bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of a New Etest for Detecting Metallo-β-Lactamases in Routine Clinical Testing
Authors: Walsh Timothy R. , Bolmström Anne , Qwärnström Anette , Gales Ana 
Published: October 2002

ABSTRACT

Several Etest (AB BIODISK, Solna, Sweden) gradient formats were developed for detection of metallo-β-lactamases based on the reduction of imipenem (IP) or ceftazidime (TZ) MICs in the presence of EDTA or 2-mercaptopropionic acid (MPA). The Etest metallo-β-lactamase (Etest MBL) strips consisted of a double-sided seven-dilution range of IP or TZ (4 to 256 μg/ml) and IP or TZ (1 to 64 μg/ml) overlaid with a constant concentration of EDTA or MPA. The prototype strips were evaluated on several agar media (brain heart infusion agar, Isosensitest agar, nutrient agar, and Mueller-Hinton agar for aerobes and brucella blood agar for anaerobes) with 138 challenge strains:
Acinetobacter
spp. (
n
= 9),
Aeromonas
spp. (
n
= 8),
Chryseobacterium
spp. (
n
= 28),
Escherichia coli
(
n
= 1),
Klebsiella pneumoniae
(
n
= 4),
Pseudomonas aeruginosa
(
n
= 14),
Proteus mirabilis
(
n
= 3),
Serratia
spp. (
n
= 10),
Stenotrophomonas maltophilia
(
n
= 43),
Sphingobacterium
spp. (
n
= 3), and
Bacteroides fragilis
group (
n
= 15). PCR analysis using specific primers for IMP-1, L1, CcrA, and
bla
B/C
confirmed the presence of the metallo-β-lactamase genes. Enzyme assays were also performed with IP as an indicator substrate followed by EDTA inhibition profiles. EDTA was found to be a better inhibitor of metallo-β-lactamases, especially for anaerobes. IP was a better than TZ. Mueller-Hinton agar was the preferred medium, particularly when compared to Isosensitest agar, which frequently produced falsely low MICs for IP. Etest IP plus IP-EDTA with Mueller-Hinton agar had a sensitivity of 94% (79 of 84) and specificity of 95% (124 of 130). The Etest MBL strip appears to be an acceptable diagnostic reagent to detect metallo-β-lactamase phenotypes in the clinical microbiology laboratory.
Evaluation of Three Techniques for Detection of Low-Level Methicillin-Resistant
Staphylococcus aureus
(MRSA): a Disk Diffusion Method with Cefoxitin and Moxalactam, the Vitek 2 System, and the MRSA-Screen Latex Agglutination Test
Authors: Felten Annie , Grandry Bernadette , Lagrange Philippe Henri , Casin Isabelle 
Published: October 2002

ABSTRACT

Very-low-level methicillin-resistant
Staphylococcus aureus
(MRSA), or class 1 MRSA, is often misdiagnosed as methicillin-susceptible
S. aureus
(MSSA). We evaluated the performances of three methods for detection of low-level methicillin resistance: the disk diffusion method using the cephamycin antibiotics cefoxitin and moxalactam, the Vitek 2 system (bioMérieux), and the MRSA-screen test (Denka). Detection of the
mecA
gene by PCR was considered to be the “gold standard.” We also determined the sensitivity of the oxacillin disk diffusion method with 5- and 1-μg disks and that of the Oxascreen agar assay with 6 mg of oxacillin liter
−1
for detection of MRSA. We compared the distributions of MICs of oxacillin and cefoxitin by the E-test (AB Biodisk), and those of moxalactam by dilutions in agar, for MRSA and MSSA isolates. The 152 clinical isolates of
S. aureus
studied were divided into 69 MSSA (
mecA
-negative) and 83 MRSA (
mecA
-positive) isolates, including 63 heterogeneous isolates and 26 class 1 isolates (low-level resistance). The cefoxitin and moxalactam disk diffusion tests detected 100% of all the MRSA classes: cefoxitin inhibition zone diameters were <27 mm, and moxalactam inhibition zone diameters were <24 mm. The Vitek 2 system and the MRSA-screen test detected 94 and 97.6% of all MRSA isolates, respectively. The sensitivities of the 5- and 1-μg oxacillin disks were 95.2 and 96.4%, respectively, whereas that of the Oxascreen agar screen assay was 94%. All of the tests except the 1-μg oxacillin disk test were 100% specific. For the class 1 MRSA isolates, the sensitivity of the Vitek 2 test was 92.3%, whereas those of the MRSA-screen test and the disk diffusion method with cefoxitin and moxalactam were 100%. Therefore, the cefoxitin and moxalactam disk diffusion methods were the best-performing tests for routine detection of all classes of MRSA.
Phylogenetic Analysis of
Salmonella
,
Shigella
, and
Escherichia coli
Strains on the Basis of the
gyrB
Gene Sequence
Authors: Fukushima Masao , Kakinuma Kenichi , Kawaguchi Ryuji 
Published: October 2002

ABSTRACT

Phylogenetic analysis of about 200 strains of
Salmonella
,
Shigella
, and
Escherichia coli
was carried out using the nucleotide sequence of the gene for DNA gyrase B (
gyrB
), which was determined by directly sequencing PCR fragments. The results establish a new phylogenetic tree for the classification of
Salmonella
,
Shigella
, and
Escherichia coli
in which
Salmonella
forms a cluster separate from but closely related to
Shigella
and
E. coli.
In comparison with 16S rRNA analysis, the
gyrB
sequences indicated a greater evolutionary divergence for the bacteria. Thus, in screening for the presence of bacteria, the
gyrB
gene might be a useful tool for differentiating between closely related species of bacteria such as
Shigella
spp. and
E. coli
. At present, 16S rRNA sequence analysis is an accurate and rapid method for identifying most unknown bacteria to the genus level because the highly conserved 16S rRNA region is easy to amplify; however, analysis of the more variable
gyrB
sequence region can identify unknown bacteria to the species level. In summary, we have shown that
gyrB
sequence analysis is a useful alternative to 16S rRNA analysis for constructing the phylogenetic relationships of bacteria, in particular for the classification of closely related bacterial species.
Direct Detection of Enterovirus 71 (EV71) in Clinical Specimens from a Hand, Foot, and Mouth Disease Outbreak in Singapore by Reverse Transcription-PCR with Universal Enterovirus and EV71-Specific Primers
Authors: Singh Sunita , Chow Vincent T. K. , Phoon M. C. , Chan K. P. , Poh Chit Laa 
Published: October 2002

ABSTRACT
A recent outbreak of hand, foot, and mouth disease in Singapore in 2000 affected several thousand children and resulted in four deaths. The aim of this study was to determine the applicability of reverse transcription-PCR (RT-PCR) with universal pan-enterovirus primers and enterovirus 71 (EV71) type-specific primers for the direct detection of enteroviruses in clinical specimens derived from this outbreak. With the universal primers, EV71 RNA sequences were successfully detected by RT-PCR and direct sequencing in 71% of positive specimens. Three pairs of EV71 type-specific primers were evaluated for rapid detection of EV71 directly from clinical specimens and cell culture isolates. By using a seminested RT-PCR strategy, specific identification of EV71 sequences directly in clinical specimens was achieved, with a detection rate of 53%. In contrast, cell culture could isolate EV71 in only 20% of positive specimens. EV71 was detected directly from brain, heart, and lung specimens of two deceased siblings. Although more than one type of enterovirus was identified in clinical specimens from this outbreak, 90% of the enteroviruses were confirmed as EV71. The data demonstrate the clinical applicability of pan-enterovirus and seminested RT-PCR for the detection of EV71 RNA directly from clinical specimens in an outbreak situation.
1998-1999 Rotavirus Seasons in Juiz de Fora, Minas Gerais, Brazil: Detection of an Unusual G3P[4] Epidemic Strain
Authors: Rosa e Silva Maria Luzia , Pires de Carvalho Iná , Gouvea Vera 
Published: October 2002

ABSTRACT

An epidemiologic survey on the rotavirus strains causing gastroenteritis in young children was conducted in Juiz de Fora, Minas Gerais, in Southern Brazil during two consecutive seasons. Rotavirus was detected in 94 of the 1,056 fecal specimens collected from January 1998 to December 1999. Among the 13 discernible long electrophoretic profiles found, one was highly prevalent (73.4%) and represented the rotavirus strain responsible for the May-August winter epidemic outbreak of 1998, as clearly shown in a three-dimensional graph. This epidemic strain, designated JF98, was characterized as subgroup II and genotype G3P[4] by the original reverse transcription-PCR typing assays. Besides the unusual combination of G and P types, this G3 strain lacked reactivity with anti-G3-specific monoclonal antibodies and presented an uncommon pattern upon digestion of its cDNA-copied VP7 gene with the
Bst
YI restriction enzyme. Strain JF98 affected primarily 6- to 24-month-old children and accounted for 85.5% of the severe rotavirus-associated dehydrating diarrhea cases that required hospitalization. As in our previous studies in neighboring Rio de Janeiro and São Paulo, a remarkably large proportion (44%) of mixed infections was detected, generating a complex set of circulating strains in the community, represented by the many distinct electropherotypes. Other common human types were detected as minor strains in single or in mixed infections, including the JF98 strain. Those were types G1, G4, G8, G9, P[8], and P[6], but not G2 or G5. One specimen contained a mixture of group A and C rotaviruses.
Differentiation of Foot-and-Mouth Disease Virus-Infected from Vaccinated Pigs by Enzyme-Linked Immunosorbent Assay Using Nonstructural Protein 3AB as the Antigen and Application to an Eradication Program
Authors: Chung Wen-Bin , Sorensen Karl Johan , Liao Pei-Chih , Yang Pin-Cheng , Jong Ming-Hwa 
Published: October 2002

ABSTRACTBaculovirus-expressed foot-and-mouth disease virus (FMDV) nonstructural protein 3AB was used as the antigen in an enzyme-linked immunosorbent assay. This assay allowed the differentiation of vaccinated from infected pigs. Serial studies were performed using sera collected from pigs in the field. Positive reactions were found in sera from fattening pigs and sows 16 weeks and 3.5 years postoutbreak, respectively. There was, however, no positive reaction in sows with at least 10 vaccinations. Maternally derived antibodies to the 3AB antigen persisted in piglets up to 13 weeks of age. A high correlation coefficient (r= 0.93) was found between the test results from sow sera and those from their offspring. Therefore, piglet serum was a good substitute for sow serum to monitor the infection status of the dam. The application of this assay to serological surveillance in an FMD eradication program in Taiwan showed that the positive reactors steadily decreased over time in both finishers and sows, indicating that the pig population risk of infection by FMDV has decreased.
Role of Corpus Gastritis and
cagA
-Positive
Helicobacter pylori
Infection in Reflux Esophagitis
Authors: Queiroz Dulciene Maria Magalhães , Rocha Gifone Aguiar , Oliveira Celso Affonso de , Rocha Andreia Maria Camargos , Santos Adriana , Cabral Mônica Maria Demas Álvares , Nogueira Ana Margarida Miguel Ferreira 
Published: October 2002

ABSTRACT

Considering that the role of
Helicobacter pylori
infection in gastroesophageal reflux and reflux esophagitis (GERD) is still controversial and that the role of virulence markers of the bacterium has not been evaluated in most studies of GERD, we investigated the association among
H. pylori
infection with
cagA
-positive and -negative strains, corpus gastritis, and GERD in a large group of patients by controlling for confounding factors. We studied prospectively 281 consecutive adult patients: 93 with GERD and 188 controls.
H. pylori
infection status was diagnosed by culture, by the preformed urease test, with a carbolfuchsin-stained smear, and by histology. The
cagA
status was determined by PCR of
H. pylori
isolates and gastric biopsy specimens.
H. pylori
infection was diagnosed in 191 (68.0%) of 281 patients. Among the 93 patients with GERD, 84 presented with mild or moderate esophagitis and 9 presented with severe esophagitis. In the multivariate analysis, the age of the patients and the degree of oxyntic gastritis were associated with GERD. Among the strains isolated from patients with GERD and from the control group, 24.4 and 66.9%, respectively, were positive for
cagA
(
P
< 0.001). Compared to infection with
cagA
-negative strains, infection with
cagA
-positive
H. pylori
strains was associated with a more intense gastritis in the corpus (
P
= 0.001).
cagA
status (odds ratio [OR] = 0.16, 95% confidence interval [CI] = 0.07 to 0.40), gastritis of the corpus (OR = 0.69, 95% CI = 0.48 to 0.99), and age (OR = 1.04, 95% CI = 1.01 to 1.07) were associated with GERD. In conclusion, the study provides evidence supporting the independent protective roles of
cagA
-positive
H. pylori
strains and the degree of corpus gastritis against GERD.
Molecular Epidemiology of Caliciviruses Causing Outbreaks and Sporadic Cases of Acute Gastroenteritis in Spain
Authors: Buesa J. , Collado B. , López-Andújar P. , Abu-Mallouh R. , Rodríguez Díaz J. , García Díaz A. , Prat J. , Guix S. , Llovet T. , Prats G. , Bosch A. 
Published: October 2002

ABSTRACT
The molecular epidemiology of human caliciviruses (HuCVs) causing sporadic cases and outbreaks of acute gastroenteritis around eastern Spain (Catalonia and the Valencian Community) was studied by reverse transcription-PCR (RT-PCR) and by sequencing part of the RNA polymerase gene in open reading frame 1. HuCVs were detected in 44 of 310 stool specimens (14.19%) negative for other enteric pathogens obtained from children with acute gastroenteritis. Norwalk-like viruses (NLVs) were the most common cause of the gastroenteritis outbreaks investigated here. They were detected in 14 out of 25 (56%) outbreaks with an identified pathogen. Genotypes producing both sporadic cases and outbreaks were diverse, with a predominance of GGII strains related to genotypes Melksham and Lordsdale. Five strains clustered with a “new variant” designated GGIIb, which was detected circulating throughout quite a few European countries in the years 2000 and 2001. The emergence mechanism of these strains might be the occurrence of intertypic recombinations between different viruses. The nucleotide sequence of part of the capsid gene (ORF2) from three of these strains demonstrated their relationship with Mexico virus.
Detection of
Bacillus anthracis
DNA by LightCycler PCR
Authors: Bell Constance A. , Uhl James R. , Hadfield Ted L. , David John C. , Meyer Richard F. , Smith Thomas F. , Cockerill Franklin R. 
Published: October 2002

ABSTRACT

Anthrax is a zoonotic disease that is also well recognized as a potential agent of bioterrorism. Routine culture and biochemical testing methods are useful for the identification of
Bacillus anthracis
, but a definitive identification may take 24 to 48 h or longer and may require that specimens be referred to another laboratory. Virulent isolates of
B. anthracis
contain two plasmids (pX01 and pX02) with unique targets that allow the rapid and specific identification of
B. anthracis
by PCR. We developed a rapid-cycle real-time PCR detection assay for
B. anthracis
that utilizes the LightCycler instrument (LightCycler
Bacillus anthracis
kit; Roche Applied Science, Indianapolis, Ind.). PCR primers and probes were designed to identify gene sequences specific for both the protective antigen (plasmid pX01) and the encapsulation B protein (plasmid pX02). The assays (amplification and probe confirmation) can be completed in less than 1 h. The gene encoding the protective antigen (
pagA
) was detected in 29 of 29 virulent
B. anthracis
strains, and the gene encoding the capsular protein B (
capB
) was detected in 28 of 29 of the same strains. Three avirulent strains containing only pX01 or pX02, and therefore only
pagA
or
pagB
genes, could be detected and differentiated from virulent strains. The assays were specific for
B. anthracis
: the results were negative for 57 bacterial strains representing a broad range of organisms, including
Bacillus
species other than
anthracis
(
n
= 31) and other non-
Bacillus
species (
n
= 26). The analytical sensitivity demonstrated with target DNA cloned into control plasmids was 1 copy per μl of sample. The LightCycler
Bacillus anthracis
assay appears to be a suitable method for rapid identification of cultured isolates of
B. anthracis
. Additional clinical studies are required to determine the usefulness of this test for the rapid identification of
B. anthracis
directly from human specimens.
Qualitative Detection of Hepatitis C Virus RNA: Comparison of Analytical Sensitivity, Clinical Performance, and Workflow of the Cobas Amplicor HCV Test Version 2.0 and the HCV RNA Transcription-Mediated Amplification Qualitative Assay
Authors: Krajden Mel , Ziermann Rainer , Khan Asphani , Mak Annie , Leung Kimmy , Hendricks David , Comanor Lorraine 
Published: October 2002

ABSTRACT
The qualitative Cobas Amplicor hepatitis C virus (HCV) version 2.0 assay (HCV PCR) and the Bayer Reference Testing Laboratory HCV RNA transcription-mediated amplification assay (HCV TMA) were compared for analytical sensitivity, clinical performance, and workflow. Limits of detection were determined by testing dilutions of the World Health Organization HCV standard in replicates of 15 at concentrations of from 1.0 to 70 IU/ml. The limit of detection of the HCV PCR assay was calculated to be 45 IU/ml on initial testing and 32 IU/ml after resolution of gray zone results. The calculated limit of detection for HCV TMA was 6 IU/ml. To compare clinical performance, 300 specimens, grouped as follows, were evaluated: 112 samples that were indeterminate in an anti-HCV enzyme immunoassay (EIA) and for which HCV RNA was not detected by HCV PCR; 79 samples that were EIA positive and for which HCV RNA was not detected by HCV PCR; and 105 samples that were both EIA and HCV PCR positive. For these groups, interassay concordance ranged from 96.2% to 100%. In addition, three HCV PCR gray zone specimens and one neonatal specimen were also evaluated. A 64-sample run (full run, 91 specimens) required 5 h for testing by HCV TMA, whereas almost 8 h were required to test a full run of 22 specimens by HCV PCR. HCV TMA demonstrated excellent concordance with HCV PCR when clinical samples were tested. However, HCV TMA was more sensitive than HCV PCR, required less time for test result completion, and had a greater throughput.
Multiplex PCR Assays for Simultaneous Detection of Six Major Serotypes and Two Virulence-Associated Phenotypes ofStreptococcus suisin Tonsillar Specimens from Pigs
Authors: Wisselink Henk J. , Joosten Jeroen J. , Smith Hilde E. 
Published: October 2002

ABSTRACTMultiplex PCR assays for the detection and identification of variousStreptococcus suisstrains in tonsillar specimens from pigs were developed and evaluated. In two separate reactions, five distinct DNA targets were amplified. Three targets, based on theS. suiscapsular polysaccharide (cps) genes specific for serotypes 1 (and 14), 7, and 9, were amplified in multiplex PCR I. Two other targets, based on the serotype 2- (and 1/2-) specificcpsgene and theepfgene, encoding the EF proteins of virulent serotype 2 and highly virulent serotype 1 strains, were amplified in multiplex PCR II. To identify false-negative results, firefly luciferase (luc) DNA and primers based on thelucgene were included in the assay. The multiplex PCR assays were evaluated with tonsillar specimens from pigs infected withS. suisstrains. The results obtained with the PCR assays were compared with the results obtained with a bacteriological examination. Most (94%) of the results obtained with multiplex PCR assays were confirmed by the bacteriological examination. The PCR method seems to be more sensitive compared to the bacteriological method, since the remaining 6% of the samples were positive by PCR and negative by bacteriological examination. These results indicate that the PCR method is highly specific for the detection ofS. suisstrains most frequently involved in clinical disease in infected pig herds. The serotypes found by PCR in tonsillar specimens from diseased pigs were compared with the serotypes of the strains isolated from the affected tissues of the same pigs. The results showed that there is significant association between carriership and clinical illness forS. suisserotype 9 and EF-positive serotype 2 strains and not for serotype 7 and EF-negative serotype 2 (or 1/2) strains.
Monitoring of Viral Load by Quantitative Plasma PCR during Active Cytomegalovirus Infection of Individual Liver Transplant Patients
Authors: Piiparinen Heli , Höckerstedt Krister , Lappalainen Maija , Suni Jukka , Lautenschlager Irmeli 
Published: October 2002

ABSTRACT

A quantitative PCR test, the Cobas Amplicor CMV Monitor, was used for the monitoring of viral load in the peripheral blood of 27 individual liver transplant patients and correlated with cytomegalovirus (CMV) pp65 antigenemia. Altogether, 243 specimens were analyzed. During the first 3 months, 20 patients showed PCR positivity which correlated with pp65 antigenemia. Of those, 13 patients developed symptomatic CMV infection 27 to 52 days after transplantation, with a significantly higher peak viral load in PCR and in pp65 assay compared with the seven asymptomatic infections (median 10,200 versus 2,240 copies/ml,
P
< 0.05, and median 100 versus 30 pp65-positive cells/50,000 leukocytes,
P
< 0.01). Five were primary infections of D+/R− cases (donor CMV seropositive and recipient seronegative) and demonstrated, except in one case, a high peak viral load (>10,000 copies/ml; range, 10,200 to 21,600 copies, and ≥50 positive cells, range, 50 to 800 cells). The peak viral loads of the six D+/R+ patients with symptomatic infection varied widely (range, 2,290 to 126,000 copies and 50 to 300 positive cells). Two D−/R+ patients developed symptomatic infection with a lower viral load (range, 1,120 to 6,510 copies and 25 to 100 positive cells). All symptomatic infections were successfully treated with ganciclovir. The asymptomatic infections all in D+/R+ patients with low copy numbers (<5,500 copies) were monitored until CMV disappeared. One of the seven PCR-negative patients had one sample with low antigenemia, but the subsequent specimens were all negative. The time-related correlation of the two methods was also good. In summary, quantitative PCR could equally well be used as the CMV pp65 assay for the monitoring of viral load in individual transplant patients.