bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Inoculum and β-Lactamase Activity in AmpC- and Extended-Spectrum β-Lactamase (ESBL)-Producing
Escherichia coli
and
Klebsiella pneumoniae
Clinical Isolates Tested by Using NCCLS ESBL Methodology
Authors: Queenan Anne Marie , Foleno Barbara , Gownley Colleen , Wira Ellyn , Bush Karen
Published: January 2004

Escherichia coli
and
Klebsiella pneumoniae
isolates with extended-spectrum β-lactamases (ESBLs) or AmpC cephalosporinases generally respond as predicted to NCCLS tests for ESBL production. However, inoculum size may affect MICs. The effect of inoculum level in clinical isolates expressing β-lactamases were studied at inocula within 0.5 log unit of the standard inoculum, using broth microdilution methodology with ceftazidime, cefotaxime, cefepime, cefpodoxime, and aztreonam. Strains with TEM-1 or no β-lactamases gave consistent MIC results with inocula of 10
5
and 10
6
CFU/ml. When the bacteria were screened for ESBL production and the lower inoculum was used, several strains with ESBLs, including CTX-M-10, TEM-3, TEM-10, TEM-12, TEM-6, SHV-18, and K1, gave false-negative results for one or more antimicrobial agents (MICs below the NCCLS screening concentration for detecting suspected ESBLs). When the higher inoculum was used, MICs of at least one antimicrobial agent increased at least fourfold in strains producing TEM-3, TEM-10, TEM-28, TEM-43, SHV-5, SHV-18, and K1. All antimicrobial agents showed an inoculum effect with at least one ESBL producer. Confirmatory clavulanate effects were seen for both inocula for all ESBL-producing strains with all antimicrobial agents tested, except for the CTX-M-10-producing
E. coli
with ceftazidime and the SHV-18-producing
K. pneumoniae
with cefotaxime. In kinetic studies, cefpodoxime and cefepime were hydrolyzed by ESBLs in a manner similar to that of cefotaxime. When total β-lactamase activity and hydrolysis parameters were evaluated, however, no single factor was predictive of inoculum effects. These results indicate that the NCCLS screening and confirmation tests are generally predictive of ESBL production, but false-negative results can arise when a lower inoculum is used in testing.
Comparative Analysis of Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis for Characterizing
Listeria monocytogenes
Strains Isolated from Environmental and Clinical Sources
Authors: Revazishvili Tamara , Kotetishvili Mamuka , Stine O. Colin , Kreger Arnold S. , Morris J. Glenn , Sulakvelidze Alexander
Published: January 2004

One hundred seventy-five
Listeria monocytogenes
strains were characterized by serotyping, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) based on loci in
actA
,
betL
,
hlyA
,
gyrB
,
pgm
, and
recA.
One hundred twenty-two sequence types (STs) were identified by MLST based on allelic profiles of the four housekeeping genes (
betL
,
gyrB
,
pgm
, and
recA
), and 34 and 38 alleles were identified for
hlyA
and
actA
, respectively. Several
actA
and
hlyA
alleles appeared to be predominantly associated with clinical isolates. MLST differentiated most of the
L. monocytogenes
strains better than did PFGE, and the discriminating ability of PFGE was better than that of serotyping. Several strains with different serotypes were found, by MLST and PFGE, to have very closely related genetic backgrounds, which suggested possible “antigen switching” among them. MLST can be a useful typing tool for differentiating
L. monocytogenes
strains (including strains undistinguishable by PFGE typing and serotyping), and it may be of value during investigations of food-borne outbreaks of listeriosis.
Assessment of Agreement between the AMPLICOR HIV-1 MONITOR Test Versions 1.0 and 1.5
Authors: Hill Charles E. , Green Alicia M. , Ingersoll Jessica , Easley Kirk A. , Nolte Frederick S. , Caliendo Angela M.
Published: January 2004

The agreement of the microwell plate AMPLICOR HIV-1 MONITOR version 1.0 (MWP 1.0), the microwell plate AMPLICOR HIV-1 MONITOR version 1.5 (MWP 1.5), and the COBAS AMPLICOR HIV-1 MONITOR version 1.5 (COBAS 1.5) tests was evaluated using clinical specimens and well-characterized control material. Two hundred patient plasma specimens and a panel of known human immunodeficiency virus type 1 (HIV-1) subtypes were tested. All data were log
10
transformed prior to analysis. The 95% limits of agreement for the three tests at the average of 3.66 log
10
copies/ml were ± 0.28 log
10
, ± 0.34 log
10
, and ± 0.34 log
10
copies/ml for MWP 1.0-MWP 1.5, MWP 1.0-COBAS 1.5, and MWP 1.5-COBAS 1.5, respectively. Ten specimens (6.1%) had differences exceeding the limits of agreement for the MWP 1.0 and MWP 1.5 tests. Correlation coefficients among the three tests were high (
r
≥ 0.96). The viral-load values obtained with the MWP 1.0 test were only 2.1% higher on average than those measured with the MWP 1.5 test and 1.6% higher than those seen with the COBAS 1.5 test. The MWP 1.5 test values were 0.8% higher than the COBAS 1.5 test values. Overall, there was less agreement among the different tests for viral-load values near the lower limit of quantification. The MWP 1.0 test underquantified subtypes A, E, F, G, and H by 1.0 to 2.0 log
10
copies/ml; this problem was not observed with the MWP 1.5 test. The close agreement among the results obtained with the different test versions and formats suggests that it is not necessary to reestablish a baseline viral load when changing AMPLICOR HIV-1 MONITOR tests, unless the patient is known to be infected with a non-B subtype.
Isolation of a Fastidious
Bergeyella
Species Associated with Cellulitis after a Cat Bite and a Phylogenetic Comparison with
Bergeyella zoohelcum
Strains
Authors: Shukla Sanjay K. , Paustian David L. , Stockwell Patrick J. , Morey Roger E. , Jordan Jean G. , Levett Paul N. , Frank Daniel N. , Reed Kurt D.
Published: January 2004

Bergeyella zoohelcum
is an uncommon zoonotic pathogen typically associated with cat or dog bites. Previously, only five cases of
B. zoohelcum
infection have been reported. We report the isolation and characterization of a fastidious
Bergeyella
species from acute cellulitis in the upper extremity of a 60-year-old woman. The organism was too fastidious for identification and susceptibility testing with traditional culture methods. The isolate was characterized further by PCR amplification and sequencing of the 16S rRNA gene with broad-range eubacterial primers. Phylogenetic analysis of the 16S ribosomal DNA sequence indicated that this isolate was a member of the species
B. zoohelcum
(previously
Weeksella zoohelcum
), a gram-negative bacillus that is rarely associated with infections in humans. Despite sharing a close genetic relationship with other
B. zoohelcum
strains, this isolate was extremely fastidious in nature, raising the possibility that similar strains from cat or dog bite wound infections have been underreported.
Utility of NCCLS Guidelines for Identifying Extended-Spectrum β-Lactamases in Non-
Escherichia coli
and Non-
Klebsiella
spp. of
Enterobacteriaceae
Authors: Schwaber Mitchell J. , Raney Patti M. , Rasheed J. Kamile , Biddle James W. , Williams Portia , McGowan John E. , Tenover Fred C.
Published: January 2004

NCCLS screening and confirmation methods for detecting extended-spectrum β-lactamases (ESBLs) apply only to
Escherichia coli
and
Klebsiella
spp., yet ESBLs have been found in other members of the family
Enterobacteriaceae
. We evaluated the effectiveness of NCCLS methods for detecting ESBLs in 690 gram-negative isolates of
Enterobacteriaceae
that excluded
E. coli
,
Klebsiella pneumoniae
, and
Klebsiella oxytoca.
Isolates were collected between January 1996 and June 1999 from 53 U.S. hospitals participating in Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology). The antimicrobial susceptibility patterns of the isolates were determined by using the NCCLS broth microdilution method (BMD), and those isolates for which the MIC of ceftazidime, cefotaxime, ceftriaxone, or aztreonam was ≥2 μg/ml or the MIC of cefpodoxime was ≥8 μg/ml (positive ESBL screen test) were further tested for a clavulanic acid (CA) effect by BMD and the disk diffusion method (confirmation tests). Although 355 (51.4%) of the isolates were ESBL screen test positive, only 15 (2.2%) showed a CA effect. Since 3 of the 15 isolates were already highly resistant to the five NCCLS indicator drugs, ESBL detection would have an impact on the reporting of only 1.7% of the isolates in the study. Only 6 of the 15 isolates that showed a CA effect contained a
bla
TEM
,
bla
SHV
,
bla
CTX-M
, or
bla
OXA
β-lactamase gene as determined by PCR (with a corresponding isoelectric focusing pattern). Extension of the NCCLS guidelines for ESBL detection to
Enterobacteriaceae
other than
E. coli
and
Klebsiella
spp. does not appear to be warranted in the United States at present, since the test has poor specificity for this population and would result in changes in categorical interpretations for only 1.7% of
Enterobacteriaceae
tested.
Evaluation of a TaqMan PCR Assay To Detect Rabies Virus RNA: Influence of Sequence Variation and Application to Quantification of Viral Loads
Authors: Hughes G. J. , Smith J. S. , Hanlon C. A. , Rupprecht C. E.
Published: January 2004

Published assays that use TaqMan PCR are consistently sensitive, rapid, and readily transferable. Here we describe a TaqMan PCR-based method for the detection of rabies virus (RV) RNA in tissue samples. We show that the method has an acceptable linear range, is both sensitive and specific, and, importantly, correlates with the concentration of infectious virus. In addition, the levels of RV-specific amplification are adjustable according to the levels of an endogenous control (β-actin mRNA), allowing the calculation of comparable quantities. We tested the capacity of this assay to cope with target sequence variations. The number of sequence mismatches between gene-specific oligonucleotides and the target sequence significantly affects amplification (
P
< 0.001), and point mutations at the center of the probe can result in false-negative results through the prevention of probe binding and subsequent fluorescence. This study demonstrates that the genetic heterogeneity of RVs may prove a serious obstacle in the development of a diagnostic assay based on TaqMan PCR; however, the quantification of RV levels may prove to be a valuable application of this assay.
Clonal and Horizontal Dissemination of
Klebsiella pneumoniae
Expressing SHV-5 Extended-Spectrum β-Lactamase in a Mexican Pediatric Hospital
Authors: Miranda Guadalupe , Castro Natividad , Leaños Blanca , Valenzuela Adriana , Garza-Ramos Ulises , Rojas Teresa , Solórzano Fortino , Chihu Lilia , Silva Jesús
Published: January 2004

One hundred eighty-four clinical isolates of
Klebsiella pneumoniae
were recovered from August 1996 to October 1997 at the Pediatric Hospital of the Instituto Mexicano del Seguro Social in Mexico City, Mexico. Most of the isolates were collected from the neonatal intensive care unit and infant wards, which are located on the same floor of the hospital. Isolates were genotypically compared by pulsed-field gel electrophoresis with
Xba
I restriction of chromosomal DNA. Of 184 clinical isolates, 91 belonged to cluster A and comprised three subtypes (A1, A2, and A3), while 93 isolates, comprising two minor clones, B (10 isolates) and C (7 isolates), and 76 unique patterns, were considered unrelated isolates (URI). Susceptibility patterns were indistinguishable in both groups. Fifty extended-spectrum β-lactamase-producing isolates, including 34 from clone A and 16 from URI, were examined for further studies. Molecular and genetic analysis showed that 47 of 50 clinical isolates expressed the SHV-5 β-lactamase. This enzyme, in combination with TEM-1, was encoded in a ≥170-kb conjugative plasmid. Results indicate that dissemination of this resistance was due to clonal and horizontal spread.
Molecular Epidemiology of Clinical Isolates of Methicillin-Resistant
Staphylococcus aureus
in Taiwan
Authors: Huang Yhu-Chering , Su Lin-Hui , Wu Tsu-Lan , Liu Chun-Eng , Young Tzuu-Guang , Chen Po-Yen , Hseuh Po-Ren , Lin Tzou-Yien
Published: January 2004

During July 2000 and October 2001, a total of 595 clinical isolates of methicillin-resistant
Staphylococcus aureus
(MRSA) were collected from six medical centers distributed in northern, central, and southern Taiwan. Specimen sources included blood (
n
= 279), pus (
n
= 173), sputum (
n
= 94), body fluids (
n
= 21), catheter tips (
n
= 20), and urine (
n
= 8). Pulsed-field gel electrophoresis (PFGE) with
Sma
I digestion was used to fingerprint these isolates. A total of 31 genotypes with 97 type-subtypes were identified. Subtypes could be identified in 7 genotypes. While there were 6 to 15 genotypes in each hospital, 433 isolates (73%) were shown to belong to a major type (genotype A, with 29 subtypes). This genotype was not only the type prevailing in all six hospitals but also the predominant clone in each hospital, accounting for 46 to 89% of all isolates in each hospital. Genotype C (16 subtypes) was the second dominant genotype, accounting for 9% of all isolates, and was distributed in five hospitals. Genotypes D (11 subtypes), E (5 subtypes), and B (6 subtypes) were distributed in five, four, and three hospitals, respectively. The other 26 types (30 type-subtypes) were minor. We conclude that the majority of MRSA clinical isolates shared a common PFGE pattern, indicating the presence of a single, epidemic MRSA clone prevailing in major hospitals in Taiwan.
Serotypes, Virulence Genes, and Intimin Types of Shiga Toxin (Verotoxin)-Producing
Escherichia coli
Isolates from Human Patients: Prevalence in Lugo, Spain, from 1992 through 1999
Authors: Blanco J. E. , Blanco M. , Alonso M. P. , Mora A. , Dahbi G. , Coira M. A. , Blanco J.
Published: January 2004

We have analyzed the prevalence of Shiga toxin-producing
Escherichia coli
(STEC) in stool specimens of patients with diarrhea or other gastrointestinal alterations from the Xeral-Calde Hospital of Lugo City (Spain). STEC strains were detected in 126 (2.5%) of 5,054 cases investigated, with a progressive increase in the incidence from 0% in 1992 to 4.4% in 1999. STEC O157:H7 was isolated in 24 cases (0.5%), whereas non-O157 STEC strains were isolated from 87 patients (1.7%). STEC strains were (after
Salmonella
and
Campylobacter
strains) the third most frequently recovered enteropathogenic bacteria. A total of 126 human STEC isolates were characterized in this study. PCR showed that 43 (34%) isolates carried
stx
1
genes, 45 (36%) possessed
stx
2
genes and 38 (30%) carried both
stx
1
and
stx
2
. A total of 88 (70%) isolates carried an
ehxA
enterohemolysin gene, and 70 (56%) isolates possessed an
eae
intimin gene (27 isolates with type γ1, 20 with type β1, 8 with type ζ, 5 with type γ2, and 3 with type ε). STEC isolates belonged to 41 O serogroups and 66 O:H serotypes, including 21 serotypes associated with hemolytic uremic syndrome and 30 new serotypes not previously reported among human STEC strains in other studies. Although the 126 STEC isolates belonged to 81 different seropathotypes (associations between serotypes and virulence genes), only four accounted for 31% of isolates. Seropathotype O157:H7
stx
1
stx
2
eae-
γ1
ehxA
was the most common (13 isolates) followed by O157:H7
stx
2
eae-
γ1
ehxA
(11 isolates), O26:H11
stx
1
eae-
β1
ehxA
(11 isolates), and O111:H-
stx
1
stx
2
eae-
γ2
ehxA
(4 isolates). Our results suggest that STEC strains are a significant cause of human infections in Spain and confirm that in continental Europe, infections caused by STEC non-O157 strains are more common than those caused by O157:H7 isolates. The high prevalence of STEC strains (both O157:H7 and non-O157 strains) in human patients, and their association with serious complications, strongly supports the utilization of protocols for detection of all serotypes of STEC in Spanish clinical microbiology laboratories.
Use of Real-Time PCR To Resolve Slide Agglutination Discrepancies in Serogroup Identification of
Neisseria meningitidis
Authors: Mothershed Elizabeth A. , Sacchi Claudio T. , Whitney Anne M. , Barnett Gwen A. , Ajello Gloria W. , Schmink Susanna , Mayer Leonard W. , Phelan Maureen , Taylor Thomas H. , Bernhardt Scott A. , Rosenstein Nancy E. , Popovic Tanja
Published: January 2004

Neisseria meningitidis
is a leading cause of bacterial meningitis and septicemia in children and young adults in the United States. Rapid and reliable identification of
N. meningitidis
serogroups is crucial for judicious and expedient response to cases of meningococcal disease, including decisions about vaccination campaigns. From 1997 to 2002, 1,298
N. meningitidis
isolates, collected in the United States through the Active Bacterial Core surveillance (ABCs), were tested by slide agglutination serogrouping (SASG) at both the ABCs sites and the Centers for Disease Control and Prevention (CDC). For over 95% of isolates, SASG results were concordant, while discrepant results were reported for 58 isolates. To resolve these discrepancies, we repeated the SASG in a blinded fashion and employed
ctrA
and six serogroup-specific PCR assays (SGS-PCR) to determine the genetic capsule type. Seventy-eight percent of discrepancies were resolved, since results of the SGS-PCR and SASG blinded study agreed with each other and confirmed the SASG result at either state health laboratories or CDC. This study demonstrated the ability of SGS-PCR to efficiently resolve SASG discrepancies and identified the main cause of the discrepancies as overreporting of these isolates as nongroupable. It also reemphasized the importance of adherence to quality assurance procedures when performing SASG and prompted prospective monitoring for SASG discrepancies involving isolates collected through ABCs in the United States.
Development of a Real-Time Reverse-Transcription PCR for Detection of Newcastle Disease Virus RNA in Clinical Samples
Authors: Wise Mark G. , Suarez David L. , Seal Bruce S. , Pedersen Janice C. , Senne Dennis A. , King Daniel J. , Kapczynski Darrell R. , Spackman Erica
Published: January 2004

A real-time reverse-transcription PCR (RRT-PCR) was developed to detect avian paramyxovirus 1 (APMV-1) RNA, also referred to as Newcastle disease virus (NDV), in clinical samples from birds. The assay uses a single-tube protocol with fluorogenic hydrolysis probes. Oligonucleotide primers and probes were designed to detect sequences from a conserved region of the matrix protein (M) gene that recognized a diverse set (
n
= 44) of APMV-1 isolates. A second primer-probe set was targeted to sequences in the fusion protein (F) gene that code for the cleavage site and detect potentially virulent NDV isolates. A third set, also directed against the M gene, was specific for the North American (N.A.) pre-1960 genotype that includes the common vaccine strains used in commercial poultry in the United States. The APMV-1 M gene, N.A. pre-1960 M gene, and F gene probe sets were capable of detecting approximately 10
3
, 10
2
, and 10
4
genome copies, respectively, with in vitro-transcribed RNA. Both M gene assays could detect approximately 10
1
50% egg infective doses (EID
50
), and the F gene assay could detect approximately 10
3
EID
50
. The RRT-PCR test was used to examine clinical samples from chickens experimentally infected with the NDV strain responsible for a recent epizootic in the southwestern United States. Overall, a positive correlation was obtained between the RRT-PCR results and virus isolation for NDV from clinical samples.
Novel Mass Spectrometry-Based Tool for Genotypic Identification of Mycobacteria
Authors: Lefmann Michael , Honisch Christiane , Böcker Sebastian , Storm Niels , von Wintzingerode Friedrich , Schlötelburg Cord , Moter Annette , van den Boom Dirk , Göbel Ulf B.
Published: January 2004

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after base-specific cleavage of PCR amplified and in vitro-transcribed 16S rRNA gene (rDNA) was used for the identification of mycobacteria. Full-length 16S rDNA reference sequences of 12 type strains of
Mycobacterium
spp. frequently isolated from clinical specimens were determined by PCR, cloning, and sequencing. For MALDI-TOF MS-based comparative sequence analysis, mycobacterial 16S rDNA signature sequences (∼500 bp) of the 12 type strains and 24 clinical isolates were PCR amplified using RNA promoter-tagged forward primers. T7 RNA polymerase-mediated transcription of forward strands in the presence of 5-methyl ribo-CTP maximized mass differences of fragments generated by base-specific cleavage. In vitro transcripts were subsequently treated with RNase T1, resulting in G-specific cleavage. Sample analysis by MALDI-TOF MS showed a specific mass signal pattern for each of the 12 type strains, allowing unambiguous identification. All 24 clinical isolates were identified unequivocally by comparing their detected mass signal pattern to the reference sequence-derived in silico pattern of the type strains and to the in silico mass patterns of published 16S rDNA sequences. A 16S rDNA microheterogeneity of the
Mycobacterium xenopi
type strain (DSM 43995) was detected by MALDI-TOF MS and later confirmed by Sanger dideoxy sequencing. In conclusion, analysis of 16S rDNA amplicons by MS after base-specific cleavage of RNA transcripts allowed fast and reliable identification of the
Mycobacterium tuberculosis
complex and ubiquitous mycobacteria (mycobacteria other than tuberculosis). The technology delivers an open platform for high-throughput microbial identification on the basis of any specific genotypic marker region.
Detection of Severe Acute Respiratory Syndrome Coronavirus in Blood of Infected Patients
Authors: Ng Lisa F. P. , Wong Michelle , Koh Susie , Ooi Eng-Eong , Tang King-Fai , Leong Hoe-Nam , Ling Ai-Ee , Agathe Lora V. , Tan Jenny , Liu Edison T. , Ren Ee-Chee , Ng Lee-Ching , Hibberd Martin L.
Published: January 2004

Severe acute respiratory syndrome (SARS) has caused major outbreaks worldwide, resulting in an urgent need to obtain sensitive and accurate diagnosis of this disease. PCR-based detection methods were developed for use on a variety of samples, including blood. Eighteen subjects were investigated, and results indicated that blood samples contain sufficient virus for detection by using quantitative real-time PCR.
Rapid Immunochromatographic Test Using Recombinant SAG2 for Detection of Antibodies against
Toxoplasma gondii
in Cats
Authors: Huang Xiaohong , Xuan Xuenan , Hirata Haruyuki , Yokoyama Naoaki , Xu Longshan , Suzuki Naoyoshi , Igarashi Ikuo
Published: January 2004

An immunochromatographic test using recombinant truncated surface antigen 2 for detection of antibodies against
Toxoplasma gondii
was developed. Evaluation of detection of the antibody in mice and cats suggests that this test is rapid, simple, accurate, relatively inexpensive, and suitable for use under field conditions.
Nissui Glucose Fermentative Gram-Negative Rod Identification System EB-20 Gives a Unique Profile for Typical Non-Sorbitol-Fermenting
Escherichia coli
O157:H7
Authors: Kodaka H. , Uesaka Y. , Kashitani F.
Published: January 2004

The 98 non-sorbitol-fermenting (NSF)
Escherichia coli
O157:H7 strains identified on a Nissui glucose fermentative gram-negative rod identification system (EB-20) gave a unique biochemical profile number that was not detected in 85 pathogenic and 13 nonpathogenic
E. coli
strains. Thus, EB-20 is useful for the identification of NSF
E. coli
O157:H7 and provides a simple, cost-effective, and reliable tool for clinical laboratories.
Development of an Immunochromatographic Test with Recombinant EMA-2 for the Rapid Detection of Antibodies against
Babesia equi
in Horses
Authors: Huang Xiaohong , Xuan Xuenan , Xu Longshan , Zhang Shoufa , Yokoyama Naoaki , Suzuki Naoyoshi , Igarashi Ikuo
Published: January 2004

An immunochromatographic test (BeICT) for the rapid detection of antibodies against
Babesia equi
was developed. It clearly differentiated
B. equi-
infected horses from
B. caballi-
infected and uninfected horses. The agreement with enzyme-linked immunosorbent assay results was 96.7% in the detection of field sera. The results suggest that BeICT is rapid, simple, reliable, and suitable for use to detect
B. equi
infection in the field.
Molecular Epidemiology of the Iron Utilization Genes of Enteroaggregative
Escherichia coli
Authors: Okeke Iruka N. , Scaletsky Isabel C. A. , Soars Elizabeth H. , Macfarlane Louissa R. , Torres Alfredo G.
Published: January 2004

Enteroaggregative
Escherichia coli
(EAEC) strains are etiologic agents of acute and persistent diarrhea. In this study, the results of phenotypic assays suggested that EAEC strains possess specialized iron acquisition systems. Genes required for the synthesis (
iucA
) or transport (
fepC
) of siderophores, and genes encoding siderophore (
fyuA
,
ireA
, and
iroN
) or heme transport (
chu
) receptors or hemoglobin proteases (
pic
and
hbp
), were sought in EAEC strains which have been characterized with respect to known virulence genes and phylogeny. The
chuA
,
iucA
,
fyuA
,
fepC
, and
pic
genes were detected in 33, 76.2, 85.7, 33, and 61.9% of these EAEC strains, respectively, and the other genes were absent. The majority of EAEC strains possessed genes encoding multiple iron transport systems, and there was no phylogenetic correlation in the distribution of the majority of these loci, as is typical for EAEC. The notable exceptions were
chuA
and
fepC
(which is associated with the
prrA-modA-fepC
pathogenicity island); these genes were restricted to the EAEC2 and DAEC2 phylogenetic groups, which could represent pathogenic subsets. When collections of EAEC strains isolated during case-control studies in Nigeria and Brazil were examined, no association of the presence of either
chuA
or
iucA
alone with diarrhea was seen, but both genes together were present in significantly more strains from cases than from controls in the Nigerian collection (
P
< 0.05). It is possible that the presence of both genes marks at least some virulent strains. The data also demonstrate geographical variation in the association of iron utilization genes with disease in EAEC.
Possible High Rate of Transmission of Nontypeable
Haemophilus influenzae
, Including β-Lactamase-Negative Ampicillin-Resistant Strains, between Children and Their Parents
Authors: Watanabe Hiroshi , Hoshino Kazuhiko , Sugita Rinya , Asoh Norichika , Watanabe Kiwao , Oishi Kazunori , Nagatake Tsuyoshi
Published: January 2004

The possible transmission of nontypeable
Haemophilus influenzae
between children and their parents was evaluated in 18 pairs of subjects from 15 families. Of the 33 isolates, 31 were found to be β-lactamase negative, including 10 β-lactamase-negative, ampicillin (AMP)-resistant (BLNAR) strains (AMP MIC, ≥1.0 μg/ml) and 2 were β-lactamase producing. Molecular typing by pulsed-field gel electrophoresis (PFGE) showed that 10 BLNAR isolates had 6 patterns, 23 non-BLNAR isolates had 13 patterns, and these patterns were different from each other, except for 1 pattern. As a result, the PFGE patterns in 14 of 18 pairs were indistinguishable and those in 4 pairs were different. These data indicate a possible high rate of intrafamilial transmission of nontypeable
H. influenzae
, including BLNAR strains, between children and their parents.
Method for Testing for Human Papillomavirus Infection in Patients with Cervical Intraepithelial Disease
Authors: Lin Hsing-Pei , Huang Yang-Yang , Wu Hsueh-Yin , Kao Jau-Tsuen
Published: January 2004

To determine whether DNA detection of high-risk human papillomavirus could represent a reliable screening technique and a useful follow-up method, we investigated the performance of the Hybrid Capture II assay in detecting high-grade cervical lesions. The test positivity at 1.86 pg/ml had a high sensitivity (94.7%) and improved specificity. It was also a suitable follow-up method for detecting the recurrence of cervical intraepithelial disease in patients.
Amplified Fragment Length Polymorphism Fingerprinting Is an Effective Technique To Distinguish
Streptococcus pneumoniae
from Other Streptococci and an Efficient Alternative to Pulsed-Field Gel Electrophoresis for Molecular Typing of Pneumococci
Authors: Neeleman Chris , Klaassen Corné H. W. , de Valk Hanneke A. , de Ruiter Maaike T. , Mouton Johan W.
Published: January 2004

Amplified fragment length polymorphism versus pulsed-field gel electrophoresis was used for fingerprinting of 85 macrolide-resistant pneumococcal isolates identified by using primarily phenotypic methods. Confirmation of identification by 16S rRNA sequencing revealed that 27 isolates were actually nonpneumococci. Amplified fragment length polymorphism but not pulsed-field gel electrophoresis offered simultaneous and accurate discrimination between pneumococci and nonpneumococcal species.
PCR-Based Genotyping of
Mycobacterium tuberculosis
with New GC-Rich Repeated Sequences and IS
6110
Inverted Repeats Used as Primers
Authors: Kotłowski Roman , Shamputa Isdore Chola , El Aila Nabil Abdullah , Sajduda Anna , Rigouts Leen , van Deun Armand , Portaels Françoise
Published: January 2004

In the present study we attempted to develop a PCR-based epidemiological tool for the differentiation of
Mycobacterium tuberculosis
isolates. Use of the designed primers Mtb1 (5′-CCG-GCG-GGG-CCG-GCG-G) and Mtb2 (5′-CGG-CGG-CAA-CGG-CGG-C) targeting frequently repeated 16-bp sequences in combination with primers sited at the inverted repeats flanking IS
6110
allowed differentiation of
M. tuberculosis
isolates.
Improving the Bacteriological Diagnosis of Tuberculous Meningitis
Authors: Thwaites Guy E. , Chau Tran Thi Hong , Farrar Jeremy J.
Published: January 2004

We made a bacteriological diagnosis of tuberculous meningitis in 107 of 132 (81%) adults with clinical tuberculous meningitis: acid-fast bacilli were seen in 77 of 132 (58%) and cultured from 94 of 132 (71%). Volume of cerebrospinal fluid, duration of symptoms, cerebrospinal fluid neutrophils, lactate, and glucose were all independently associated with bacteriological confirmation.
Evaluation of the NCCLS M44-P Disk Diffusion Method for Determining Susceptibilities of 276 Clinical Isolates of
Cryptococcus neoformans
to Fluconazole
Authors: Pfaller M. A. , Messer S. A. , Boyken L. , Rice C. , Tendolkar S. , Hollis R. J. , Diekema D. J.
Published: January 2004

We evaluated the NCCLS M44-P fluconazole disk diffusion method in comparison with the NCCLS M27-A2 broth microdilution method for determining the susceptibility of 276 isolates of
Cryptococcus neoformans
. Disk diffusion testing was performed using Mueller-Hinton agar supplemented with 2% glucose and 0.5 μg of methylene blue/ml. Among the 276 isolates, 259 (93.8%) were susceptible, 16 (5.8%) were susceptible—dose dependent, and 1 (0.4%) was resistant to fluconazole as determined by the NCCLS broth microdilution method. The overall categorical agreement between the two methods was 86%, with 0% very major errors, 2% major errors, and 12% minor errors. The disk diffusion method using Mueller-Hinton agar supplemented with glucose and methylene blue appears to be a useful approach for determining the fluconazole susceptibility of C.
neoformans.
Helicobacter
Species in the Intestinal Mucosa of Patients with Ulcerative Colitis
Authors: Oliveira Adriana Gonçalves , Sanna Maria das Graças Pimenta , Rocha Gifone Aguiar , Rocha Andreia Maria Camargos , Santos Adriana , Dani Renato , Marinho Frederico Passos , Moreira Liano Sia , Ferrari Maria de Lourdes Abreu , Moura Sílvia Beleza , Castro Lúcia Porto Fonseca , Queiroz Dulciene Maria Magalhães
Published: January 2004

In a search for
Helicobacter
species in the intestinal mucosae of 42 patients with ulcerative colitis (UC) and 74 without UC, only
H. pylori
was found. Although the bacterium was detected in UC patients by culture (7.1%) and nested PCR (19.0%), its presence was not associated with the disease (
P
= 0.13).
Susceptibilities to Different Antibiotics of
Helicobacter pylori
Strains Isolated from Patients at the Pediatric Medical Center of Tehran, Iran
Authors: Falsafi Tahereh , Mobasheri Fatemeh , Nariman Farahnaz , Najafi Mehri
Published: January 2004

Antibiotic susceptibility testing of 70 pediatric
Helicobacter pylori
isolates was performed by using screening agar and disk diffusion methods. Resistance to metronidazole and tinidazole was 72 to 79% and 71 to 81% by modified disk diffusion and 77% and 78% by screening agar, respectively. Susceptibilities to amoxicillin, ampicillin, clarithromycin, tetracycline, erythromycin, and ciprofloxacin were 58, 69, 75, 68, 68, and 65%, respectively.
Mutations Including IS
6110
Insertion in the Gene Encoding the MPB64 Protein of Capilia TB-Negative
Mycobacterium tuberculosis
Isolates
Authors: Hirano Kazue , Aono Akio , Takahashi Mitsuyoshi , Abe Chiyoji
Published: January 2004

A simple immunochromatographic assay, Capilia TB, using anti-MPB64 monoclonal antibodies, is a kit for discriminating between the
Mycobacterium tuberculosis
complex and mycobacteria other than tubercle bacilli. The sensitivity of the kit was estimated to be 99.2% (381 of 384 samples). The sequencing analysis revealed that all of the Capilia TB-negative isolates had mutations within the
mpb64
gene, leading to the production of an incomplete protein as a result of a deletion of the C-terminal region of the protein.
Eight-Year Survey of Human Rotavirus Strains Demonstrates Circulation of Unusual G and P Types in Hungary
Authors: Bányai Krisztián , Gentsch Jon R. , Glass Roger I. , Új Mária , Mihály Ilona , Szücs György
Published: January 2004

Between 1992 and 2000, a total of 4,173 rotavirus-positive samples were collected from two areas of Hungary. Of these, 2,020 specimens (48.4%) were analyzed for G serotype, using monoclonal antibody-based immunoassay and reverse transcription-PCR. By the two methods, 1,789 samples were specified as G1 (62%), G2 (12.2%), G3 (1.4%), G4 (6.4%), G6 (1.0%), G9 (2.9%), or mixed infection (2.6%), and the remaining 231 (11.4%) could not be G typed. The linkage between G and P type, subgroup specificity, and RNA profile was investigated with a sample subset. Among these specimens, we identified both the four globally common strains (P[8],G1 subgroup II (sgII); P[4],G2 sgI; P[8],G3 sgII; and P[8],G4 sgII) and six uncommon strains (P[6],G4 sgII; P[9],G3 sgI; P[9],G6 sgI; P[14],G6 sgI; P[8],G9 sgII; and P[8],G9 sgI). All strains with P[8], P[6], P[9], and P[14] specificities had a long electropherotype, whereas most of those carrying a P[4] specificity were associated with a short electropherotype. Although once considered to be rare, P[9],G6 and P[8],G9 rotavirus strains represent potentially important new serotypes in Hungary.
Reduced Etiological Role for Enteropathogenic
Escherichia coli
in Cases of Diarrhea in Brazilian Infants
Authors: Rodrigues Josias , Thomazini Cristiane M. , Morelli Alexandra , de Batista Georgea C. M.
Published: January 2004

Previously common in Brazil, enteropathogenic
Escherichia coli
(EPEC) strains of serogroups O55, O111, and O119 are now rare, while enteroadherent strains other than EPEC, belonging to serogroups such as O125, were prevalent among 126 diarrheic infants less than 1 year old who were surveyed. None of these strains had the EPEC bundle-forming pilus (
bfpA
) gene.
Use of Genome Selected Repeated Sequences Increases the Sensitivity of PCR Detection of
Tropheryma whipplei
Authors: Fenollar Florence , Fournier Pierre-Edouard , Robert Catherine , Raoult Didier
Published: January 2004

The availability of the
Tropheryma whipplei
genome offers the putative possibility of choosing logical DNA targets. We applied a PCR assay (targeting repeated sequences of
T. whipplei
) to samples from patients with Whipple's disease and to those from members of a control group. When compared to the results seen with regular PCR, the sensitivity of repeat PCR was significantly enhanced (
P
= 0.02) without alteration of its specificity.
Specific Oligonucleotide Primers for Identification ofCladophialophora carrionii, a Causative Agent of Chromoblastomycosis
Authors: Abliz Paride , Fukushima Kazutaka , Takizawa Kayoko , Nishimura Kazuko
Published: January 2004

Cladophialophora carrioniiis one of the relatively common causative agents of chromoblastomycosis. We have developed the specific oligonucleotide primer set based on the internal transcribed spacer regions of ribosomal DNA for the rapid identification of this pathogen. PCR with this primer set amplified a 362-bp amplicon fromC. carrioniistrains. From other relevant dematiaceous species, including medically important dematiaceous fungi, such asFonsecaea pedrosoi,Phialophora verrucosa, andExophiala dermatitidis, and eight species of medically important yeasts, such asCandida albicansandCryptococcus neoformansvar.neoformans, the primer set did not produce any amplicon. PCR with this primer set may be a useful tool for the identification ofC. carrionii.
Impact of DNA Polymerases and Their Buffer Systems on Quantitative Real-Time PCR
Authors: Wolffs Petra , Grage Halfdan , Hagberg Oskar , Rådström Peter
Published: January 2004

An investigation of the influence of five DNA polymerase-buffer systems on real-time PCR showed that the choice of both DNA polymerase and the buffer system affected the amplification efficiency as well as the detection window. The analytical repeatability of the data for different systems changed clearly, leading us to conclude that basing quantitative measurements on single-data-set standard curves can lead to significant errors.
Survival of
Streptococcus pneumoniae
,
Haemophilus influenzae
, and
Moraxella catarrhalis
Frozen in Skim Milk- Tryptone-Glucose-Glycerol Medium
Authors: Kaijalainen Tarja , Ruokokoski Esa , Ukkonen Pentti , Herva Elja
Published: January 2004

In STGG (skim milk, tryptone, glucose, glycerol) medium at −80°C,
Streptococcus pneumoniae
,
Haemophilus influenzae
, and
Moraxella catarrhalis
isolates survived for at least 3 years, and the same species have survived in nasopharyngeal swabs for at least 1.5 years. At −20°C,
S. pneumoniae
and
M. catarrhalis
survived for 1.5 years, but
H. influenzae
survived for only 2 months.
Prevalence and Antimicrobial Susceptibility of Serogroup D Nontyphoidal
Salmonella
in a University Hospital in Taiwan
Authors: Chiu Cheng-Hsun , Su Lin-Hui , Hung Ching-Chi , Chen Kuo-Lung , Chu Chishih
Published: January 2004

The incidence of serogroup D
Salmonella
has been increasing in Taiwan. Most of these isolates belonged to
Salmonella enterica
serovar Enteritidis and showed a relatively higher rate of resistance to sulfamethoxazole-trimethoprim than to other antimicrobial agents. The results of molecular experiments indicated that genes responsible for the resistance were located on plasmids. The resistance may occur via horizontal gene transfer. Furthermore, the first identification of ciprofloxacin and ceftriaxone resistance in serogroup D
Salmonella
in our hospital is also than they did to other antimicrobial agents cause for concern.
Evaluation of Four Commercial Test Systems for Identification of
Actinomyces
and Some Closely Related Species
Authors: Santala Anne-Marie , Sarkonen Nanna , Hall Val , Carlson Petteri , Jousimies-Somer Hannele , Könönen Eija
Published: January 2004

We evaluated four commercially available kits for rapid identification of
Actinomyces
and related species. The kits identified correctly 26 to 65% of “classical”
Actinomyces
strains to the species level and 13 to 49% of newly described
Actinomyces
strains to the genus level, thus indicating relatively poor applicability and a need to update these kits.
Strengths and Limitations of Commercial Tests for Hepatitis C Virus RNA Quantification
Authors: Morishima Chihiro , Chung Minjun , Ng Ka Wing , Brambilla Donald J. , Gretch David R.
Published: January 2004

The sensitivity of the Roche COBAS Monitor v.2.0 test was slightly better than that of the Bayer bDNA 3.0 test, but the Monitor test underquantified specimens by 2.5- to 10.6-fold even at relatively low hepatitis C virus RNA concentrations. Dilution prior to assay minimally decreased the reproducibility of the Monitor assay, leading to the recommendation for all specimens to be diluted 1:100 prior to testing by this assay.
Prevalence of Human Immunodeficiency Virus Drug Resistance Mutations and Subtypes in Drug-Naive, Infected Individuals in the Army Health Service of Rio de Janeiro, Brazil
Authors: Pires Ivone L. , Soares Marcelo A. , Speranza Francisco A. B. , Ishii Solange K. , Vieira Maria C. G. , Gouvêa Maria I. F. S. , Guimarães Maria A. A. M. , de Oliveira Fátima E. , Magnanini Monica M. F. , Brindeiro Rodrigo M. , Tanuri Amilcar
Published: January 2004

The prevalence of mutations that confer resistance to antiretroviral drugs was examined in 56 drug-naive, human immunodeficiency virus type 1 (HIV-1)-infected individuals from the Army Health Service in Rio de Janeiro, Brazil. No primary protease inhibitor mutations were found, but secondary mutations were observed in 51.2% of the samples. Fourteen percent of the viruses had reverse transcriptase inhibitor-associated mutations. Comparative analysis of protease secondary mutations from four different time periods in drug-naive patients in the city of Rio de Janeiro has indicated constant rates for particular mutations. Changes in CD4 cell counts and HIV viral load over time in subtype B- and non-B-infected drug-naive patients were not significantly different.
Molecular Epidemiology of Disease Due to
Mycobacterium bovis
in Humans in the United Kingdom
Authors: Gibson Andrea L. , Hewinson Glyn , Goodchild Tony , Watt Brian , Story Alistair , Inwald Jacqueline , Drobniewski Francis A.
Published: January 2004

Mycobacterium bovis
is the causative agent of bovine tuberculosis, with a wide host range. Fifty human
M. bovis
isolates were typed using spoligotyping and variable number tandem repeats (VNTR). Fifteen of these spoligotypes have not yet been recorded in cattle. The predominant spoligotype in humans and cattle was subdivided by VNTR.
Efficacy of Galactomannan Antigen in the Platelia
Aspergillus
Enzyme Immunoassay for Diagnosis of Invasive Aspergillosis in Liver Transplant Recipients
Authors: Kwak Eun Jeong , Husain Shahid , Obman Asia , Meinke Lisa , Stout Janet , Kusne Shimon , Wagener Marilyn M. , Singh Nina
Published: January 2004

The utility of galactomannan antigen for diagnosing invasive aspergillosis was evaluated in 154 liver transplant recipients. Sample agreement was 98.5%, and patient specificity was 87%. Galactomannan positivity correlated with mortality, even when controlled for the number of tests performed. Whether galactomannan positivity identifies a subgroup at risk for poor outcome warrants further evaluation.
Automated Procedure for Improving the RNA Isolation Step in Viral Load Testing for Human Immunodeficiency Virus
Authors: Xu Min , Chan Yung , Fischer Steven H. , Remaley Alan T.
Published: January 2004

An automated RNA isolation procedure on the Qiagen BioRobot is described for performing viral load tests for human immunodeficiency virus (HIV) with the Amplicor HIV type 1 test. The new procedure improves the precision of the assay and requires significantly less labor than the presently used manual RNA isolation procedure.
Detection of an Unusual Human Rotavirus Strain with G5P[8] Specificity in a Cameroonian Child with Diarrhea
Authors: Esona Mathew D , Armah George E. , Geyer Annelise , Steele A. Duncan
Published: January 2004

Rotavirus strains detected as part of ongoing strain surveillance in Cameroon, and whose first-round reverse transcription-PCR product could not be genotyped by using conventional genotyping primers, were subjected to sequence analysis for strain characterization. We detected for the first time in Africa a human rotavirus with G5 specificity. The Cameroonian G5 strain had a short electrophoretic pattern and was of VP6 subgroup I specificity and a VP4 P[8] type. The VP7 gene shared a higher nucleic acid and amino acid homology with the porcine G5 strain CC117 (90 and 96%, respectively) than with human G5 strain IAL-28 (86 and 92%, respectively). Phylogenetic analysis showed Cameroonian strain MRC3105 clustered together in the same lineage as two other reported porcine G5 strains. The Cameroonian G5 strain, the first to be reported in humans outside of Latin America, may be a natural reassortant between animal and human rotavirus strains.
Antimicrobial Susceptibility and Epidemiology of a Worldwide Collection of
Chryseobacterium
spp.: Report from the SENTRY Antimicrobial Surveillance Program (1997-2001)
Authors: Kirby Jeffrey T. , Sader Helio S. , Walsh Timothy R. , Jones Ronald N.
Published: January 2004

Limited data are available on
Chryseobacterium
spp. leading to an evaluation of the patient demographics and susceptibility patterns for
Chryseobacterium
spp. collected in the first 5 years of the SENTRY Antimicrobial Surveillance Program (1997 to 2001). Fifty isolates (24
Chryseobacterium meningosepticum
, 20
Chryseobacterium indologenes
, two
Chryseobacterium gleum
, and 4
Chryseobacterium
spp. isolates) were collected. The highest
Chryseobacterium
prevalence was detected among the elderly. The most active antimicrobials were the newer quinolones (garenoxacin, gatifloxacin, and levofloxacin, each with a MIC at which 90 percent of the isolates are inhibited [MIC
90
] of 1 μg/ml and 98.0% susceptibility) followed by rifampin (MIC
90
, 2 μg/ml and 85.7% susceptibility). Trimethoprim-sulfamethoxazole, ciprofloxacin, and piperacillin-tazobactam also showed reasonable activity; vancomycin showed poor potency.
Serodiagnosis of Chronic and Acute Chagas' Disease with
Trypanosoma cruzi
Recombinant Proteins: Results of a Collaborative Study in Six Latin American Countries
Authors: Umezawa Eufrosina S. , Luquetti Alejandro O. , Levitus Gabriela , Ponce Carlos , Ponce Elisa , Henriquez Diana , Revollo Susana , Espinoza Bertha , Sousa Octavio , Khan Baldip , da Silveira José Franco
Published: January 2004

An enzyme-linked immunosorbent assay to diagnose Chagas' disease by a serological test was performed with
Trypanosoma cruzi
recombinant antigens (JL8, MAP, and TcPo). High sensitivity (99.4%) and specificity (99.3%) were obtained when JL8 was combined with MAP (JM) and tested with 150 serum samples from chagasic and 142 nonchagasic individuals. Moreover, JM also diagnosed 84.2% of patients in the acute phase of
T. cruzi
infection.
Multiplex Real-Time PCR Assay for Detection of Influenza and Human Respiratory Syncytial Viruses
Authors: Boivin Guy , Côté Stéphanie , Déry Pierre , De Serres Gaston , Bergeron Michel G.
Published: January 2004

A multiplex real-time PCR assay was developed with a LightCycler instrument for detection of influenza viruses A and B and the human respiratory syncytial virus (HRSV). Detection of each viral product and of an internal control was based on determination of specific melting temperatures by the LightCycler software. The lower limit of detection in the multiplex PCR assay was found to be 50 copies for each viral target. In an evaluation of nasopharyngeal samples collected from hospitalized children (ages, 0 to 3 years) with acute respiratory tract infections during the winter of 2001 to 2002, a viral pathogen was detected by the multiplex PCR test in 139 (66.8%) of 208 cases, including 45 (21.6%) influenza A virus infections, no (0%) influenza B virus infections, 106 (51%) HRSV infections, and 12 (5.8%) coinfections. The multiplex PCR test was compared to rapid antigen detection assays for influenza viruses A and B (Directigen; Becton Dickinson, Sparks, Md.) and HRSV (RSV TestPack; Abbott Laboratories, Abbott Park, Ill.) in 172 and 204 samples, respectively. After resolution of discrepant test results by use of additional PCR assays targeting other viral genes, the sensitivity (Se) and specificity (Sp) of the multiplex PCR assay for influenza A virus were 100 and 97.7% compared to 43.6 and 98.5% for the antigenic test. Similarly, the Se and Sp of the multiplex PCR assay for HRSV were 94.5 and 98.9% compared to 81.6 and 94.7% for the antigenic test. In conclusion, our multiplex real-time PCR assay combines both rapidity and sensitivity for detecting the most important respiratory viral pathogens in children.