caused by Streptococcus pneumoniae Serotypes 5 and 8 in Calgary, Canada 
PLoS ONE  2011;6(12):e28547.
Outbreaks of invasive pneumococcal disease (IPD) typically occur within institutions. Beginning in 2005, we detected an increase in serotype (ST) 5 and ST8 IPD cases, predominantly in homeless persons living in an open community.
Methodology/Principal Findings
CASPER (Calgary Area S. pneumoniae Epidemiology Research) surveillance study of all IPD (sterile site isolates) in our region (pop ∼1,100,000). Interviews and chart reviews of all cases and all isolates phenotypically analyzed and selected isolated tested by multi-locus sequence typing (MLST).
During 2005–2007, 162 cases of ST5 IPD and 45 cases of ST8 IPD were identified. The isolates demonstrated phenotypic and genotypic clonality. The ST5 isolates were sequence type (ST) 289 and demonstrated intermediate susceptibility to TMP-SMX. The ST8 isolates were predominantly ST1268, with a susceptible antimicrobial susceptibility profile. Individuals with ST5 IPD were more likely to be middle aged (OR 2.6), homeless (OR 4.4), using illicit drugs(OR 4.8), and asthmatic(OR 2.6). Those with ST8 were more likely to be male (OR 4.4), homeless (OR 2.6), aboriginal (OR7.3), and a current smoker (OR 2.5). Overlapping outbreaks of ST5 and ST8 IPD occurred in an open community in Calgary, Canada and homelessness was a predominant risk factor. Homelessness represents a unique community in which pneumococcal outbreaks can occur.
PMCID: PMC3246448  PMID: 22216100
16.  Comparison of the RealTime HIV-1, COBAS TaqMan 48 v1.0, Easy Q v1.2, and Versant v3.0 assays for Determination of HIV-1 Viral Loads in a Cohort of Canadian Patients with Diverse HIV Subtype Infections▿  
Journal of Clinical Microbiology  2010;49(1):118-124.
HIV clinics in Canada provide care to an increasing number of patients born outside of Canada with HIV-1 non-B subtype infections. Because the Easy Q HIV-1 v1.2 assay (EQ; bioMérieux) failed to detect some non-B subtype infections, a multiassay HIV-1 viral load (VL) study was conducted with patients with diverse HIV subtype infections. Patients were enrolled from the Southern Alberta HIV Clinic (SAC), Calgary, Alberta, Canada (n = 349) and the McGill HIV Clinic (MHC), Montreal, Quebec, Canada (n = 20) and had four or five tubes of blood drawn for testing by EQ and three other commercial HIV VL assays: (i) the Versant 3.0 HIV-1 test, with the Versant 440 instrument (branched DNA [bDNA]; Siemens), (ii) the RealTime HIV-1 test, with the m2000rt instrument (m2000rt; Abbott Molecular Diagnostics), and (iii) the COBAS AmpliPrep TaqMan HIV-1 48 test (CAP-CTM; Roche Molecular Diagnostics). Blood was processed according to the individual manufacturer's requirements and stored frozen at −86°C. The HIV subtype was known for patients who had undergone HIV genotypic resistance testing (Virco, Belgium). Data analyses were done using standard statistical methods within Stata 9.0 (StataCorp, College Station, TX). A total of 371 samples were tested on 369 patients, of whom 291 (81%) had a Virco genotype result of B (195; 53%) or non-B (96; 26%) subtypes A to D and F to K, as well as circulating recombinant forms (CRFs) (i.e., CRF01_AE and CRF02_AG). Most (58/78; 74%) patients of unknown subtype were recent African emigrants who likely have non-subtype B infection. Overall bias was small in pairwise Bland-Altman plots, but the limits of agreement between assays were wide. Discordant viral load results occurred for 98 samples and were due to missing values, false negatives, and significant underquantification that varied by HIV subtype. Results were obtained for all 371 samples with m2000rt, but for only 357 (97%) with CAP-CTM, 338 (92%) with EQ, and 276 (75%) with bDNA due to errors/equipment failures. False-negative results (nondetection of viral RNA versus other assay results) occurred for all platforms, as follows: for m2000rt, 8 (2%) [B(4) and non-B(4) subtypes], CAP-CTM, 9 (2.5%) [B(6) and non-B(3) subtypes]; EQ, 20 (6%) [B(7) and non-B(13) subtypes]; bDNA, 5 (2%) [B(1) and C(4)]. EQ and bDNA had the highest rates of underquantification by ≥1.0 log10 copies/ml, mainly for HIV non-B subtypes. Performance significantly varied between HIV VL platforms according to subtype. HIV viral diversity in the population being tested must be considered in selection of the viral load platform.
PMCID: PMC3020439  PMID: 21084515
17.  Antimicrobial susceptibility of invasive and lower respiratory tract isolates of Streptococcus pneumoniae, 1998 to 2007 
Previous surveys of antimicrobial resistance in Streptococcus pneumoniae have found differences depending on source of isolate (eg, higher resistance in lower respiratory tract [LRT] versus invasive isolate) and age (higher resistance in children versus adults). Susceptibility profiles in the Calgary Health Region (approximately 1.25 million population) over a 10-year period were studied. Prospective laboratory-based population surveillance for S pneumoniae disease has been conducted since 1998. Patient demographics and susceptibility testing were analyzed. In total, 2382 patient isolates were available for analysis from 1998 to 2007. Of these, 1170 isolates were invasive while 496 were LRT. Patient age distribution was: younger than five years, 14%; five to 17 years, 6%; 18 to 64 years, 56%; and 65 years or older, 24%. Mean patient age was 44.8 years and 60.0% were male. The overall incidence of nonsusceptibility was: penicillin, 8.2%; amoxicillin, 0.3%; cefuroxime, 6.2%; ceftriaxone, 1.7%; erythromycin, 8.8%; trimethoprim-sulfamethoxazole (TMP-SMX), 25.6%; clindamycin, 2.3%; and levofloxacin, 0.2%. Overall resistance rates were stable, except for increasing erythromycin resistance from 5.4% (1998) to a high of 14.2% (2004) (P=0.007). Isolates that were nonsusceptible to penicillin or TMP-SMX were more likely to be multidrug resistant (P<0.001) compared with penicillin- or TMP-SMX-susceptible isolates. Compared with invasive isolates, LRT isolates showed more resistance to penicillin, TMP-SMX, cefuroxime and erythromycin, and were more likely to be multidrug resistant. Isolates from children younger than five years of age are more likely to be multidrug resistant and resistant to erythromycin and cefotaxime. Ongoing surveillance of S pneumoniae isolates is important because resistance rates vary by source and patient age among health care regions.
PMCID: PMC2807249  PMID: 21119791
Antimicrobial susceptibility; Lower respiratory tract infection; Streptococcus pneumoniae
18.  Higher levels of Zidovudine resistant HIV in the colon compared to blood and other gastrointestinal compartments in HIV infection 
Retrovirology  2010;7:74.
The gut-associated lymphoid tissue (GALT) is the largest lymphoid organ infected by human immunodeficiency virus type 1 (HIV-1). It serves as a viral reservoir and host-pathogen interface in infection. This study examined whether different parts of the gut and peripheral blood lymphocytes (PBL) contain different drug-resistant HIV-1 variants.
Gut biopsies (esophagus, stomach, duodenum and colon) and PBL were obtained from 8 HIV-1 infected preHAART (highly active antiretroviral therapy) patients at three visits over 18 months. Patients received AZT, ddI or combinations of AZT/ddI. HIV-1 Reverse transcriptase (RT)-coding sequences were amplified from viral DNA obtained from gut tissues and PBL, using nested PCR. The PCR fragments were cloned and sequenced. The resulting sequences were subjected to phylogenetic analyses, and antiretroviral drug mutations were identified.
Phylogenetic and drug mutation analyses revealed differential distribution of drug resistant mutations in the gut within patients. The level of drug-resistance conferred by the RT sequences was significantly different between different gut tissues and PBL, and varied with antiretroviral therapy. The sequences conferring the highest level of drug-resistance to AZT were found in the colon.
This study confirms that different drug-resistant HIV-1 variants are present in different gut tissues, and it is the first report to document that particular gut tissues may select for drug resistant HIV-1 variants.
PMCID: PMC2949729  PMID: 20836880
19.  Human granulocytic anaplasmosis: First reported case in Canada 
Human granulocytic anaplasmosis (HGA) is a tick-borne rickettsial infection of peripheral blood neutrophils caused by Anaplasma phagocytophilum. While this infection is increasingly recognized as endemic throughout much of the United States, no Canadian cases have been previously described, despite the agent being identified in Canadian ticks. Herein we present a case of HGA acquired in an urban Alberta centre. Canadian physicians must be aware of the possibility of tick-borne rickettsial diseases as etiology of fever in individuals presenting with leukopenia/lymphopenia, thrombocytopenia and elevated transaminases during periods of tick activity. Prompt recognition and treatment are important in minimizing resultant morbidity and mortality.
PMCID: PMC2770309  PMID: 20808448
Anaplasma phagocytophilum; Ehrlichia; Ixodes; Leukopenia; Thrombocytopenia; Ticks
20.  Pyogenic ventriculitis complicating Aggregatibacter aphrophilus infective endocarditis: A case report and literature review 
Pyogenic ventriculitis (PV) is an uncommon, but frequently fatal infection that results from inflammation of the ventricular ependymal lining associated with a purulent ventricular system. PV has been rarely reported as a secondary complication of infective endocarditis. Prompt diagnosis and treatment with appropriate culture-directed antibiotics with adequate central nervous system penetration is crucial when managing patients who are suspected of having PV. The present study reports on a fatal case of a previously well 42-year-old alcoholic woman with infective endocarditis caused by Aggregatibacter aphrophilus, with secondary brain abscess and spontaneous rupture into the ventricles causing PV.
PMCID: PMC2770311  PMID: 20808450
Aggregatibacter aphrophilus; Community-acquired; HACEK; Haemophilus aphrophilus; Infective endocarditis; Meningitis
21.  Clinical outcome of empiric antimicrobial therapy of bacteremia due to extended-spectrum beta-lactamase producing Escherichia coli and Klebsiella pneumoniae 
BMC Research Notes  2010;3:116.
Prompt administration of adequate empiric antimicrobial therapy is a major determinant influencing the outcome of serious infections. The objective of this study was to describe empiric antimicrobial therapy employed and assess its effect on the outcome of patients bacteremic with extended-spectrum beta-lactamase (ESBL) producing Escherichia coli and Klebsiella pneumoniae.
A retrospective surveillance study of all patients with bacteremias caused by ESBL-producing E. coli and K. pneumoniae (EK-ESBL) from 2000-2007 in the Calgary Health Region was conducted. Data were available for 79 episodes of bacteremia among 76 patients. Forty-four (56%) were male, the median age was 70.0 yrs [interquartile range (IQR) 60.6-70.1 yrs], and 72 (91%) episodes were E. coli. Seventy-four episodes (94%) were treated with empiric therapy within the first 48 hours. A non-statistically significant increased mortality occurred in those treated empirically with a beta-lactam/beta-lactamase inhibitor combination (6/16; 38% vs. 10/53; 18%; p = 0.063) while empiric carbapenem therapy was associated with lower mortality (0/10 died vs. 16/53 (30%), p = 0.089). Only 42 (53%) episodes received adequate therapy within the first 48 hours. The median time to first adequate antibiotic therapy was 41.0 hours [IQR 5.8-59.5] (n = 75). The case-fatality rate was not different among those that received adequate compared to inadequate therapy by 48 hours as compared to inadequate empiric therapy (9/42; 21% vs. 7/37; 19%; p = 1.0).
Inadequate empiric therapy is common among patients with EK-ESBL bacteremia in our region but was not associated with adverse mortality outcome.
PMCID: PMC2877056  PMID: 20423493
22.  Molecular Characteristics of Travel-Related Extended-Spectrum-β-Lactamase-Producing Escherichia coli Isolates from the Calgary Health Region▿  
Extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli has recently emerged as a major risk factor for community-acquired, travel-related infections in the Calgary Health Region. Molecular characterization was done on isolates associated with infections in returning travelers using isoelectric focusing, PCR, and sequencing for blaCTX-Ms, blaTEMs, blaSHVs, blaOXAs, and plasmid-mediated quinolone resistance determinants. Genetic relatedness was determined with pulsed-field gel electrophoresis using XbaI and multilocus sequence typing (MLST). A total of 105 residents were identified; 6/105 (6%) presented with hospital-acquired infections, 9/105 (9%) with health care-associated community-onset infections, and 90/105 (86%) with community-acquired infections. Seventy-seven of 105 (73%) of the ESBL-producing E. coli isolates were positive for blaCTX-M genes; 55 (58%) produced CTX-M-15, 13 (14%) CTX-M-14, six (6%) CTX-M-24, one (1%) CTX-M-2, one (1%) CTX-M-3, and one (1%) CTX-M-27, while 10 (10%) produced TEM-52, three (3%) TEM-26, 11 (11%) SHV-2, and four (4%) produced SHV-12. Thirty-one (30%) of the ESBL-producing E. coli isolates were positive for aac(6′)-Ib-cr, and one (1%) was positive for qnrS. The majority of the ESBL-producing isolates (n = 95 [90%]) were recovered from urine samples, and 83 (87%) were resistant to ciprofloxacin. The isolation of CTX-M-15 producers belonging to clone ST131 was associated with travel to the Indian subcontinent (India, Pakistan), Africa, the Middle East, and Europe, while clonally unrelated strains of CTX-M-14 and -24 were associated with travel to Asia. Our study suggested that clone ST131 coproducing CTX-M-15, OXA-1, TEM-1, and AAC(6′)-Ib-cr and clonally unrelated CTX-M-14 producers have emerged as important causes of community-acquired, travel-related infections.
PMCID: PMC2687226  PMID: 19364876
23.  Using a Commercial DiversiLab Semiautomated Repetitive Sequence-Based PCR Typing Technique for Identification of Escherichia coli Clone ST131 Producing CTX-M-15▿  
Journal of Clinical Microbiology  2009;47(4):1212-1215.
A study was designed to evaluate the ability of the DiversiLab fingerprinting kit, a type of repetitive element PCR (rep-PCR), to identify Escherichia coli clone ST131 producing β-lactamase CTX-M-15. A set of 53 nonduplicate isolates of extended-spectrum β-lactamase-producing E. coli underwent rep-PCR, pulsed-field gel electrophoresis, and multilocus sequence typing. The DiversiLab system successfully identified E. coli clone ST131 producing CTX-M-15 and provides a simple standardized typing protocol for monitoring the spread of this clone.
PMCID: PMC2668328  PMID: 19204095
24.  Rationale for and protocol of a multi-national population-based bacteremia surveillance collaborative 
BMC Research Notes  2009;2:146.
Bloodstream infections are frequent causes of human illness and cause major morbidity and death. In order to best define the epidemiology of these infections and to track changes in occurrence, adverse outcome, and resistance rates over time, population based methodologies are optimal. However, few population-based surveillance systems exist worldwide, and because of differences in methodology inter-regional comparisons are limited. In this report we describe the rationale and propose first practical steps for developing an international collaborative approach to the epidemiologic study and surveillance for bacteremia.
The founding collaborative participants represent six regions in four countries in three continents with a combined annual surveillance population of more than 8 million residents.
Future studies from this collaborative should lead to a better understanding of the epidemiology of bloodstream infections.
PMCID: PMC2721840  PMID: 19624839

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