bluuebunny/crossref_metadata_2025 huggingface dataset:
Diagnostic Yield of a PCR Assay in Focal Complications of Brucellosis
Authors: Morata P. , Queipo-Ortuño M. I. , Reguera J. M. , Miralles F. , Lopez-Gonzalez J. J. , Colmenero J. D.
Published: July 2002

In order to evaluate the diagnostic yield of a PCR assay for patients with focal complications of brucellosis, we studied by PCR and by conventional microbiological techniques 34 nonblood samples from 32 patients with different focal forms of brucellosis. The samples from patients with brucellosis were paired to an equal number of control samples from the same locations of patients whose illnesses had different etiologies. Thirty-three of the 34 nonblood samples (97%) from the brucellosis patients were positive by PCR, whereas
Brucella
spp. were isolated from only 29.4% of the conventional cultures. For 11.4% of the patients, the confirmatory serological tests were either negative or showed titers below the diagnostic range. Two patients (6.2%) from the control group, both with tuberculous vertebral osteomyelitis, had a positive PCR result. The brucella PCR of blood from these two patients was also positive, and the two strains of
Mycobacterium tuberculosis
isolated were analyzed by the brucella PCR, with no evidence of amplification. These results show that the PCR assay is far more sensitive than conventional cultures, and this, coupled with its speed and reduction in risk to laboratory workers, makes this technique a very useful tool for the diagnosis of focal complications of brucellosis.
Rapid Detection of Enterovirus RNA in Cerebrospinal Fluid Specimens with a Novel Single-Tube Real-Time Reverse Transcription-PCR Assay
Authors: Verstrepen Walter A. , Kuhn Sofie , Kockx Mark M. , Van De Vyvere Martine E. , Mertens An H.
Published: July 2002

A single-tube real-time reverse transcription-PCR (RT-PCR) assay for enterovirus detection in cerebrospinal fluid (CSF) was developed based on a fluorogenic probe and primers directed to highly conserved sequences in the 5′ untranslated region of the enterovirus genome. Quantitative detection of enterovirus genome was demonstrated in a linear range spanning at least 5 logs. Endpoint titration experiments revealed that the in-tube detection limit of the assay was 11.8 enterovirus genome equivalents (95% detection rate) corresponding in our current extraction protocol to 592 enterovirus genome equivalents per ml of CSF. Twenty CSF specimens not suspected of viral meningitis were all found to be negative, and no cross-reactivity with herpes simplex virus type 1 and type 2, varicella-zoster virus, rhinovirus type 53, and influenza viruses A and B was observed. Nineteen CSF specimens from 70 patients suspected of viral meningitis were determined to be positive by PCR (27.1%), whereas only 17 were found to be positive by viral culture (24.3%). The sensitivity of the assay was 100% and the specificity was 96.2% compared to viral culture. Data from the real-time RT-PCR assay were available within 4 h. Our data suggest that the novel real-time RT-PCR assay may offer a reliable but significantly faster alternative to viral culture. Owing to the elimination of postamplification detection steps, its conduct required considerably less hands-on time and was associated with a substantially reduced carryover risk compared to previously described PCR-based enterovirus detection assays.
Outbreak of Infection with Multidrug-Resistant
Klebsiella pneumoniae
Carrying
bla
IMP-8
in a University Medical Center in Taiwan
Authors: Yan Jing-Jou , Ko Wen-Chien , Tsai Shu-Huei , Wu Hsiu-Mei , Wu Jiunn-Jong
Published: July 2002

Klebsiella pneumoniae
strains with the transferable carbapenem-hydrolyzing metallo-β-lactamases, which include IMP- and VIM-type enzymes, remain extremely rare. To investigate whether IMP- or VIM-producing
K. pneumoniae
isolates had spread at a university medical center in Taiwan, a total of 3,458 clinical isolates of
K. pneumoniae
consecutively collected in 1999 and 2000 were tested by the agar diffusion method, colony hybridization, PCR, and nucleotide sequencing. A total of 40 isolates (1.2%), or 17 nonrepetitive isolates, from 16 patients were found to carry
bla
IMP-8
, a metallo-β-lactamase gene recently identified from a
K. pneumoniae
strain in Taiwan. Carriage of
bla
VIM
or other
bla
IMP
genes was detected in none of the remaining isolates. Of the 17 nonrepetitive
bla
IMP-8
-positive isolates, 15 isolates (88.2%) appeared susceptible to imipenem (MICs, ≤4 μg/ml) and meropenem (MICs, ≤1 μg/ml), indicating the difficulty in detecting
bla
IMP-8
in
K. pneumoniae
by routine susceptibility tests; 14 isolates (82.4%) produced SHV-12 as well; and 14 isolates (82.4%) were also resistant to fluoroquinolones. The organisms caused wound infections in eight patients and bloodstream infections in three patients. They were not directly associated with the death of nine patients. Before the recovery of the
bla
IMP-8
-positive isolates, all 16 patients had undergone various surgical procedures, and 15 patients had been admitted to the surgical intensive care unit, suggesting a nosocomial outbreak. Two major patterns were observed by pulsed-field gel electrophoresis for 14 of the 17 nonrepetitive isolates, indicating that the clonal spread was mainly responsible for the outbreak.
Evaluation of BACTEC Mycobacteria Growth Indicator Tube (MGIT 960) Automated System for Drug Susceptibility Testing of
Mycobacterium tuberculosis
Authors: Ardito Fausta , Posteraro Brunella , Sanguinetti Maurizio , Zanetti Stefania , Fadda Giovanni
Published: July 2002

The reliability of the BACTEC MGIT 960 system, an automated version of the Mycobacteria Growth Indicator Tube (MGIT), for antimicrobial susceptibility testing of
Mycobacterium tuberculosis
was evaluated on 78 clinical isolates. Rifampin (RMP), isoniazid (INH), streptomycin (SM), and ethambutol (EMB) were tested at the following concentrations: 1.0 μg/ml for RMP, 0.1 and 0.4 μg/ml for INH, 1.0 and 4.0 μg/ml for SM, and 5.0 and 7.5 μg/ml for EMB. Results were compared with those obtained by the BACTEC 460 TB radiometric system. Initially the reproducibility study showed 99.5% agreement on repeat testing with all the four drugs. With susceptibility testing of clinical isolates, excellent agreement between the two systems was found for all the drugs. A total of nine major errors were observed for only three isolates, resistant according to BACTEC MGIT 960 and susceptible according to BACTEC 460 TB, to SM (4.0 μg/ml), INH (0.1 μg/ml), and EMB (5.0 μg/ml) (one isolate) and to SM (1.0 μg/ml), INH (0.4 μg/ml), and EMB (5.0 μg/ml) (two isolates). When these isolates were tested by using the conventional proportion method on Löwenstein-Jensen medium, agreement with BACTEC MGIT 960 was found for five results and with BACTEC 460 TB for the remainder. The time to report results was 7.9 days by MGIT 960 and 7.3 days by BACTEC 460 TB, which was not found statistically significant (
P
> 0.05). In conclusion, the performance of BACTEC MGIT 960 was found similar to that of BACTEC 460 TB and this new system can be considered a good alternative to the radiometric method for routine susceptibility testing of
M. tuberculosis
.
Analysis of Growth Characteristics of Filamentous Fungi in Different Nutrient Media
Authors: Meletiadis Joseph , Meis Jacques F. G. M. , Mouton Johan W. , Verweij Paul E.
Published: July 2002

A microbroth kinetic model based on turbidity measurements was developed in order to analyze the growth characteristics of three species of filamentous fungi (
Rhizopus microsporus, Aspergillus fumigatus
, and
Scedosporium prolificans
) characterized by different growth rates in five nutrient media (antibiotic medium 3, yeast nitrogen base medium, Sabouraud broth, RPMI 1640 alone, and RPMI 1640 with 2% glucose). In general, five distinct phases in the growth of filamentous fungi could be distinguished, namely, the lag phase, the first transition period, the log phase, the second transition period, and the stationary phase. The growth curves were smooth and were characterized by the presence of long transition periods. Among the different growth phases distinguished, the smallest variability in growth rates among the strains of each species was found during the log phase in all nutrient media. The different growth phases of filamentous fungi were barely distinguishable in RPMI 1640, in which the poorest growth was observed for all fungi even when the medium was supplemented with 2% glucose.
R. microsporus
and
A. fumigatus
grew better in Sabouraud and yeast nitrogen base medium than in RPMI 1640, with growth rates three to four times higher. None of the media provided optimal growth of
S. prolificans
. The germination of
Rhizopus
spores and
Aspergillus
and
Scedosporium
conidia commenced after 2 and 5 h of incubation, respectively. The elongation rates ranged from 39.6 to 26.7, 25.4 to 20.2, and 16.9 to 9.9 μm/h for
Rhizopus, Aspergillus
, and
Scedoporium
hyphae, respectively. The germination of conidia and spores and the elongation rates of hyphae were enhanced in antibiotic medium 3 and delayed in yeast nitrogen base medium. In conclusion, the growth curves provide a useful tool to gain insight into the growth characteristics of filamentous fungi in different nutrient media and may help to optimize the methodology for antifungal susceptibility testing.
Epidemiological Patterns of Rotaviruses Causing Severe Gastroenteritis in Young Children throughout Australia from 1993 to 1996
Authors: Bishop Ruth F. , Masendycz Paul J. , Bugg Helen C. , Carlin John B. , Barnes Graeme L.
Published: July 2002

Rotavirus strains that caused severe diarrhea in 4,634 (2,533 male) children aged less than 5 years and admitted to major hospitals in eight centers throughout Australia from 1993 to 1996 were subject to antigenic and genetic analyses. The G serotypes of rotaviruses were identified in 81.9% (3,793 of 4,634) children. They included 67.8% (from 3,143 children) serotype G1 isolates (containing 46 electropherotypes), 11.5% (from 531 children) serotype G2 isolates (27 electropherotypes), 0.8% (from 39 children) serotype G3 isolates (8 electropherotypes), and 1.6% (from 76 children) serotype G4 isolates (9 electropherotypes). G6 (two strains) and G8 (two strains) isolates were identified during the same period. G1 serotypes were predominant in all centers, with intermittent epidemics of G2 serotypes and sporadic detection of G3 and G4 strains. With the exception of two strains (typed as G1P2A[6] and G2P2A[6]) all serotype G1, G3, and G4 strains were P1A[8] and all serotype G2 strains were P1B[4]. Two contrasting epidemiological patterns were identified. In all temperate climates rotavirus incidence peaked during the colder months. The genetic complexity of strains (as judged by electropherotype) was greatest in centers with large populations. Identical electropherotypes appeared each winter in more than one center, apparently indicating the spread of some strains both from west to east and from east to west. Centers caring for children in small aboriginal communities showed unpredictable rotavirus peaks unrelated to climate, with widespread dissemination of a few rotavirus strains over distances of more than 1,000 km. Data from continued comprehensive etiological studies of genetic and antigenic variations in rotaviruses that cause severe disease in young children will serve as baseline data for the study of the effect of vaccination on the incidence of severe rotavirus disease and on the emergence of new strains.
Multisite Comparison of Reproducibility and Recovery from the Standard and Ultrasensitive Roche AMPLICOR HIV-1 MONITOR Assays
Authors: Brambilla Donald J. , Granger Suzanne , Jennings Cheryl , Bremer James W.
Published: July 2002

Reproducibility and recovery from the standard and ultrasensitive Roche AMPLICOR HIV-1 MONITOR kits were compared in 19 laboratories. The results were generally similar, but the consistently low level of recovery from the ultrasensitive assay in one laboratory points to the need to include external controls in order to track assay performance.
Comparison between the LCx Probe System and the COBAS AMPLICOR System for Detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
Infections in Patients Attending a Clinic for Treatment of Sexually Transmitted Diseases in Amsterdam, The Netherlands
Authors: van Doornum G. J. J. , Schouls L. M. , Pijl A. , Cairo I. , Buimer M. , Bruisten S.
Published: July 2002

Two assays for the detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
were compared: the LCx Probe system (the LCx system; Abbott Diagnostic Laboratories, North Chicago, Ill.) and the COBAS AMPLICOR
C. trachomatis/N. gonorrhoeae
system (the COBAS AMPLICOR system; Roche Diagnostic Systems, Branchburg, N.J.). Endocervical swab specimens, male urethral swab specimens, and female and male urine specimens were collected from 503 female and 498 male visitors attending a sexually transmitted diseases clinic in Amsterdam, The Netherlands. Prevalences for
C. trachomatis
were 12.5% (63 of 503) and 10.0% (50 of 498) in females and males, respectively. The prevalences for
N. gonorrhoeae
were 1.2% (6 of 503) and 4.2% (21 of 498) in females and males, respectively. Both assays showed high values for sensitivity and specificity with regard to the detection of
C. trachomatis
in endocervical swab specimens, male urethral swab specimens, and female and male urine specimens. The sensitivities for the LCx system were 92.1, 90.0, 88.9, and 94.0% for each type of specimen, respectively; and the sensitivies for the COBAS AMPLICOR system were 96.8, 98.0, 82.5, and 92.0% for each type of specimen, respectively. Specificities ranged between 98.4 and 100%. The sensitivity of the LCx system for the detection of
N. gonorrhoeae
was 100% for female cervical swab and urine specimens and male urethral swab specimens, while for male urine specimens the sensitivity was 95.2%; the specificity was 100% for all types of specimens. For the detection of
N. gonorrhoeae
by the COBAS AMPLICOR assay, the sensitivity for female cervical swab and male urethral swab specimens was 100%, that for female urine specimens was 66.7%, and that for male urine specimens was 95.2%. However, the predictive values of a positive test for female cervical swab specimens and urine specimens were 31.6 and 36.4%, respectively. Sequence analysis of the amplimers obtained by an in-house 16S rRNA PCR of the solely COBAS AMPLICOR system-positive swab specimens revealed neither
N. gonorrhoeae
nor other
Neisseria
spp. The COBAS AMPLICOR assay was considered not suitable for screening for infections with
N. gonorrhoeae
. If this assay is used for detection of
N. gonorrhoeae
, confirmation of positive results by a reliable test is mandatory.
Comparative Performance of Three Viral Load Assays on Human Immunodeficiency Virus Type 1 (HIV-1) Isolates Representing Group M (Subtypes A to G) and Group O: LCx HIV RNA Quantitative, AMPLICOR HIV-1 MONITOR Version 1.5, and Quantiplex HIV-1 RNA Version 3.0
Authors: Swanson Priscilla , Soriano Vincent , Devare Sushil G. , Hackett John
Published: July 2002

The performance of the LCx HIV RNA Quantitative (LCx HIV), AMPLICOR HIV-1 MONITOR version 1.5 (MONITOR v1.5), and Quantiplex HIV-1 RNA version 3.0 (bDNA v3.0) viral load assays was evaluated with 39 viral isolates (3 A, 7 B, 6 C, 4 D, 8 E, 4 F, 1 G, 4 mosaic, and 2 group O). Quantitation across the assay dynamic ranges was assessed using serial fivefold dilutions of the viruses. In addition, sequences of
gag
-encoded p24 (
gag
p24),
pol
-encoded integrase, and
env
-encoded gp41 were analyzed to assign group and subtype and to assess nucleotide mismatches at primer and probe binding sites. For group M isolates, quantification was highly correlated among all three assays. In contrast, only the LCx HIV assay reliably quantified group O isolates. The bDNA v3.0 assay detected but consistently underquantified group O viruses, whereas the MONITOR v1.5 test failed to detect group O viruses. Analysis of target regions revealed fewer primer or probe mismatches in the LCx HIV assay than in the MONITOR v1.5 test. Consistent with the high level of nucleotide conservation is the ability of the LCx HIV assay to quantify efficiently human immunodeficiency virus type 1 group M and the genetically diverse group O.
Detection of
Mycobacterium bovis
in Bovine Clinical Specimens Using Real-Time Fluorescence and Fluorescence Resonance Energy Transfer Probe Rapid-Cycle PCR
Authors: Taylor Malcolm James , Hughes Mary Siobhan , Skuce Robin Alfred , Neill Sydney Donnelly
Published: July 2002

Nucleic acid sequence capture extraction was coupled with LightCycler PCR amplification and product detection using real-time fluorescence for rapid, definitive detection of
Mycobacterium bovis
in lymph node specimens from 38 cattle with bovine tuberculosis lesions. PCR amplification of sequence-captured DNA using both a conventional heating block thermocycler and a LightCycler thermocycler was compared with culture and histopathological analyses. Conventional PCR enabled detection of 26 of 28 culture-positive specimens (93%) in approximately 9 h, and the LightCycler PCR detected 20 of 28 culture-positive specimens (71%) in only 30 min. Specific confirmation of
Mycobacterium tuberculosis
complex DNA was achieved by LightCycler PCR amplification using Syb Green 1 and an
M. tuberculosis
complex-specific Cy5-labeled fluorescence resonance energy transfer probe. The system described here enabled rapid and specific laboratory confirmation of bovine tuberculosis, and this is the first report of the detection of
M. bovis
in tissues using LightCycler PCR. The fluorescence technology used in the study has potential to allow development of a high-throughput molecular diagnostic test for bovine tuberculosis.
Fluconazole and Voriconazole Multidisk Testing of
Candida
Species for Disk Test Calibration and MIC Estimation
Authors: Kronvall Göran , Karlsson Inga
Published: July 2002

Fluconazole and voriconazole MICs were determined for 114 clinical
Candida
isolates, including isolates of
Candida albicans, Candida glabrata, Candida krusei, Candida lusitaniae, Candida parapsilosis
, and
Candida tropicalis
. All strains were susceptible to voriconazole, and most strains were also susceptible to fluconazole, with the exception of
C. glabrata
and
C. krusei
, the latter being fully fluconazole resistant. Single-strain regression analysis (SRA) was applied to 54 strains, including American Type Culture Collection reference strains. The regression lines obtained were markedly different for the different
Candida
species. Using an MIC limit of susceptibility to fluconazole of ≤8 μg/ml, according to NCCLS standards, the zone breakpoint for susceptibility for the 25-μg fluconazole disk was calculated to be ≥18 mm for
C. albicans
and ≥22 mm for
C. glabrata
and
C. krusei
. SRA results for voriconazole were used to estimate an optimal disk content according to rational criteria. A 5-μg disk content of voriconazole gave measurable zones for a tentative resistance limit of 4 μg/ml, whereas a 2.5-μg disk gave zones at the same MIC level for only three of the species. A novel SRA modification, multidisk testing, was also applied to the two major species,
C. albicans
and
C. glabrata
, and the MIC estimates were compared with the true MICs for the isolates. There was a significant correlation between the two measurements. Our results show that disk diffusion methods might be useful for azole testing of
Candida
isolates. The method can be calibrated using SRA. Multidisk testing gives direct estimations of the MICs for the isolates.
Application and Evaluation of the Interlaboratory Reproducibility of tRNA Intergenic Length Polymorphism Analysis (tDNA-PCR) for Identification of
Streptococcus
Species
Authors: Baele Margo , Storms Virginie , Haesebrouck Freddy , Devriese Luc A. , Gillis Monique , Verschraegen Gerda , de Baere Thierry , Vaneechoutte Mario
Published: July 2002

The discriminatory power, speed, and interlaboratory reproducibility of tRNA intergenic length polymorphism analysis (tDNA-PCR) combined with capillary electrophoresis was evaluated for the identification of streptococci. This method was carried out in three different laboratories under highly standardized conditions for 54 strains belonging to 18 different species. It was concluded that interlaboratory reproducibility of tDNA fingerprints produced by means of capillary electrophoresis was sufficiently high to permit the exchange between different laboratories and the construction of common libraries which can be consulted for comparison with fingerprints obtained independently in separate laboratories. In a second step, 17 other species were included in the study and examined in one of the participating laboratories. All
Streptococcus
species studied, except
S. mitis, S. oralis, S. parasanguinis, S. pneumoniae, S. thermophilus
, and
S. vestibularis
, showed distinguishable tDNA fingerprints. A database of well-characterized strains was constructed to enable computer-aided identification of unknown streptococcal isolates.
Serum Is the Preferred Clinical Specimen for Diagnosis of Human Brucellosis by PCR
Authors: Zerva L. , Bourantas K. , Mitka S. , Kansouzidou A. , Legakis N. J.
Published: July 2002

Human brucellosis poses a significant public health problem in many developing countries and requires fast and accurate diagnosis. A PCR assay amplifying part of the 31-kDa
Brucella abortus
antigenic protein gene sequence was developed and applied to whole-blood and serum samples from 31 brucellosis patients and 45 healthy individuals. All patients except one had detectable
Brucella
DNA in either whole blood or serum (combined sensitivity, 97%), but the assay sensitivity was higher with serum samples (94%) than with whole-blood samples (61%). The assay specificity was excellent (100%). A confirmatory PCR assay targeting another
Brucella
gene region (
omp-2
) was also developed but lacked sensitivity. Serum is the optimal specimen for the diagnosis of brucellosis by PCR, a choice that leads to assay simplification and shortens turnaround time.
Three Days of Incubation May Be Sufficient for Routine Blood Cultures with BacT/Alert FAN Blood Culture Bottles
Authors: Bourbeau Paul P. , Pohlman Janice K.
Published: July 2002

BacT/Alert FAN blood culture bottles have been shown to enhance the recovery of bacteria and yeast from blood compared with standard BacT/Alert bottles. It is well established that standard BacT/Alert blood culture bottles require no more than 5 days of incubation for the detection of routine bacteria and yeast. It is less clear, however, whether FAN bottles also routinely require 5 days of incubation. To address this question, we recently reviewed the results of 17,887 blood culture sets collected in FAN blood culture bottles at Geisinger Medical Center. Of these cultures, 1,780 were positive for bacteria or yeast, yielding a total of 1,242 clinically significant isolates. The numbers of isolates recovered on days 1, 2, 3, 4, and 5 were as follows: (values in parentheses are percentages of total significant isolates): 877 (71%), 269 (22%), 65 (5%), 18 (1%) and, 13 (1%), respectively. In total, 97.5% of all clinically significant isolates were detected in the first 3 days of incubation. Of the 31 significant isolates detected on day 4 or 5 of incubation, 17 were detected in concurrent blood cultures within the first 3 days of incubation. Chart reviews were conducted for the 13 patients with the remaining 14 isolates detected on day 4 or 5 to determine whether therapy was changed due to this blood culture result. Therapy was changed for only 1 patient. These results suggest that it may not be necessary to routinely incubate FAN blood culture bottles for more than 3 days.
Evaluation of a Modified Sanitary Napkin as a Sample Self-Collection Device for the Detection of Genital Chlamydial Infection in Women
Authors: Alary Michel , Poulin Céline , Bouchard Céline , Fortier Michel , Murray Gilles , Gingras Suzanne , Aubé Michel , Morin Carol
Published: July 2002

A modified sanitary napkin was compared with endocervical swab and urine specimens for the detection of urogenitalChlamydia trachomatisinfection. Endocervical swabs and/or first-catch urine were collected from 510 women at medical or community settings in Quebec City. Participants were also asked to wear a modified sanitary napkin (Ezy-Detek) during 4 consecutive hours and to bring it back to the clinic or mail it to the laboratory. Endocervical and urine specimens were tested using the Cobas Amplicor CT/NG assay (Roche Diagnostic Systems) according to the manufacturer's instructions, as were specimens collected with the napkin after adequate preparation. If the PCR test result was positive on the endocervical sample or on any two samples, a woman was considered to be infected. PCR testing results on paired samples were identical for 493 (96.6%) of 510 women. According to the definition given above, 58 (11.3%; 95% confidence interval [CI], 8.7 to 14.5%) women were infected withC. trachomatis. The sensitivity and specificity of PCR testing on modified sanitary napkin specimens were, respectively, 93.1% (54 of 58; 95% CI, 83.3 to 98.1%) and 98.9% (447 of 452; 95% CI, 97.4 to 99.6%) compared to 81.0% (47 of 58; 95% CI, 68.6 to 90.1%) and 100% (451 of 451; 95% CI, 99.2 to 100%) for urine specimens. The positive and negative predictive values were, respectively, 91.5% (54 of 59) and 99.1% (447 of 451) for the sanitary napkin specimens compared to 100% (47 of 47) and 97.6% (451 of 462) for urine samples. These results suggest that a modified sanitary napkin represents an effective noninvasive device for self-collection of specimens to detect urogenitalC. trachomatisinfection.
Identification of Rifampin-Resistant
Mycobacterium tuberculosis
Strains by Hybridization, PCR, and Ligase Detection Reaction on Oligonucleotide Microchips
Authors: Mikhailovich Vladimir , Lapa Sergey , Gryadunov Dimitry , Sobolev Alexander , Strizhkov Boris , Chernyh Nikolai , Skotnikova Olga , Irtuganova Olga , Moroz Arkadii , Litvinov Vitalii , Vladimirskii Mikhail , Perelman Mikhail , Chernousova Larisa , Erokhin Vladislav , Zasedatelev Alexander , Mirzabekov Andrei
Published: July 2002

Three new molecular approaches were developed to identify drug-resistant strains of
Mycobacterium tuberculosis
using biochips with oligonucleotides immobilized in polyacrylamide gel pads. These approaches are significantly faster than traditional bacteriological methods. All three approaches—hybridization, PCR, and ligase detection reaction—were designed to analyze an 81-bp fragment of the gene
rpoB
encoding the β-subunit of RNA polymerase, where most known mutations of rifampin resistance are located. The call set for hybridization analysis consisted of 42 immobilized oligonucleotides and enabled us to identify 30 mutant variants of the
rpoB
gene within 24 h. These variants are found in 95% of all mutants whose rifampin resistance is caused by mutations in the 81-bp fragment. Using the second approach, allele-specific on-chip PCR, it was possible to directly identify mutations in clinical samples within 1.5 h. The third approach, on-chip ligase detection reaction, was sensitive enough to reveal rifampin-resistant strains in a model mixture containing 1% of resistant and 99% of susceptible bacteria. This level of sensitivity is comparable to that from the determination of
M. tuberculosis
drug resistance by using standard bacteriological tests.
Endophthalmitis Caused by
Listeria monocytogenes
Authors: Betriu Carmen , Fuentemilla Santos , Méndez Rosalı́a , Picazo Juan J. , Garcı́a-Sánchez Julián
Published: July 2002

Listeria monocytogenes
was isolated from the aqueous chamber of an immunodepressed patient with acute hypertensive uveitis, who developed a dark hypopyon and pigment dispersion. No extraocular septic focus was found. Treatment was successful with intravitreal vancomycin, anterior chamber irrigation with vancomycin, orally administered ciprofloxacin, and topical fortified vancomycin.
Identification of 54 Mycobacterial Species by PCR-Restriction Fragment Length Polymorphism Analysis of the
hsp65
Gene
Authors: Brunello Francesca , Ligozzi Marco , Cristelli Emanuela , Bonora Stefano , Tortoli Enrico , Fontana Roberta
Published: July 2002

A total of 121 reference and clinical strains of both slowly and rapidly growing mycobacteria belonging to 54 species were studied for restriction fragment length polymorphism of a PCR-amplified 439-bp segment of the gene encoding the 65-kDa heat shock protein. Restriction digests were separated by 10% polyacrylamide gel electrophoresis (PAGE). By including a size standard in each sample, the restriction fragment profile was calculated using a computer-aided comparison program. An algorithm describing these 54 species (including 22 species not previously described) is proposed. We found that this assay based on 10% PAGE provided a more precise estimate than that based on agarose gel electrophoresis of the real size of restriction fragments as deduced from the sequence analysis and allowed identification of mycobacteria whose PCR-restriction fragment length polymorphism analysis patterns were unequivocally identified by fragments shorter than 60 bp.
Detection and Quantification of Infectious Hypodermal and Hematopoietic Necrosis Virus and White Spot Virus in Shrimp Using Real-Time Quantitative PCR and SYBR Green Chemistry
Authors: Dhar Arun K. , Roux Michelle M. , Klimpel Kurt R.
Published: July 2002

A rapid and highly sensitive real-time PCR detection and quantification method for infectious hypodermal and hematopoietic necrosis virus (IHHNV), a single-stranded DNA virus, and white spot virus (WSV), a double-stranded DNA (dsDNA) virus infecting penaeid shrimp (
Penaeus
sp.), was developed using the GeneAmp 5700 sequence detection system coupled with SYBR Green chemistry. The PCR mixture contains a fluorescence dye, SYBR Green, which upon binding to dsDNA exhibits fluorescence enhancement. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. A linear relationship was observed between the amount of input plasmid DNA and cycle threshold (

C
T

) values over a range of 1 to 10
5
copies of the viral genome. To control the variation in sampling and processing among samples, the shrimp β-actin gene was amplified in parallel with the viral DNA. The

C
T

values of IHHNV- and WSV-infected samples were used to determine absolute viral copy numbers from the standard

C
T

curves of these viruses. For each virus and its β-actin control, the specificity of amplification was monitored by using the dissociation curve of the amplified product. Using genomic DNA as a template, SYBR Green PCR was found to be 100- to 2000-fold more sensitive than conventional PCR, depending on the virus, for the samples tested. The results demonstrate that SYBR Green PCR can be used as a rapid and highly sensitive detection and quantification method for shrimp viruses and that it is amenable to high-throughout assay.
Transmission of Hepatitis C Virus in a Gynecological Surgery Setting
Authors: Massari Marco , Petrosillo Nicola , Ippolito Giuseppe , Solforosi Laura , Bonazzi Lucio , Clementi Massimo , Manzin Aldo
Published: July 2002

A cluster of hepatitis C virus (HCV) infections among gynecological patients who underwent surgical intervention in the same setting is described. An epidemiological investigation was conducted to identify the cases, the likely source of infection, and the route of transmission. Four recent HCV infections were identified. Based on molecular fingerprinting analysis and epidemiological investigation, transmission between the putative source patient (an HCV-positive woman who was the first patient of the surgical session) and outbreak patients was highly suggestive. All patients, including the source patient, were infected with HCV type 1b. Molecular characterization of HCV clones by sequence analysis of both structural envelope regions (20 clones from the source patient and 58 from the outbreak patients) and the nonstructural NS5 region of the viral genome (12 clones from the source patient and 32 from the outbreak patients) showed close homology between the viral isolates from the source and those from the outbreak patients that was higher than that observed between the viral isolates from the source and those from four unrelated, HCV type 1b-infected patients from the same geographical area (in the latter case, 33 clones were sequenced for the envelope regions and 30 were sequenced for the NS5 region). The mean percent divergence between clones was 4.69 for the envelope and 3.71 for the NS5 region in the source patient and the outbreak patients compared with 6.76 (
P
= 0.001) and 5.22 (
P
= 0.01) in the source patient and control patients, respectively. Among the risk factors investigated, only that of having undergone surgery in the morning session of the same day reached statistical significance (
P
= 0.003). The investigation showed that the source patient and outbreak patients shared only the administration of propofol in multidose vials. The study documents the risk of nosocomial transmission of HCV and the importance of infection control procedures in the operating room and highlights the crucial role of molecular strategies, especially sequence-based phylogenetic analysis of cloned viral isolates, in the investigation of HCV outbreaks.
Detection of Antibodies to U.S. Isolates of Avian Pneumovirus by a Recombinant Nucleocapsid Protein-Based Sandwich Enzyme-Linked Immunosorbent Assay
Authors: Gulati Baldev R. , Munir Shirin , Patnayak Devi P. , Goyal Sagar M. , Kapur Vivek
Published: July 2002

The nucleocapsid (N) protein of subgroup C (United States-specific) avian pneumovirus (APV/US) was expressed in
Escherichia coli
, and antibodies to the recombinant N protein were shown to specifically recognize the ≈47-kDa N protein of APV/US by Western immunoblot analysis. The recombinant APV/US N protein was used in a sandwich-capture enzyme-linked immunosorbent assay (ELISA), and the resulting assay was found to be more sensitive and specific than the routine indirect ELISA for the detection of APV/US antibodies in turkey sera.
Evidence of Extrahepatic Sites of Replication of the Hepatitis E Virus in a Swine Model
Authors: Williams T. P. E. , Kasorndorkbua C. , Halbur P. G. , Haqshenas G. , Guenette D. K. , Toth T. E. , Meng X. J.
Published: July 2002

Hepatitis E virus (HEV) is the major cause of enterically transmitted non-A, non-B hepatitis in many developing countries and is also endemic in many industrialized countries. Due to the lack of an effective cell culture system and a practical animal model, the mechanisms of HEV pathogenesis and replication are poorly understood. Our recent identification of swine HEV from pigs affords us an opportunity to systematically study HEV replication and pathogenesis in a swine model. In an early study, we experimentally infected specific-pathogen-free pigs with two strains of HEV: swine HEV and the US-2 strain of human HEV. Eighteen pigs (group 1) were inoculated intravenously with swine HEV, 19 pigs (group 2) were inoculated with the US-2 strain of human HEV, and 17 pigs (group 3) were used as uninoculated controls. The clinical and pathological findings have been previously reported. In this expanded study, we aim to identify the potential extrahepatic sites of HEV replication using the swine model. Two pigs from each group were necropsied at 3, 7, 14, 20, 27, and 55 days postinoculation (DPI). Thirteen different types of tissues and organs were collected from each necropsied animal. Reverse transcriptase PCR (RT-PCR) was used to detect the presence of positive-strand HEV RNA in each tissue collected during necropsy at different DPI. A negative-strand-specific RT-PCR was standardized and used to detect the replicative, negative strand of HEV RNA from tissues that tested positive for the positive-strand RNA. As expected, positive-strand HEV RNA was detected in almost every type of tissue at some time point during the viremic period between 3 and 27 DPI. Positive-strand HEV RNA was still detectable in some tissues in the absence of serum HEV RNA from both swine HEV- and human HEV-inoculated pigs. However, replicative, negative-strand HEV RNA was detected primarily in the small intestines, lymph nodes, colons, and livers. Our results indicate that HEV replicates in tissues other than the liver. The data from this study may have important implications for HEV pathogenesis, xenotransplantation, and the development of an in vitro cell culture system for HEV.
Multicenter Proficiency Testing of Nucleic Acid Amplification Methods for the Detection of Enteroviruses
Authors: Van Vliet Karin E. , Muir Peter , Echevarria Jose M. , Klapper Paul E. , Cleator Graham M. , Van Loon Anton M.
Published: July 2002

A multicenter study of molecular detection of enteroviruses was conducted using a proficiency panel. Of 70 data sets, 46 (66%) reported correct results for samples containing at least 1 50% infective dose per ml and for negative samples. Variation in performance between laboratories demonstrates the need for ongoing quality control.
Development and Evaluation of a Quantitative, Touch-Down, Real-Time PCR Assay for Diagnosing
Pneumocystis carinii
Pneumonia
Authors: Larsen Hans Henrik , Masur Henry , Kovacs Joseph A. , Gill Vee J. , Silcott Victoria A. , Kogulan Palaniandy , Maenza Janine , Smith Margo , Lucey Daniel R. , Fischer Steven H.
Published: July 2002

A rapid (time to completion, <4 h, including DNA extraction) and quantitative touch-down (QTD) real-time diagnostic
Pneumocystis carinii
PCR assay with an associated internal control was developed, using fluorescence resonance energy transfer (FRET) probes for detection. The touch-down procedure significantly increased the sensitivity of the assay compared to a non-touch-down procedure. Tenfold serial dilutions of a cloned target were used as standards for quantification.
P. carinii
DNA has been detected in respiratory specimens from patients with
P. carinii
pneumonia (PCP) and from patients without clinical evidence of PCP. The latter probably represents colonization or subclinical infection. It is logical to hypothesize that quantification might prove helpful in distinguishing between infected and colonized patients: the latter group would have lower copy numbers than PCP patients. A blinded retrospective study of 98 respiratory samples (49 lower respiratory tract specimens and 49 oral washes), from 51 patients with 24 episodes of PCP and 34 episodes of other respiratory disease, was conducted. PCR-positive samples from colonized patients contained a lower concentration of
P. carinii
DNA than samples from PCP patients: lower respiratory tract samples from PCP and non-PCP patients contained a median of 938 (range, 2.4 to 1,040,000) and 2.6 (range, 0.3 to 248) (
P
< 0.0004) copies per tube, respectively. Oral washes from PCP and non-PCP patients contained a median of 49 (range, 2.1 to 2,595) and 6.5 (range, 2.2 to 10) (
P
< 0.03) copies per tube, respectively. These data suggest that this QTD PCR assay can be used to determine if
P. carinii
is present in respiratory samples and to distinguish between colonization and infection.
Detection of
seg
,
seh
, and
sei
genes in
Staphylococcus aureus
Isolates and Determination of the Enterotoxin Productivities of
S. aureus
Isolates Harboring
seg
,
seh
, or
sei
Genes
Authors: Omoe Katsuhiko , Ishikawa Machiko , Shimoda Yu , Hu Dong-Liang , Ueda Shigeko , Shinagawa Kunihiro
Published: July 2002

To investigate the distribution of staphylococcal enterotoxin (SE) A to I (SEA to SEI) genes (
sea
to
sei
) in
Staphylococcus aureus
, 146 isolates obtained in Japan from humans involved in and samples from food poisoning outbreaks, healthy humans, cows with mastitis, and bovine raw milk were analyzed by multiplex PCR. One hundred thirteen (77.4%)
S. aureus
isolates were found to be positive for one or more
se
genes. The
se
genotype was classified into 14 genotypes.
seg
and
sei
coexisted in the same
S. aureus
strain. The newly developed sandwich enzyme-linked immunosorbent assay showed that most
seh
-harboring
S. aureus
isolates were able to produce a significant amount of SEH. However, most of the
S. aureus
isolates harboring
seg
and about 60% of the isolates harboring
sei
did not produce a detectable level of SEG or SEI, while reverse transcription-PCR analysis proved that the mRNAs of SEG and SEI were transcribed in
S. aureus
strains harboring
seg
and
sei
genes. These results suggest the importance of quantitative assessment of SEG and SEI production in foods in order to clarify the relationship between these new SEs and food poisoning.
Real-Time PCR for Detection and Quantitation of
Leishmania
in Mouse Tissues
Authors: Nicolas Luc , Prina Eric , Lang Thierry , Milon Geneviève
Published: July 2002

Leishmania
spp. are intracellular protozoan parasites that cause a wide spectrum of diseases in humans and dogs worldwide. However, monitoring of the
Leishmania
burden in its different hosts is still based on cumbersome and poorly sensitive methods. Here we have developed a highly accurate real-time PCR assay with which to reproducibly detect and quantify the relative
Leishmania major
burden in mouse tissue samples. The assay is performed with the LightCycler system using SYBR Green I and primers amplifying a ca. 120-bp fragment from minicircles of the kinetoplast DNA (kDNA). The assay was able to detect as little as 100 fg of
L. major
DNA per reaction, which is equivalent to 0.1 parasite. The standard curve designed for quantitation of parasites showed linearity over an at least 6-log DNA concentration range, corresponding to 0.1 to 10
4
parasites per reaction, with a correlation coefficient of 0.979. The assay also proved to have a detection range of the same magnitude as that used for detection of
L. donovani
and
L. amazonensis
, but it was 100-fold less sensitive for
L. mexicana
. When applied to tissues from experimentally infected mice, the real-time PCR assay is not only as sensitive as a conventional PCR assay for detection of
Leishmania
kDNA but also more rapid. Results indicate that this assay is compatible with the clinical diagnosis of leishmaniasis and will be a great help to scientists who use animals to monitor the efficacy of antileishmanial drugs or vaccines or decipher the unique properties of the life cycle of
Leishmania
spp
.
Variable Sensitivity of CCR5-Tropic Human Immunodeficiency Virus Type 1 Isolates to Inhibition by RANTES Analogs
Authors: Torre Vincent S. , Marozsan Andre J. , Albright Jamie L. , Collins Kalonji R. , Hartley Oliver , Offord Robin E. , Quiñones-Mateu Miguel E. , Arts Eric J.
Published: July 2002

Aminooxypentane (AOP)-RANTES efficiently and specifically blocks entry of non-syncytium-inducing (NSI), CCR5-tropic (R5) human immunodeficiency virus type 1 (HIV-1) into host cells. Inhibition appears to be mediated by increased intracellular retention of the CCR5 coreceptor- AOP-RANTES complex and/or competitive binding of AOP-RANTES with NSI R5 HIV-1 isolates for CCR5. Although AOP-RANTES and other β-chemokine analogs are potent inhibitors, the extreme heterogeneity of the HIV-1 envelope glycoproteins (gp120 and gp41) and variable coreceptor usage may affect the susceptibility of variant HIV-1 strains to these drugs. Using the same peripheral blood mononuclear cells (PBMC) with all isolates, we observed a significant variation in AOP-RANTES inhibition of 13 primary NSI R5 isolates; 50% inhibitory concentrations (IC
50
) ranged from 0.04 nM with HIV-1
A-92RW009
to 1.3 nM with HIV-1
B-BaL
. Experiments performed on the same isolate (HIV-1
B-BaL
) with PBMC from different donors revealed no isolate-specific variation in AOP-RANTES IC
50
values but did show a considerable difference in virus replication efficiency. Exclusive entry via the CCR5 coreceptor by these NSI R5 isolates suggests that variable inhibition by AOP-RANTES is not due to alternative coreceptor usage but rather differential CCR5 binding. Analysis of the envelope V3 loop sequence linked a threonine or arginine at position 319 (numbering based on the HXB2 genome) with AOP-RANTES resistance. With the exception of one isolate, A319 was associated with increased sensitivity to AOP-RANTES inhibition. Distribution of AOP-RANTES IC
50
values with these isolates has promoted ongoing screens for new CCR5 agonists that show broad inhibition of HIV-1 variants.
Identification of the
Lymantria dispar
Nucleopolyhedrovirus Envelope Fusion Protein Provides Evidence for a Phylogenetic Division of the
Baculoviridae
Authors: Pearson Margot N. , Groten Christoph , Rohrmann George F.
Published: July 2002

The complete genome sequences of a number of diverse members of the
Baculoviridae
including both nucleopolyhedroviruses (NPVs) and granuloviruses (GVs) revealed that they lack a homolog of GP64, the envelope fusion protein of the budded form of
Autographa californica
multinucleocapsid NPV (Ac
M
NPV) and its close relatives. Computer-assisted analyses of the genome of one of these viruses,
Lymantria dispar M
NPV (Ld
M
NPV), revealed a single open reading frame (
ld130
) whose product had the predicted properties of a membrane protein. Characterization of the localization of the products of the full-length
ld130
gene and of an
ld130
-enhanced green fluorescent protein gene (
egfp
) fusion using both immunofluorescence and fluorescence microscopy revealed that LD130 accumulates at the plasma membranes of cells infected with Ld
M
NPV or transfected with
ld130-egfp
. In addition, cells transfected with either
ld130
or
ld130-egfp
or infected with wild-type virus undergo membrane fusion at pH 5. Western blot analyses indicate that LD130 is present in infected cells as an 83-kDa protein and is also present in budded virions as a protein doublet containing bands of 81 and 83 kDa. Tunicamycin treatment of infected cells resulted in an immunoreactive band of about 72 kDa, indicating that LD130 is N-glycosylated. Whereas the distribution of
gp64
appears to be confined to a relatively closely related group of NPVs, homologs of
ld130
are present in a diverse number of both NPVs and GVs. This suggests that LD130 may be the primordial baculovirus envelope fusion protein.
Evolution of Lamivudine Resistance in Human Immunodeficiency Virus Type 1-Infected Individuals: the Relative Roles of Drift and Selection
Authors: Frost Simon D. W. , Nijhuis Monique , Schuurman Rob , Boucher Charles A. B. , Brown Andrew J. Leigh
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) rapidly develops resistance to lamivudine during monotherapy, typically resulting in the appearance at position 184 in reverse transcriptase (RT) of isoleucine instead of the wild-type methionine (M184I) early in therapy, which is later replaced by valine (M184V). M184V reduces viral susceptibility to drug in vitro by approximately 100-fold, but also results in a lower processivity of RT. We show that a drop in absolute viral fitness associated with the outgrowth of M184V results in a drop in viral load only in individuals with high CD4
+
counts, from whom we estimate the relative fitness of M184V in the presence of drug to be approximately 10% of that of the wild type prior to therapy. The timing of emergence of the M184V mutant varies widely between infected individuals. From analysis of the frequency of M184I and M184V mutants determined at multiple time points in seven individuals during lamivudine therapy, we estimated the fitness advantage of M184V over M184I during therapy to be approximately 23% on average. We have also estimated the average ratio of the frequencies of the two mutants prior to therapy to be 0.2:1, with a range from 0.12:1 to 0.33:1. We have found that the differences between individuals in the rate of evolution of lamivudine resistance arise due to genetic drift affecting the relative frequency of M184I and M184V prior to therapy. These results show that stochastic effects can be significant in HIV evolution, even when there is large fitness difference between mutant and wild-type variants.