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Biomedical subjects

L Louie

Publications and source records attributed to L Louie.

At least 19 recordsLinked to original sources
PubMed2006

Detection of severe acute respiratory syndrome coronavirus in stool specimens by commercially available real-time reverse transcriptase PCR assays.

Three commercially available real-time reverse transcriptase PCR assays (the Artus RealArt HPA coronavirus LightCycler, the Artus RealArt HPA coronavirus Rotor-Gene, and the EraGen severe acute respiratory syndrome coronavirus POL assay) and three RNA extraction methodologies were evaluated for the detection of severe acute respiratory syndrome coronavirus RNA from 91 stool specimens. The assays' sensitivities were highest (58% to 75%) for specimens obtained 8 to 21 days after symptom onset. The assays were less sensitive when specimens were obtained less than 8 days or more than 21 days after the onset of symptoms. All assays were 100% specific.

Feces↗
PubMed2006

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.

Feces↗
PubMed2002

Rapid detection of methicillin-resistant staphylococci from blood culture bottles by using a multiplex PCR assay.

Rapid detection and accurate identification of methicillin-resistant staphylococci are critical for the effective management of infections caused by these organisms. We describe a multiplex PCR-based assay for the direct detection of methicillin-resistant staphylococci from blood culture bottles (BacT/Alert; Organon-Teknika, Durham, N.C.). A simple lysis method followed by a multiplex PCR assay designed to detect the nuc, mecA, and bacterial 16S rRNA genes was performed. A total of 306 blood culture specimens were collected over a period of 10 months from June 1998 to April 1999, consisting of 236 blood cultures growing staphylococci (including 124 methicillin-resistant Staphylococcus spp.), 50 positive blood cultures which grew organisms other than staphylococci, and 20 blood cultures that were negative for bacterial and fungal pathogens after 5 days of incubation and terminal subculture. DNA extraction, PCR, and detection could be completed in 2.5 h. Of the positive blood cultures with staphylococci, the multiplex PCR assay had a sensitivity and specificity of 99.2% and 100%, respectively. Our results show that rapid, direct detection of methicillin-resistant staphylococci is possible, allowing clinicians to make prompt and effective decisions for the management of patients with staphylococcal bacteremia.

Bacteremia↗
PubMed2001

Spontaneous entrapment of polynucleotides upon electrostatic interaction with ethanol-destabilized cationic liposomes.

This study describes the effect of ethanol and the presence of poly(ethylene) glycol (PEG) lipids on the interaction of nucleotide-based polyelectrolytes with cationic liposomes. It is shown that preformed large unilamellar vesicles (LUVs) containing a cationic lipid and a PEG coating can be induced to entrap polynucleotides such as antisense oligonucleotides and plasmid DNA in the presence of ethanol. The interaction of the cationic liposomes with the polynucleotides leads to the formation of multilamellar liposomes ranging in size from 70 to 120 nm, only slightly bigger than the parent LUVs from which they originated. The degree of lamellarity as well as the size and polydispersity of the liposomes formed increases with increasing polynucleotide-to-lipid ratio. A direct correlation between the entrapment efficiency and the membrane-destabilizing effect of ethanol was observed. Although the morphology of the liposomes is still preserved at the ethanol concentrations used for entrapment (25-40%, v/v), entrapped low-molecular-weight solutes leak rapidly. In addition, lipids can flip-flop across the membrane and exchange rapidly between liposomes. Furthermore, there are indications that the interaction of the polynucleotides with the cationic liposomes in ethanol leads to formation of polynucleotide-cationic lipid domains, which act as adhesion points between liposomes. It is suggested that the spreading of this contact area leads to expulsion of PEG-ceramide and triggers processes that result in the formation of multilamellar systems with internalized polynucleotides. The high entrapment efficiencies achieved at high polyelectrolyte-to-lipid ratios and the small size and neutral character of these novel liposomal systems are of utility for liposomal delivery of macromolecular drugs.

Cations↗
PubMed2001

Genetic variability and linkage disequilibrium within the HLA-DP region: analysis of 15 different populations.

In order to understand the forces governing the evolution of the genetic diversity in the HLA-DP molecule, polymerase chain reaction (PCR)-based methods were used to characterize genetic variation at the DPA1 and DPB1 loci encoding this heterodimer on 2,807 chromosomes from 15 different populations including individuals of African, Asian, Amerindian, Indian and European origin. These ethnically diverse samples represent a variety of population substructures and include small, isolated populations as well as larger, presumably admixed populations. Ten DPA1 and 39 DPB1 alleles were identified and observed on 87 distinct DP haplotypes, 34 of which were found to be in significant positive linkage disequilibrium in at least one population. Some haplotypes were found in all ethnic groups while others were confined to a single ethnic group or population. Strong positive global linkage disequilibrium (Wn) between DPA1 and DPB1 was present in all 15 populations. The African populations displayed the lowest values of Wn whereas the Amerindian populations displayed near absolute disequilibrium. Analysis of the distribution of haplotypes using the normalized deviate of the Ewens-Watterson homozygosity statistic, F, suggests that DP haplotypes encoding the functional heterodimer are subject to much lower degrees of balancing selection than other loci within the HLA region. Finally, neighbor joining tree analyses demonstrate the power of haplotype diversity for inferring the relationships between the different populations.

Alleles↗
PubMed2001

Genotyping TAP2 variants in North American Caucasians, Brazilians, and Africans.

The protein forms of transporter associated with antigen processing, subunit 2 (TAP2), differ either by amino acid substitutions (Thr374Ala, Ile379Val, Ile467Val, Thr565Ala, Val577Met, Cys651Arg, and Ala665Thr) or by a truncation (Gln687Stop) of 17 amino acid residues at the C-terminus. Nonsynonymous single nucleotide polymorphisms (N-SNPs) causing these amino acid variations except 577Val were detected in genomic DNA samples from North American Caucasians (n = 76), Brazilians (n = 148), Rwandans (n = 285), and Zambians (n = 117). Exclusive (100%) and nearly exclusive (>95%) linkage disequilibrium was seen with a number of N-SNPs. The average heterozygosity at any given dimorphic site ranged from 7.3% to 44.6%, and at least four N-SNPs showed clear population specificity. N-SNP combinations alone led to the identification of 16 relatively common alleles, which appeared to form at least three lineages. Further analyses of 101 cDNA samples from Brazilians detected nine expressed TAP2 alleles, four of which matched the official assignments. Genetic complexity at the TAP2 locus was further enhanced by two out of five synonymous SNPs (S-SNPs), especially the GGT386GGG (Gly) that had similar heterozygosity rates in Caucasians (28.9%), Rwandans (33.3%), and Zambians (33.3%). Overall, distribution of both synonymous and nonsynonymous SNPs in the various ethnic groups examined here conformed well to the Hardy-Weinberg equilibrium, and between 57.9% and 77.0% of subjects in each ethnic group were heterozygous with two TAP2 alleles predicted to differ by at least one amino acid residue. Such complexity of TAP2 polymorphisms, in the form of SNPs as well as alleles, is likely to complicate the analyses of disease associations and haplotype structures in the HLA class II region.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Development of a Canadian standardized protocol for subtyping methicillin-resistant Staphylococcus aureus using pulsed-field gel electrophoresis.

A panel of 24 methicillin-resistant Staphylococcus aureus strains was distributed to 15 laboratories in Canada to evaluate their in-house pulsed-field gel electrophoresis (PFGE) protocols and interpretation criteria. Attempts to compare fingerprint images using computer-aided analysis were not successful due to variability in individual laboratory PFGE protocols. In addition, individual site interpretation of the fingerprint patterns was inadequate, as 7 of 13 sites (54%) made at least one error in interpreting the fingerprints from the panel. A 2-day standardized PFGE protocol (culture to gel image) was developed and distributed to all of the sites. Each site was requested to use the standardized protocol on five strains from the original panel. Thirteen sites submitted gel images for comparisons. The protocol demonstrated excellent reproducibility and allowed interlaboratory comparisons with Molecular Analyst DST software (Bio-Rad) and 1.5% band tolerance.

Bacterial Typing Techniques↗
PubMed2001

Evaluation of a latex agglutination test (MRSA-Screen) for detection of oxacillin resistance in coagulase-negative Staphylococci.

The MRSA-Screen (Denka-Seiken, Tokyo, Japan) latex agglutination test was evaluated for its ability to detect PBP 2a from 200 clinical isolates of coagulase-negative staphylococci (CoNS; 84 mecA-positive strains and 116 mecA-negative strains) consisting of 108 Staphylococcus epidermidis, 37 S. saprophyticus, 15 S. haemolyticus, 11 S. hominis, 10 S. capitis, 10 S. warneri, and 3 S. lugdunensis species as well as 6 other species of CoNS. The assay was compared with susceptibility testing with an agar screen plate with oxacillin at 6 microg/ml (OXA6), by oxacillin disk diffusion (DD), by broth microdilution (BMDIL), by the E test, and with Vitek GPS-SV and Vitek GPS-107 susceptibility cards. PCR for the detection of the mecA gene was used as the "gold standard." The sensitivities and specificities for the methods evaluated were as follows: MRSA-Screen, 100 and 100%, respectively; OXA6, 100 and 99%, respectively; DD, 98 and 62%, respectively; BMDIL, 100 and 60%, respectively; E test, 100 and 51%, respectively; Vitek GPS-SV susceptibility card, 98 and 87%, respectively; and Vitek GPS-107 susceptibility card, 100 and 61%, respectively. The MRSA-Screen test accurately and rapidly detected oxacillin resistance in CoNS.

Bacterial Proteins↗
PubMed2000

The role of DNA amplification technology in the diagnosis of infectious diseases.

Nucleic acid amplification and detection methods developed in the past decade are useful for the diagnosis and management of a variety of infectious diseases. The most widely used of these methods is the polymerase chain reaction (PCR). PCR assays can detect rapidly and accurately the presence of fastidious and slow-growing microorganisms, such as Chlamydia, mycoplasmas, mycobacteria, herpesviruses and enteroviruses, directly from clinical specimens. Commercial PCR assays for the diagnosis of tuberculosis and genital C. trachomatis infection are now routinely used in many diagnostic laboratories. Assays have also been developed that can detect antimicrobial resistance and are used to identify the cause of infection by organisms that cannot be cultivated. The value of viral load measurement by nucleic acid amplification in the management of patients with HIV infection or hepatitis C has also been well established. However, evaluations of this technology for rapid microbial diagnosis have generally been limited by small samples, and the cost of these assays may be as high as Can$125 per test. As nucleic acid amplification methods continue to evolve, their role in the diagnosis and management of patients with infectious diseases and their impact on clinical outcomes will become better defined.

Communicable Diseases↗
PubMed2000

PCR detection of Escherichia coli O157:H7 directly from stools: evaluation of commercial extraction methods for purifying fecal DNA.

Rapid identification of Escherichia coli O157:H7 is important for patient management and for prompt epidemiological investigations. We evaluated one in-house method and three commercially available kits for their ability to extract E. coli O157:H7 DNA directly from stool specimens for PCR. Of the 153 stool specimens tested, 107 were culture positive and 46 were culture negative. The sensitivities and specificities of the in-house enrichment method, IsoQuick kit, NucliSens kit, and QIAamp kit were comparable, as follows: 83 and 98%, 85 and 100%, 74 and 98%, and 86 and 100%, respectively. False-negative PCR results may be due to the presence of either inherent inhibitors or small numbers of organisms. The presence of large amounts of bacteria relative to the amount of the E. coli O157:H7 target may result in the lower sensitivities of the assays. All commercial kits were rapid and easy to use, although DNA extracted with the QIAamp kit did not require further dilution of the DNA template prior to PCR.

Culture Media↗
PubMed2000

Evaluation of three rapid methods for detection of methicillin resistance in Staphylococcus aureus.

The probe-based Velogene Rapid MRSA Identification Assay (ID Biomedical Corp., Vancouver, British Columbia, Canada) and the latex agglutination MRSA-Screen (Denka Seiken Co., Tokyo, Japan) were evaluated for their ability to identify methicillin-resistant Staphylococcus aureus (MRSA) and to distinguish strains of MRSA from borderline oxacillin-resistant S. aureus (BORSA; mecA-negative, oxacillin MICs of 2 to 8 microgram/ml). The Velogene is a 90-min assay using a chimeric probe to detect the mecA gene. MRSA-Screen is a 15-min latex agglutination test with penicillin-binding protein 2a antibody-sensitized latex particles. We compared these assays with the BBL Crystal MRSA ID System (Becton Dickinson, Cockeysville, Md.) and with PCR for mecA gene detection. A total of 397 clinical isolates of S. aureus were tested, consisting of 164 methicillin-susceptible strains, 197 MRSA strains, and 37 BORSA strains. All assays performed well for the identification of MRSA with sensitivities and specificities for Velogene, MRSA-Screen, and BBL Crystal MRSA ID of 98.5 and 100%, 98.5 and 100%, and 98.5 and 98%, respectively. Three MRSA strains were not correctly identified by each of the Velogene and MRSA-Screen assays, but repeat testing with a larger inoculum resolved the discrepancies. The BBL Crystal MRSA ID test misclassified four BORSA strains as MRSA. Both the Velogene and the MRSA-Screen assays are easy to perform, can accurately differentiate BORSA isolates from MRSA isolates, and provide a rapid alternative for the detection of methicillin resistance in S. aureus in clinical laboratories, especially when mecA PCR gene detection is unavailable.

Bacterial Proteins↗
PubMed1999

Human immunodeficiency virus type 1-specific cytotoxic T lymphocyte activity is inversely correlated with HIV type 1 viral load in HIV type 1-infected long-term survivors.

HIV-1-specific cytotoxic T cell (CTL) activity has been suggested to correlate with protection from progression to AIDS. We have examined the relationship between HIV-specific CTL activity and maintenance of peripheral blood CD4+ T lymphocyte counts and control of viral load in 17 long-term survivors (LTSs) of HIV-1 infection. Longitudinal analysis indicated that the LTS cohort demonstrated a decreased rate of CD4+ T cell loss (18 cells/mm3/year) compared with typical normal progressors (approximately 60 cells/mm3/year). The majority of the LTSs had detectable, variable, and in some individuals, quite high (>10(4) RNA copies/ml) plasma viral load during the study period. In a cross-sectional analysis, HIV-specific CTL activity to HIV Gag, Pol, and Env proteins was detectable in all 17 LTSs. Simultaneous analysis of HIV-1 Gag-Pol, and Env-specific CTLs and virus load in protease inhibitor-naive individuals showed a significant inverse correlation between Pol-specific CTL activity and plasma HIV-1 RNA levels (p = 0.001). Furthermore, using a mixed linear effects model the combined effects of HIV-1 Pol- and Env-specific CTL activity on the viral load were significantly stronger than the effects of HIV-1 Pol-specific CTL activity alone on predicted virus load. These data suggest that the presence of HIV-1-specific CTL activity in HIV-1-infected long-term survivors is an important component in the effective control of HIV-1 replication.

CD4 Lymphocyte Count↗
PubMed1999

Characterization and proposed nomenclature of epidemic strains of methicillin-resistant Staphylococcus aureus in Canada.

We hope that standardized nomenclature for identifying epidemic MRSA strains prevalent in Canadian hospitals will be helpful to physicians and infection control practitioners attempting to understand and control the spread of the organism in health-care facilities. It is anticipated that as MRSA continues to evolve in Canadian health-care facilities other strains may be recognized as "epidemic"; as these strains become better characterized they may be added to those designated above. Laboratory physicians and infection control personnel are invited to submit strains that may warrant characterization and designation as a Canadian epidemic strain to the Laboratory Centre for Disease Control, Health Canada, Winnipeg, Manitoba.

Canada↗
PubMed1999

Molecular typing methods to investigate transmission of Escherichia coli O157:H7 from cattle to humans.

The utility of phage typing, pulsed-field gel electrophoresis (PFGE), and plasmid profile analysis was compared, to differentiate between Canadian Escherichia coli O157:H7 strains of human (n = 27) and cattle (n = 24) origin. The diversity indices for phage typing, plasmid analysis and PFGE were 0.85, 0.69 and 0.93, respectively. PFGE and phage typing were also applied to study the role of direct transmission of E. coli O157:H7 from cattle to humans on isolates collected from two separate farm outbreaks. PFGE showed that more than one E. coli O157:H7 strain with varying PFGE DNA subtype profiles, may be responsible for an outbreak, and that more than one E. coli O157:H7 subtype may be circulating on a particular farm at any one time. To our knowledge, this is one of the first reports where PFGE typing was used to verify the direct transmission of E. coli O157:H7 from cattle to humans.

Animals↗
PubMed1999

Molecular analysis of the genetic variation among penicillin-susceptible and penicillin-resistant Streptococcus pneumoniae serotypes in Canada.

Few studies have examined the genetic relatedness within and between serotypes of Streptococcus pneumoniae. Penicillin-susceptible S. pneumoniae (PSSP) isolates (n=170) and penicillin-resistant S. pneumoniae (PRSP) isolates (n=48) belonging to 13 different serotypes from across Canada were studied to further understand the molecular epidemiology of S. pneumoniae. PRSP of serotypes 6B, 14, and 19F appeared to be novel clones, differentiated by pulsed-field gel electrophoresis from the recognized international penicillin-resistant clones belonging to these serotypes. In addition, not all PRSP strains shared similar genetic backgrounds with PSSP strains of the same serotype, suggesting that in Canada, certain PRSP strains have emerged from new unique lineages of PRSP. Although there was significant heterogeneity among S. pneumoniae of different serotypes, certain serotypes (3, 7F, and 22F) appeared to be more clonally related despite having originated from different geographic regions.

Cluster Analysis↗
PubMed1999

Synergy testing of vancomycin-resistant Enterococcus faecium against quinupristin-dalfopristin in combination with other antimicrobial agents.

Using checkerboard and time-kill assays, we evaluated the in vitro activity of quinupristin-dalfopristin (RP 59500) alone and in combination with five other antimicrobial agents against 12 clinical strains of vancomycin-resistant Enterococcus faecium (VREF). In time-kill studies, six VREF strains exhibited synergism with the combination of quinupristin-dalfopristin and doxycycline and three exhibited synergism with quinupristin-dalfopristin plus ampicillin-sulbactam. Combinations of quinupristin-dalfopristin with these and other agents warrant further clinical evaluation for the treatment of serious VREF infections.

Anti-Bacterial Agents↗
PubMed1998

Comparison of six rapid agglutination tests for the identification of Staphylococcus aureus, including methicillin-resistant strains.

Six rapid agglutination tests for the identification of Staphylococcus aureus were evaluated by testing 416 strains of staphylococci. The sensitivities and specificities of the tests were as follows: Staphyloslide, 97.7% and 99.3% respectively; Prolex Staph Latex, 96.9% and 93.6%; Staphaurex, 95.4% and 94.2%; Staphaurex Plus, 100% and 93.4%; Slidex Staph Plus, 98.9% and 99.3%; and Staphytect Plus, 99.6% and 88.2%. The three tests utilizing latex particles coated with staphylococcal capsular antibodies were better able to identify methicillin-resistant S. aureus, but only Staphyloslide and Slidex Staph Plus had adequate specificity.

Agglutination Tests↗