bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative Study Using Type Strains and Clinical and Food Isolates To Examine Hemolytic Activity and Occurrence of the
cyl
Operon in Enterococci
Authors: Semedo Teresa , Almeida Santos Margarida , Martins Paula , Silva Lopes Maria Fátima , Figueiredo Marques José J. , Tenreiro Rogério , Barreto Crespo Maria Teresa
Published: June 2003

The hemolytic ability, the presence of
cyl
genes, and the diagnostic accuracy of cytolysin molecular detection were investigated in the genus
Enterococcus
by using 164 strains from 20 different species (26 reference strains, 42 clinical isolates from human and veterinary origin, and 96 isolates from ewe cheese and milk). Hemolysis was assayed with sheep and horse erythrocytes and under aerobic or anaerobic conditions. Screening of cytolysin genes (
cylL

L

,
cylL

S

,
cylM
,
cylB
, and
cylA
) was performed with new specific primers and the anaerobic assay of beta-hemolysis was used as the “gold standard” for the evaluation of
cyl
gene-based PCRs. Since beta-hemolysis and
cyl
genes were found in 10 and 14 species, respectively, the hemolytic ability seems to be spread throughout the genus
Enterococcus
. Beta-hemolysis was observed in 6 of 26 (23%) reference strains, 14 of 42 (33%) clinical isolates, and 6 of 96 (6%) food isolates. The presence of
cyl
genes was detected in 15 of 26 (58%) reference strains, 37 of 42 (88%) clinical isolates, and 67 of 96 (70%) food isolates. These data indicate a virulence potential in food isolates, reinforcing the need of their safety assessment. Analysis of phenotypic-genotypic congruence suggests a divergent sequence evolution of
cyl
genes and the effect of environmental factors in the regulation of cytolysin expression. Evaluation of the diagnostic accuracy of cytolysin molecular detection points to
cylL

L

-based PCR and
cylL

L

L

S

MBA
-based PCR as the most reliable approaches. Nevertheless, the low sensitivity (46%) and gene variability indicated by our study strongly recommend the phenotypic assay for the assessment of hemolytic ability in enterococci, followed by the molecular screening of
cyl
genes in nonhemolytic strains to evaluate their virulence potential.
Characterization of the 8-Kilodalton Antigens of
Taenia solium
Metacestodes and Evaluation of Their Use in an Enzyme-Linked Immunosorbent Assay for Serodiagnosis
Authors: Hancock Kathy , Khan Azra , Williams Fatima B. , Yushak Melinda L. , Pattabhi Sowmya , Noh John , Tsang Victor C. W.
Published: June 2003

The Western blot for cysticercosis, which uses lentil lectin purified glycoprotein (LLGP) antigens extracted from the metacestode of
Taenia solium
, has been the “gold standard” serodiagnostic assay since it was first described in 1989. We report that the diagnostic antigens at 14, 18, and 21 kDa, as well as some larger disulfide-bonded antigens, are actually all members of a very closely related family of proteins, the 8-kDa antigens. The genes for 18 unique, mature proteins have been identified. Nine of these were chemically synthesized and tested in an enzyme-linked immunosorbent assay with a battery of defined serum samples, including 32 cysticercosis-positive serum samples reactive with the 8-kDa antigens of LLGP on Western blotting, 34 serum samples from patients with other parasitic infections, and 15 normal human serum samples. One of the 8-kDa antigens, TsRS1, is 100% sensitive and 100% specific. TsRS1 will be one component of a cocktail of three to four synthetic or recombinant antigens, based on the diagnostic bands of the Western blot, which will be used for the serodiagnosis of cysticercosis.
Phenotypic Description and Antimicrobial Susceptibilities of
Aerococcus sanguinicola
Isolates from Human Clinical Samples
Authors: Facklam Richard , Lovgren Marguerite , Shewmaker Patricia Lynn , Tyrrell Gregory
Published: June 2003

This report describes the clinical sources and phenotypic characterization of 16 isolates of
Aerococcus sanguinicola
. Sixteen conventional tests were used to describe and differentiate the 16 isolates of
A. sanguinicola
from 30 strains of
Aerococcus viridans
, 27 strains of
Aerococcus urinae
, and a single strain each of
Aerococcus christensenii
and
Aerococcus urinaehominis
. The phenotypic characterizations of the type strains for each species and 14
A. sanguinicola
isolates were also compared in the two reference laboratories.
A. sanguinicola
are catalase-negative, vancomycin-susceptible, gram-positive cocci arranged in clusters and tetrads, as are all
Aerococcus
species except
A. christensenii
(which is arranged in short chains). All 16 isolates of
A. sanguinicola
were leucine aminopeptidase and pyrrolidonylarylamidase positive, which is unique to this species among the aerococci. All
A. sanguinicola
isolates grew in broth containing 6.5% NaCl, hydrolyzed hippurate, and were variable in the bile-esculin test. None of the isolates deaminated arginine or were Voges-Proskauer positive. The type strain of
A. sanguinicola
was isolated from a blood culture of a patient living in Denmark. Seven additional isolates were from patients living in Canada, all with urinary tract infections (six were female). Eight isolates were from patients living in five different states in the United States; five were from patients with urinary tract infections, and three were from blood cultures of one patient each with pneumonia, suspected endocarditis, and unknown clinical conditions. The antimicrobial susceptibility patterns were unremarkable; all isolates tested were susceptible to penicillin, amoxicillin, cefotaxime, cefuroxime, erythromycin, chloramphenicol, vancomycin, quinupristin-dalfopristin (Synercid), rifampin, linezolid, and tetracycline. Six of the 15 cultures were resistant to ciprofloxacin and levofloxacin, but all 15 strains were susceptible to sparfloxacin. High-level resistance was detected for meropenem (2 strains) and trimethoprim-sulfamethonazole (1 strain). Intermediate resistance was detected for trimethoprim-sulfamethoxazole (10 strains) and clindamycin (3 strains).
Prevalence of Human T-Lymphotropic Virus Type 1 among Blood Donors from Mashhad, Iran
Authors: Abbaszadegan Mohammad Reza , Gholamin Mehran , Tabatabaee Abbas , Farid Reza , Houshmand Massoud , Abbaszadegan Morteza
Published: June 2003

The city of Mashhad is the capital of Khorasan, the northeastern province of Iran, which has been recognized as an area where human T-lymphotropic virus type 1 (HTLV-1) infection is endemic. All serum samples from blood donors are routinely screened for HTLV-1 by using enzyme-linked immunosorbent assay (ELISA). In the present study, 28,926 donors (81.86% male and 18.14% female) with a mean age of 32 years (range, 18 to 65 years) were screened in a 6 months period (July to December 1999). Of these donors in the primary screening, 228 (0.78%) tested positive by ELISA. The positive samples were confirmed by Western blot (WB) analysis. The WB results indicated that, of 228 positive ELISA specimens, 91.2% (208 specimens) were HTLV-1, 4.82% (11 specimens) were HTLV, 3.5% (8 specimens) were indeterminate, and 0.44% (1 specimen) was not confirmed. HTLV refers to samples in which the complete viral antigen banding patterns on WB strips were not present. In order to further evaluate the detection methodologies used, the HTLV-1-seropositive samples, the indeterminant samples, and/or HTLV samples were examined and confirmed by PCR. The HTLV samples were determined to be HTLV-1, the remaining samples were indeterminant, and the negative sample could not be confirmed for HTLV-1 by PCR. The prevalence of HTLV-1 infection in our study was 0.77% among blood bank donors, which reconfirms the city of Mashhad as an area where the virus is endemic compared to other regions in the world. The incidence was correlated with increasing age, and it was higher in females than in males.
Antimicrobial Susceptibility Testing of Porcine
Brachyspira
(
Serpulina
) Species Isolates
Authors: Karlsson M. , Fellström C. , Gunnarsson A. , Landén A. , Franklin A.
Published: June 2003

No standardized method for susceptibility testing of
Brachyspira
spp. is currently available. A broth dilution procedure was evaluated and used to test the activities of six antimicrobial agents for 108 isolates of Swedish porcine
Brachyspira
spp. representing biochemical groups I, II, and III. Group I corresponds to
Brachyspira hyodysenteriae
, group II corresponds to
B
.
intermedia
, and group III corresponds to
B
.
murdochii
and
B
.
innocens
. A panel was designed with the antimicrobial agents dried in tissue culture trays with wells that allowed a liquid volume of 0.5 ml in each and agitation of the broth when incubated on a shaker. The MICs were determined by using brain heart infusion broth with 10% fetal calf serum. For 10 isolates, the results obtained in broth were compared to the MICs obtained on two different types of agar. Different inoculum densities and incubation times were also compared. The concentrations at which 90% of the
B
.
hyodysenteriae
isolates (
n
= 72) were inhibited in the broth dilution test by tiamulin (0.25 μg/ml), tylosin (>256 μg/ml), erythromycin (>256 μg/ml), clindamycin (>4 μg/ml), virginiamycin (4 μg/ml), and carbadox (0.06 μg/ml) were determined. The MICs tended to be lower in broth than on agar. Differences in inoculum densities and incubation times had little influence on the MICs. The evaluated broth dilution test was simple to perform, the end points were easily read, and the results were reproducible and reliable. No isolates with decreased susceptibility to tiamulin were found among the Swedish isolates tested.
Detection and Identification of
Mycobacterium
Species Isolates by DNA Microarray
Authors: Fukushima Masao , Kakinuma Kenichi , Hayashi Hiroshi , Nagai Hiroko , Ito Kunihiko , Kawaguchi Ryuji
Published: June 2003

Rapid identification of
Mycobacterium
species isolates is necessary for the effective management of tuberculosis. Recently, analysis of DNA gyrase B subunit (
gyrB
) genes has been identified as a suitable means for the identification of bacterial species. We describe a microarray assay based on
gyrB
gene sequences that can be used for the identification of
Mycobacteria
species. Primers specific for a
gyrB
gene region common to all mycobacteria were synthesized and used for PCR amplification of DNA purified from clinical samples. A set of oligonucleotide probes for specific
gyrB
gene regions was developed for the identification of 14
Mycobacterium
species. Each probe was spotted onto a silylated glass slide with an arrayer and used for hybridization with fluorescently labeled RNA derived from amplified sample DNA to yield a pattern of positive spots. This microarray produced unique hybridization patterns for each species of mycobacteria and could differentiate closely related bacterial species. Moreover, the results corresponded well with those obtained by the conventional culture method for the detection of mycobacteria. We conclude that a
gyrB-
based microarray can rapidly detect and identify closely related mycobacterial species and may be useful in the diagnosis and effective management of tuberculosis.
Loop-Mediated Isothermal Amplification for Direct Detection of
Mycobacterium tuberculosis
Complex,
M. avium
, and
M. intracellulare
in Sputum Samples
Authors: Iwamoto Tomotada , Sonobe Toshiaki , Hayashi Kozaburo
Published: June 2003

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency, and rapidity. We used LAMP for detection of
Mycobacterium tuberculosis
complex,
Mycobacterium avium
, and
Mycobacterium intracellulare
directly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT; Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) or on a solid medium (Ogawa's medium). Species-specific primers were designed by targeting the
gyrB
gene, and their specificities were validated on 24 mycobacterial species and 7 nonmycobacterial species. The whole procedure is quite simple, starting with the mixing of all reagents in a single tube, followed by an isothermal reaction during which the reaction mixture is held at 63°C. The resulting amplicons are visualized by adding SYBR Green I to the reaction tube. The only equipment needed for the amplification reaction is a regular laboratory water bath or heat block that furnishes a constant temperature of 63°C. The assay had a detection limit of 5 to 50 copies of purified DNA with a 60-min incubation time. The reaction time could be shortened to 35 min for the species identification of
M. tuberculosis
complex,
M. avium
, and
M. intracellulare
from a solid-medium culture. Residual DNA lysates prepared for the Amplicor assay (Roche Diagnostics GmbH) from 66 sputum specimens were tested in the LAMP assay. Although the sample size used for the latter assay was small, 2.75 μl of the DNA lysates, it showed a performance comparable with that of the Amplicor assay, which required 50 μl of the lysates. This LAMP-based assay is simple, rapid, and sensitive; a result is available in 35 min for a solid-medium culture and in 60 min for a liquid-medium culture or for a sputum specimen that contains a corresponding amount of DNA available for testing.
Genetic Diversity among Clinical Isolates of
Acremonium strictum
Determined during an Investigation of a Fatal Mycosis
Authors: Novicki Thomas J. , LaFe Karen , Bui Lynda , Bui Uyen , Geise Robert , Marr Kieren , Cookson Brad T.
Published: June 2003

Primarily saprophytic in nature, fungi of the genus
Acremonium
are a well-documented cause of mycetoma and other focal diseases. More recently, a number of
Acremonium
spp. have been implicated in invasive infections in the setting of severe immunosuppression. During the course of routine microbiological studies involving a case of fatal mycosis in a nonmyeloablative hematopoietic stem cell transplant patient, we identified a greater-than-expected variation among strains previously identified as
Acremonium strictum
by clinical microbiologists. Using DNA sequence analysis of the ribosomal DNA intergenic transcribed spacer (ITS) regions and the D1-D2 variable domain of the 28S ribosomal DNA gene (28S), the case isolate and four other clinical isolates phenotypically identified as
A. strictum
were found to have <99% homology to the
A. strictum
type strain, CBS 346.70, at the ITS and 28S loci, while a sixth isolate phenotypically identified only as
Acremonium
sp
.
had >99% homology to the type strain at both loci. These results suggest that five out of the six clinical isolates belong to species other than
A. strictum
or that the
A. strictum
taxon is genetically diverse. Based upon these sequence data, the clinical isolates were placed into three genogroups.
Direct Isolation of
Candida
spp. from Blood Cultures on the Chromogenic Medium CHROMagar Candida
Authors: Horvath Lynn L. , Hospenthal Duane R. , Murray Clinton K. , Dooley David P.
Published: June 2003

CHROMagar Candida is a selective and differential chromogenic medium that has been shown to be useful for identification of
Candida albicans
,
Candida krusei
,
Candida tropicalis
, and perhaps
Candida glabrata
. Colony morphology and color have been well defined when CHROMagar Candida has been used to isolate yeast directly from clinical specimens, including stool, urine, respiratory, vaginal, oropharyngeal, and esophageal sources. Direct isolation of yeast on CHROMagar Candida from blood cultures has not been evaluated. We evaluated whether the color and colony characteristics produced by
Candida
spp. on CHROMagar Candida were altered when yeasts were isolated directly from blood cultures. Fifty clinical isolates of
Candida
were inoculated into aerobic and anaerobic blood culture bottles and incubated at 35°C in an automated blood culture system. When growth was detected, an aliquot was removed and plated onto CHROMagar Candida. As a control, CHROMagar Candida plates were inoculated with the same isolate of yeast grown on Sabouraud dextrose agar simultaneously. No significant difference was detected in color or colony morphology between the blood and control isolates in any of the tested organisms. All
C. albicans
(
n
= 12),
C. tropicalis
(
n
= 12),
C. glabrata
(
n
= 9), and
C. krusei
(
n
= 5) isolates exhibited the expected species-specific colony characteristics and color, whether isolated directly from blood or from control cultures. CHROMagar Candida can be reliably used for direct isolation of yeast from blood cultures. Direct isolation could allow mycology laboratories to more rapidly identify
Candida
spp., enable clinicians to more quickly make antifungal agent selections, and potentially decrease patient morbidity and mortality.
Herpesvirus DNA Is Consistently Detected in Lungs of Patients with Idiopathic Pulmonary Fibrosis
Authors: Tang Yi-Wei , Johnson Joyce E. , Browning Philip J. , Cruz-Gervis Roberto A. , Davis Angela , Graham Barney S. , Brigham Kenneth L. , Oates John A. , Loyd James E. , Stecenko Arlene A.
Published: June 2003

On the basis of earlier reports associating Epstein-Barr Virus (EBV) with half of the cases of idiopathic pulmonary fibrosis (IPF), we hypothesized that chronic infection with EBV or a closely related herpesvirus would be detected in all cases of IPF. We tested lung specimens from 33 IPF patients (8 patients with familial IPF and 25 patients with sporadic IPF) and 25 patients with other diseases as controls for the presence of eight herpesviruses using PCR-based techniques. One or more of four herpesviruses (cytomegalovirus [CMV], EBV, human herpesvirus 7 [HHV-7], and HHV-8) were detected in 32 of 33 (97%) subjects with IPF and in 9 of 25 (36%) controls (
P
< 0.0001). CMV, EBV, and HHV-8 were found more frequently in IPF patients than in controls (
P
< 0.05,
P
< 0.001, and
P
< 0.01 respectively). Two or more herpesviruses were detected in 19 of 33 (57%) IPF patients and in 2 of 25 (8%) controls (
P
< 0.001). Two or more herpesviruses and HHV-8 were found more frequently in patients with sporadic IPF than in patients with familial IPF (
P
< 0.05 for both comparisons), and CMV was found less frequently in patients with sporadic IPF than in patients with familial IPF (
P
< 0.05). Immunohistochemistry for EBV or HHV-8 antigen showed viral antigen primarily in airway epithelial cells. These data support the concept that a herpesvirus could be a source of chronic antigenic stimulation in IPF.
Diversity of Tn
1546-
Like Elements in Vancomycin-Resistant Enterococci Isolated from Humans and Poultry in Korea
Authors: Yu Hak-Sun , Seol Sung-Yong , Cho Dong-Taek
Published: June 2003

To investigate the possible spread of vancomycin resistance among enterococci through horizontal gene transfer between two sources of vancomycin-resistant enterococci (VRE) isolated from farm animals and humans in Korea, molecular characterization of VRE isolated from poultry (20 isolates) and humans (35 isolates) was performed by PCR-restriction fragment length polymorphism typing of the
vanA
gene cluster (Tn
1546
). PCR mapping of Tn
1546
finally distinguished seven different transposon types (types A to G). Type A was observed only in the poultry isolates, while the other six types were present only in the human isolates. An insertion sequence and deleted sequence were detected in most of the human isolates in the
orf2-vanR
and
vanX-vanY
regions in the Tn
1546-
like element, but not in the poultry isolates. Tn
1546
-like elements were found in conjugal plasmids of most human VRE, whereas they were detected in the chromosomes of all poultry VRE. Accordingly, no evidence was found of any recent transmission of vancomycin resistance genes between poultry and humans in Korea.
Use of Colony Morphology To Distinguish Different Enterococcal Strains and Species in Mixed Culture from Clinical Specimens
Authors: Qamer Shabnam , Sandoe Jonathan A. T. , Kerr Kevin G.
Published: June 2003

Colony morphology on kanamycin esculin azide agar was investigated as a means of selecting different species and strains of enterococci from clinical specimens. Four representative colonies of each morphotype were indistinguishable by pulsed-field gel electrophoresis, biotype, and antibiogram analysis. The optimum time for identification of different colony morphotypes was 72 h.
Utility of an In-House Mycobacteriophage-Based Assay for Rapid Detection of Rifampin Resistance in
Mycobacterium tuberculosis
Clinical Isolates
Authors: Galí N. , Domínguez J. , Blanco S. , Prat C. , Quesada M. D. , Matas L. , Ausina V.
Published: June 2003

A rapid in-house mycobacteriophage-based assay to identify multidrug resistance by detecting the rifampin susceptibility of
Mycobacterium tuberculosis
in a microtiter plate format was evaluated. The sensitivity, specificity, and overall accuracy of the assay were 100%. This test is rapid to perform and suitable for widespread implementation.
Evaluation of the Duopath Verotoxin Test for Detection of Shiga Toxins in Cultures of Human Stools
Authors: Park C. H. , Kim H. J. , Hixon D. L. , Bubert A.
Published: June 2003

The Duopath Verotoxin test (Merck KgaA, Darmstadt, Germany) is a newly developed immunochromatographic test for the confirmation of Shiga toxin (Stx)-producing
Escherichia coli
(STEC) strains from food products. This test detects both Stx 1 (Stx1)-positive and Stx2-positive samples individually with the same device. By modification of the original protocol, the present study evaluated its performance and feasibility for clinical application with human stool samples, consisting of 41 frozen samples known to contain STEC isolates (O157:H7 and non-O157 serotypes) and 250 fresh specimens. The test specimens were polymyxin B extracts of colony sweeps taken from overnight sorbitol-MacConkey agar cultures of stools containing STEC isolates and other bacteria. All 41 frozen STEC-positive stool samples were positive by the Duopath Verotoxin test, as were 2 fresh stool samples with culture-confirmed STEC O157 infection. Thus, 100% sensitivity and no false-positive results were obtained when the Premier EHEC assay (Meridian Bioscience, Cincinnati, Ohio) was used as the “gold standard.” The Duopath Verotoxin test is simple to perform and easy to interpret, providing a turnaround time of 24 h. Despite its original intended use, the Duopath Verotoxin test has a great potential for clinical application.
Ischemic Stroke and Splenic Rupture in a Case ofStreptococcus bovisEndocarditis
Authors: Stöllberger Claudia , Finsterer Josef , Pratter Angelika , Kopsa Wolfgang , Preiser Julius , Valentin Andreas
Published: June 2003

A 58-year-old man with an acute stroke suffered from splenic rupture.Streptococcus boviswas found in blood cultures, and gram-negative cocci were found in the infarcted spleen. Hemorrhagic transformation of the stroke occurred. Echocardiography showed aortic endocarditis. Cardiac surgery was not performed because of concern about cerebral bleeding. The patient died due to cerebral rehemorrhage after 3 weeks.
Molecular Epidemiology of Macrolide Resistance in Neonatal Bloodstream Isolates of Group B Streptococci
Authors: Diekema Daniel J. , Andrews Janet I. , Huynh Holly , Rhomberg Paul R. , Doktor Stella R. , Beyer Jill , Shortridge Virginia D. , Flamm Robert K. , Jones Ronald N. , Pfaller Michael A.
Published: June 2003

Pulsed-field gel electrophoresis (PFGE) was performed on 122 neonatal bloodstream isolates of group B streptococci (GBS) to further examine the relationship between macrolide resistance and serotype V GBS (GBS-V). Over one-third (35%) of macrolide-resistant GBS belonged to a single PFGE subtype of GBS-V, which was also the most common GBS-V subtype noted in previous Centers for Disease Control and Prevention surveillance studies. Erm methylase (
ermA
and
ermB
) was the most common resistance mechanism detected, present in 12 of 20 macrolide-resistant GBS.
Comparison of Washing and Swabbing Procedures for Collecting Genital Fluids To Assess Cervicovaginal Shedding of Herpes Simplex Virus Type 2 DNA
Authors: Ndjoyi-Mbiguino Angélique , Ozouaki Francis , Legoff Jérôme , Mbopi-Kéou François-Xavier , Si-Mohamed Ali , Onas Isabelle Ndombi , Avoune Evelyne , Bélec Laurent
Published: June 2003

Asymptomatic genital shedding of herpes simplex virus type 2 (HSV-2) DNA was evidenced by real-time PCR in 25 (13.2%) of 188 cervicovaginal lavage samples and in only 13 (6.9%) paired cervicovaginal samples from 188 HSV-2-seropositive, nonpregnant childbearing-aged human immunodeficiency virus-seronegative women living in Gabon. These observations demonstrate that cervicovaginal washing is more suitable than endocervicovaginal swabbing for detecting and quantifying HSV-2 DNA by PCR in female genital secretions.
Comparison of Results Obtained by Testing with Three Different Agar Media and by the NCCLS M27-A Method for In Vitro Testing of Fluconazole against
Candida
spp
Authors: Rubio M. Carmen , Gil Joaquina , de Ocáriz Inmaculada Ramírez , Benito Rafael , Rezusta Antonio
Published: June 2003

Fluconazole susceptibilities of 150
Candida
isolates were determined by a 25-μg fluconazole disk diffusion agar test and compared with the microdilution NCCLS M27-A method. The agar test used three different media and was read at 24 and 48 h. When only the susceptible and nonsusceptible categories were used, disk diffusion with Müeller-Hinton agar supplemented with 2% glucose and 0.5 μg of methylene blue (MHGM) per ml had a 95.37% correlation with the MIC method at 24 h, followed by RPMI 1640-2% of glucose agar (correlation, 94%) and Shadomy medium (SHDM) (correlation, 92.6%). The growth of microcolonies inside the inhibition zones was common (>63%) in the RPMI and SHDM media and minimal with MHGM (8.7%). At 48 h, MHGM and SHDM still had a >91% correlation with the MIC, while RPMI results had dropped to 75%. The best overall agreement was obtained with
C. dubliniensis
(100%).
Multiplex PCR Assay for Identification of Human Diarrheagenic
Escherichia coli
Authors: Toma Claudia , Lu Yan , Higa Naomi , Nakasone Noboru , Chinen Isabel , Baschkier Ariela , Rivas Marta , Iwanaga Masaaki
Published: June 2003

A multiplex PCR assay for the identification of human diarrheagenic
Escherichia coli
was developed. The targets selected for each category were
eae
for enteropathogenic
E
.
coli
,
stx
for Shiga toxin-producing
E
.
coli
,
elt
and
est
for enterotoxigenic
E
.
coli
,
ipaH
for enteroinvasive
E
.
coli
, and
aggR
for enteroaggregative
E
.
coli
. This assay allowed the categorization of a diarrheagenic
E
.
coli
strain in a single reaction tube.
Evaluation of Genotype MTBC Assay for Differentiation of Clinical
Mycobacterium tuberculosis
Complex Isolates
Authors: Richter Elvira , Weizenegger Michael , Rüsch-Gerdes Sabine , Niemann Stefan
Published: June 2003

A commercially available DNA strip assay was evaluated for the ability to differentiate
Mycobacterium tuberculosis
complex species.
M
.
bovis
subsp.
bovis
,
M
.
bovis
subsp.
caprae
,
M
.
bovis
BCG,
M
.
africanum
subtype I, and
M
.
microti
were unequivocally identified.
M
.
tuberculosis
,
M
.
canetti
, and
M
.
africanum
subtype II showed a unique hybridization pattern.
Epitope-Blocking Enzyme-Linked Immunosorbent Assays for Detection of West Nile Virus Antibodies in Domestic Mammals
Authors: Blitvich Bradley J. , Bowen Richard A. , Marlenee Nicole L. , Hall Roy A. , Bunning Michel L. , Beaty Barry J.
Published: June 2003

We evaluated the ability of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) to detect West Nile virus (WNV) antibodies in domestic mammals. Sera were collected from experimentally infected horses, cats, and pigs at regular intervals and screened in ELISAs and plaque reduction neutralization tests. The diagnostic efficacies of these techniques were similar.
Increased Prevalence of G1P[4] Genotype among Children with Rotavirus-Associated Gastroenteritis in Metropolitan Detroit
Authors: Abdel-Haq Nahed M. , Thomas Robert A. , Asmar Basim I. , Zacharova Viera , Lyman William D.
Published: June 2003

The G and P genotypes of rotavirus stool isolates from 100 children were determined by reverse transcription-PCR and nucleotide sequencing. G1P[4] was the most prevalent genotype(41%), followed by G1P[8] (16%) and G4P[4] (14%). The G genoypes detected were G1 (73%), G4 (17.4%), G9 (6.3%), and G2 (2.8%). The P genotypes were P[4] (71%) and P[8] (29%). Coinfection with more than one G genotype occurred in 12 patients, and coinfection with more than one P genotype occurred in 11 patients.
Evaluation of the Epidemiologic Utility of Secondary Typing Methods for Differentiation ofMycobacterium tuberculosisIsolates
Authors: Kwara Awewura , Schiro Ronald , Cowan Lauren S. , Hyslop Newton E. , Wiser Mark F. , Roahen Harrison Stephanie , Kissinger Patricia , Diem Lois , Crawford Jack T.
Published: June 2003

Spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis (MIRU-VNTR) were evaluated for the ability to differentiate 64Mycobacterium tuberculosisisolates from 10 IS6110-defined clusters. MIRU-VNTR performed slightly better than spoligotyping in reducing the number of clustered isolates and the sizes of the clusters. All epidemiologically related isolates remained clustered by MIRU-VNTR but not by spoligotyping.
Evaluation of Copan Swabs with Liquid Transport Media for Use in the Gen-Probe Group A Strep Direct Test
Authors: Bourbeau Paul P. , Heiter Barbara J.
Published: June 2003

The Gen-Probe Group A Streptococcus Direct Test (GASDT), which detects the presence or absence of group A streptococci directly from pharyngeal specimens, utilizes a specific relative light unit (RLU) cutoff of 4,500 to differentiate between positive and negative test results. In response to a report by a manufacturer that the background RLU values for the Copan rayon swabs with liquid media were higher than the RLU values typically observed with Culturette swabs, we tested multiple lots of Copan rayon swabs with liquid media and determined that the swabs are unacceptable for routine use in the GASDT. The high background RLU values for the Copan rayon swabs appear to be a direct result of the gamma irradiation used to sterilize the swabs. We also performed a comparative clinical evaluation of Copan Dacron swabs with liquid media and Culturette swabs for use in the GASDT. Overall, there was 97.5% agreement between the results obtained with the Copan Dacron swabs and those obtained with the Culturette swabs. Compared to Culturette swabs, the Copan Dacron swabs had a sensitivity and a specificity of 97 and 98%, respectively. Copan Dacron swabs with liquid media are an acceptable alternative to the swabs currently qualified for use with the GASDT, but Copan rayon swabs with liquid transport media should not be used in the GASDT.
Molecular Characterization of Isolates of Waterborne
Cryptosporidium
spp. Collected during an Outbreak of Gastroenteritis in South Burgundy, France
Authors: Dalle Frédéric , Roz Pascale , Dautin Guillaume , Di-Palma Marc , Kohli Evelyne , Sire-Bidault C. , Fleischmann Marie George , Gallay Anne , Carbonel Sylvia , Bon Fabienne , Tillier Claude , Beaudeau Pascal , Bonnin Alain
Published: June 2003

In September 2001, a waterborne outbreak of gastroenteritis occurred in eastern France. Of 31 fecal samples from symptomatic individuals, 19 tested positive for
Cryptosporidium
with two PCRs targeting the Hsp70 and the 18S rRNA genes of
Cryptosporidium
. Sequencing of the PCR fragments produced sequences identical to that of
Cryptosporidium parvum
genotype 1.
Stability of Repetitive-Sequence PCR Patterns with Respect to Culture Age and Subculture Frequency
Authors: Kang Hyunseok Peter , Dunne W. Michael
Published: June 2003

To examine the stability of repetitive-sequence (rep) PCR profiles, six species of bacteria were subcultured to blood agar plates and DNA was extracted from the cultures after 24, 48, and 72 h of incubation at 35°C. In addition, the same species were subcultured to fresh blood plates daily and DNA was extracted from the cultures after growth of 5, 10, and 15 subcultures, respectively. rep PCR analysis demonstrated that all rep PCR fingerprints from a single species were identical.
Identification of
Neisseria meningitidis
Serogroups Y and W135 by
siaD
Nucleotide Sequence Analysis
Authors: Lewis C. , Clarke S. C.
Published: June 2003

Rapid serogrouping of meningococci is essential for the effective public health management of cases of the disease and the contacts of infected patients. Here we describe an accurate nucleotide-sequencing method for the confirmation of serogroup Y and W135 meningococci by
siaD
gene analysis from cultures of
Neisseria meningitidis
.
Predominance of
Chlamydia trachomatis
Serovars Associated with Urogenital Infections in Females in New Delhi, India
Authors: Singh Vineeta , Salhan Sudha , Das B. C. , Mittal A.
Published: June 2003

Screening for
Chlamydia trachomatis
was done for 280 endocervical swab samples by PCR specific for endogenous plasmid. Age dependency was seen in symptomatic patients, with a high chlamydial prevalence rate (28%) found in younger women. Genotyping by restriction fragment length polymorphism analysis of
omp1
PCR-positive samples showed serovars D, E, and F to be the most prevalent.
Comparison of Microscan Broth Microdilution, Synergy Quad Plate Agar Dilution, and Disk Diffusion Screening Methods for Detection of High-Level Aminoglycoside Resistance in
Enterococcus
Species
Authors: Murdoch David R. , Mirrett Stanley , Harrell Lizzie J. , Donabedian Susan M. , Zervos Marcus J. , Reller L. Barth
Published: June 2003

We compared the dried MicroScan microdilution panel, Synergy Quad plate agar dilution, and high-potency disk diffusion screening methods for the detection of high-level aminoglycoside resistance in 815 enterococcal bloodstream isolates. Agreement between the three methods was 99% when testing for high-level gentamicin resistance and 96% when testing for high-level streptomycin resistance.
Molecular and Conventional Epidemiology of Tuberculosis in Hong Kong: a Population-Based Prospective Study
Authors: Chan-Yeung Moira , Tam Cheuk-Ming , Wong Harriet , Leung Chi-Chiu , Wang Julie , Yew Wing-Wai , Lam Chak-Wah , Kam Kai-Man
Published: June 2003

A prospective population-based molecular and conventional epidemiological study of 65.4% of bacteriologically confirmed cases of tuberculosis was carried out on the island of Hong Kong from May 1999 to Oct 2000 by the IS
6110
-based restriction fragment length polymorphism technique. Eleven of the isolates had five or fewer bands; 24.5% of the remaining 691 isolates belonged to clusters. The estimated proportion of recently transmitted disease was 15 to 20%.
Detection of
Pneumocystis carinii
and Characterization of Mutations Associated with Sulfa Resistance in Bronchoalveolar Lavage Samples from Human Immunodeficiency Virus-Infected Subjects
Authors: Zingale Anna , Carrera Paola , Lazzarin Adriano , Scarpellini Paolo
Published: June 2003

One hundred ninety-four bronchoalveolar specimens were evaluated by microscopic examination and by amplification of a sequence of a
Pneumocystis carinii
dihidropteroate synthase gene for identification of mutations linked to sulfa resistance. PCR sensitivity and specificity were 100 and 86.7%, respectively, compared to results of microscopic examination. However, 7 out of 19 microscopy-negative, PCR-positive samples were collected from subjects with a clinically high probability of
P. carinii
pneumonia, suggesting that PCR may be more sensitive than microscopic examination, although the absolute performance of PCR cannot be determined. Mutations were identified in 28 out of 70 (40%) PCR-positive specimens and were significantly more common in patients exposed to sulfa drugs (21 out of 29 [72.4%]) than in those not exposed to sulfa drugs (4 out of 35 [11.4%]).
Interruption of Nonnucleoside Reverse Transcriptase Inhibitor (NNRTI) Therapy for 2 Months Has No Effect on Levels of Human Immunodeficiency Virus Type 1 in Plasma of Patients Harboring Viruses with Mutations Associated with Resistance to NNRTIs
Authors: Wirden Marc , Simon Anne , Schneider Luminita , Tubiana Roland , Paris Luc , Marcelin Anne Genevieve , Delaugerre Constance , Legrand Mayeule , Herson Serge , Peytavin Gilles , Katlama Christine , Calvez Vincent
Published: June 2003

A 2-month interruption of only nonnucleoside reverse transcriptase inhibitors (NNRTIs) for patients carrying mutations associated with resistance to NNRTIs was followed by no change in either viral load or CD4 cell counts. These data suggest that these compounds have lost all of their in vivo antiviral activity in such cases.
Bovine Non-O157 Shiga Toxin 2-Containing
Escherichia coli
Isolates Commonly Possess
stx
2-EDL933
and/or
stx
2vhb
Subtypes
Authors: Brett Kim N. , Hornitzky Michael A. , Bettelheim Karl A. , Walker Mark J. , Djordjevic Steven P.
Published: June 2003

stx
2
genes from 138 Shiga toxin-producing
Escherichia coli
(STEC) isolates, of which 127 were of bovine origin (58 serotypes) and 11 of human origin (one serotype; O113:H21), were subtyped. The bovine STEC isolates from Australian cattle carried
ehxA
and/or
eaeA
and predominantly possessed
stx
2-EDL933
(103 of 127; 81.1%) either in combination with
stx
2vhb
(32 of 127; 25.2%) or on its own (52 of 127; 40.4%). Of 22 (90.9%) bovine isolates of serotype O113:H21, a serotype increasingly recovered from patients with hemolytic uremic syndrome (HUS) or hemorrhagic colitis, 20 contained both
stx
2-EDL933
and
stx
2vhb
; 2 isolates contained
stx
2vhb
only. Although 7 of 11 (63.6%) human O113:H21 isolates associated with diarrhea possessed
stx
2-EDL933
, the remaining 4 isolates possessed a combination of
stx
2-EDL933
and
stx
2vhb
. Three of the four were from separate sporadic cases of HUS, and one was from an unknown source.
Characterization of the Manila Family of
Mycobacterium tuberculosis
Authors: Douglas James T. , Qian Lishi , Montoya Jaime C. , Musser James M. , Van Embden Jan D. A. , Van Soolingen Dick , Kremer Kristin
Published: June 2003

Forty-eight
Mycobacterium tuberculosis
strains were obtained from patients living in metropolitan Manila, Republic of the Philippines. Three molecular typing methods, IS
6110
restriction fragment length polymorphism, spoligotyping, and DNA sequencing of the
oxyR
,
gyrA
, and
katG
loci, established that these strains have restricted diversity and are members of a related genetic group of organisms. Comparison of the DNA fingerprint patterns with those in international databases confirmed the uniqueness of this group of isolates, which we designate the Manila family of
M. tuberculosis
.
In Vitro Detection of Dissimilar Amounts of Hepatitis C Virus (HCV) Subtype-Specific RNA Genomes in Mixes Prepared from Sera of Persons Infected with a Single HCV Genotype
Authors: Quarleri Jorge F. , Bussy María V. , Mathet Verónica L. , Ruiz Vanesa , Iácono Rubén , Lu Ling , Robertson Betty H. , Oubiña José R.
Published: June 2003

The level of in vitro detection of viral genomes in mixes with two different hepatitis C virus (HCV) subtypes was investigated by artificially mixing previously measured subtype-specific HCV RNA genomes. The RNAs in these mixtures were reverse transcribed and then PCR amplified by using two sets of primers corresponding to the 5′ untranslated region and digested with endonucleases to analyze the restriction fragment length polymorphism patterns. This approach facilitated detection of a wider range of type-specific HCV genomes than originally described, beyond equimolar concentrations of contributing HCV subtypes. Moreover, by using computerized image analysis, this study also demonstrated that the true contribution of each virus type—and consequently of mixed infections—may be underestimated when only visual observation is carried out. These results may be useful for comparing data obtained from this and other currently used methodologies.
Detection ofChlamydophila felisand Feline Herpesvirus by Multiplex Real-Time PCR Analysis
Authors: Helps Chris , Reeves Nicky , Egan Kathy , Howard Pam , Harbour Dave
Published: June 2003

Chlamydophila felisand feline herpesvirus (FHV) are pathogens commonly associated with feline respiratory and ocular disease. A real-time multiplex PCR assay was developed to allow detection of these organisms, together with feline 28S ribosomal DNA, in a single tube. Of 538 ocular swab samples tested, 123 were positive for FHV, 97 were positive forC.felis, and 16 were positive for both pathogens.
Genetic Diversity of
Gallibacterium anatis
Isolates from Different Chicken Flocks
Authors: Bojesen Anders Miki , Torpdahl Mia , Christensen Henrik , Olsen John Elmerdahl , Bisgaard Magne
Published: June 2003

Amplified fragment length polymorphisms (AFLPs) were used to characterize the genotypic diversity of a total of 114
Gallibacterium anatis
isolates originating from a reference collection representing 15 biovars from four countries and isolates obtained from tracheal and cloacal swab samples of chickens from an organic, egg-producing flock and a layer parent flock. A subset of strains was also characterized by pulsed-field gel electrophoresis and biotyping. The organic flock isolates were characterized by more than 94% genetic similarity, indicating that only a single clone was apparent in the flock. The layer parent flock isolates were grouped into two subclusters, each with similarity above 90%. One subcluster contained only tracheal isolates, while the other primarily included cloacal isolates. In conclusion, we show that AFLP analysis enables fingerprinting of
G. anatis
, which seems to have a clonal population structure within natural populations. There was further evidence of clonal lineages, which may have adapted to different sites within the same animal.
Interpreting the Results of the Amplified
Mycobacterium tuberculosis
Direct Test for Detection of
M. tuberculosis
rRNA
Authors: Middleton Andrew M. , Cullinan Paul , Wilson Robert , Kerr Jonathan R. , Chadwick Maureen V.
Published: June 2003

The Amplified
Mycobacterium tuberculosis
Direct (AMTD) test detects
M. tuberculosis
rRNA. By using culture of
M. tuberculosis
as a gold standard, a number of different diagnostic indices were examined in an attempt to determine the diagnostic performance of the AMTD test and demonstrate how it might usefully be interpreted during the early management of disease.
Subgenotype Analysis of
Cryptosporidium
Isolates from Humans, Cattle, and Zoo Ruminants in Portugal
Authors: Alves Margarida , Xiao Lihua , Sulaiman Irshad , Lal Altaf A. , Matos Olga , Antunes Francisco
Published: June 2003

Cryptosporidium parvum
and
Cryptosporidium hominis
isolates from human immunodeficiency virus-infected patients, cattle, and wild ruminants were characterized by PCR and DNA sequencing analysis of the 60-kDa glycoprotein gene. Seven alleles were identified, three corresponding to
C. hominis
and four corresponding to
C. parvum
. One new allele was found (IId), and one (IIb) had only been found in Portugal. Isolates from cattle and wild ruminants clustered in two alleles. In contrast, human isolates clustered in seven alleles, showing extensive allelic diversity.
Sequence Analysis of the Matrix (M2) Protein Gene of Avian Pneumovirus Recovered from Turkey Flocks in the United States
Authors: Dar Arshud M. , Munir Shirin , Goyal Sagar M. , Kapur Vivek
Published: June 2003

We here report the comparative sequence and phylogenetic analysis of the avian pneumovirus subgroup C (APV C) matrix (M2) gene of cell culture-adapted isolates and clinical samples. Limited heterogeneity was observed among the M2 sequences, suggesting that diagnostic tests and vaccines against APV C are likely to exhibit broad cross-reactivity.
Desulfovibrio desulfuricans
Bacteremia and Review of Human
Desulfovibrio
Infections
Authors: Goldstein Ellie J. C. , Citron Diane M. , Peraino Victoria A. , Cross Sally A.
Published: June 2003

One case of primary
Desulfovibrio desulfuricans
bacteremia in an immunocompetent man is presented, and 15 other reported cases are reviewed. While most isolates have not been identified to the species level,
Desulfovibrio fairfieldensis
and
D. desulfuricans
have been associated with incidents of bacteremia and
D. vulgaris
has been associated with intra-abdominal infections. In vitro studies suggest that empirical therapy with either imipenem or metronidazole should be considered.
Microbiologic Surveillance Using Nasal Cultures Alone Is Sufficient for Detection of Methicillin-Resistant
Staphylococcus aureus
Isolates in Neonates
Authors: Singh Kamaljit , Gavin Patrick J. , Vescio Thomas , Thomson, Richard B. , Deddish Ruth B. , Fisher Adrienne , Noskin Gary A. , Peterson Lance R.
Published: June 2003

During an outbreak of methicillin-resistant
Staphylococcus aureus
(MRSA) in the neonatal intensive care units at two hospitals, we assessed several sites for detection of MRSA colonization. Nasal cultures found 32 of 33 MRSA-colonized patients (97%). Rectal cultures detected 29% of 24 MRSA-colonized patients identified by paired rectal and nasal samples and axillary samples found 22% of 9 MRSA-colonized patients identified by axillary samples paired with nasal swabs. There were no positive umbilical samples.
Plasma versus Serum for Detection of Herpes Simplex Virus Type 2-Specific Immunoglobulin G Antibodies with a Glycoprotein G2-Based Enzyme Immunoassay
Authors: Cherpes Thomas L. , Meyn Leslie A. , Hillier Sharon L.
Published: June 2003

To evaluate the consonance between plasma and serum for the detection of herpes simplex virus type 2-specific immunoglobulin G antibodies, we compared results from concurrently obtained plasma and sera in 710 sexually active women by using a glycoprotein G2-based enzyme-linked immunosorbent assay (Focus Technologies, Cypress, Calif.) and found 98.9% agreement between the two specimen types. Plasma appears to be an acceptable matrix for use with this assay.
Emergence of Novel Human Group A Rotavirus G12 Strains in India
Authors: Das Soma , Varghese V. , Chaudhury S. , Barman P. , Mahapatra S. , Kojima K. , Bhattacharya S. K. , Krishnan T. , Ratho R. K. , Chhotray G. P. , Phukan A. C. , Kobayashi N. , Naik T. N.
Published: June 2003

Three rare human G12 strains were detected from diarrheic clinical samples of children (<8 months of age) in Calcutta during a routine surveillance study of rotaviral diarrhea in India. The VP7 genes of G12 strains and their products showed maximum homology (97 to 99% at the nucleotide level and 98% at the amino acid level, respectively) with those of two recently reported G12 strains (from the United States and Thailand) but lesser homology with those of prototype G12 strain L26.
Oncogenic Human Papillomavirus DNA Loads in Human Immunodeficiency Virus-Positive Women with High-Grade Cervical Lesions Are Strongly Elevated
Authors: Weissenborn S. J. , Funke A. M. , Hellmich M. , Mallmann P. , Fuchs P. G. , Pfister H. J. , Wieland U.
Published: June 2003

Human papillomavirus (HPV) DNA loads of six oncogenic HPV types were measured by real-time PCR in cervical scrapes of human immunodeficiency virus (HIV)-infected and uninfected women. In both groups, HPV loads increased with the grade of cervical disease. HIV infection did not affect HPV loads in low-grade lesions but was associated with significantly higher HPV loads in severe dysplasia; highest loads were found in advanced HIV disease. Our data reflect the aggressive course of HPV infection in HIV-positive women.