bluuebunny/crossref_metadata_2025_split huggingface dataset:
International Proficiency Study of a Consensus L1 PCR Assay for the Detection and Typing of Human Papillomavirus DNA: Evaluation of Accuracy and Intralaboratory and Interlaboratory Agreement
Authors: Kornegay Janet R. , Roger Michel , Davies Philip O. , Shepard Amanda P. , Guerrero Nayana A. , Lloveras Belen , Evans Darren , Coutlée François 
Published: March 2003

ABSTRACT
The PGMY L1 consensus primer pair combined with the line blot assay allows the detection of 27 genital human papillomavirus (HPV) genotypes. We conducted an intralaboratory and interlaboratory agreement study to assess the accuracy and reproducibility of PCR for HPV DNA detection and typing using the PGMY primers and typing amplicons with the line blot (PGMY-LB) assay. A test panel of 109 samples consisting of 29 HPV-negative (10 buffer controls and 19 genital samples) and 80 HPV-positive samples (60 genital samples and 20 controls with small or large amounts of HPV DNA plasmids) were tested blindly in triplicate by three laboratories. Intralaboratory agreement ranged from 86 to 98% for HPV DNA detection. PGMY-LB assay results for samples with a low copy number of HPV DNA were less reproducible. The rate of intralaboratory agreement excluding negative results for HPV typing ranged from 78 to 96%. Interlaboratory reliability for HPV DNA positivity and HPV typing was very good, with levels of agreement of >95% and kappa values of >0.87. Again, low-copy-number samples were more prone to generating discrepant results. The accuracy varied from 91 to 100% for HPV DNA positivity and from 90 to 100% for HPV typing. HPV testing can thus be accomplished reliably with PCR by using a standardized written protocol and quality-controlled reagents. The use of validated HPV DNA detection and typing assays demonstrating excellent interlaboratory agreement will allow investigators to better compare results between epidemiological studies.
Evaluation of Etest Method for Determining Fluconazole and Voriconazole MICs for 279 Clinical Isolates of
Candida
Species Infrequently Isolated from Blood
Authors: Maxwell M. J. , Messer S. A. , Hollis R. J. , Boyken L. , Tendolkar S. , Diekema D. J. , Pfaller M. A. 
Published: March 2003

ABSTRACT

The performance of Etest in fluconazole and voriconazole testing of 279 isolates of uncommon
Candida
spp. was assessed in comparison with the National Committee for Clinical Laboratory Standards (NCCLS)-approved standard broth microdilution (BMD) method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. Etest MICs were determined with RPMI agar containing 2% glucose and were read after incubation for 48 h at 35°C. The isolates include
Candida krusei
,
C. lusitaniae
,
C. guilliermondii
,
C. kefyr
,
C. rugosa
,
C. lipolytica
,
C. pelliculosa
,
C. dubliniensis
,
C. famata
,
C. zeylanoides
,
C. inconspicua
, and
C. norvegensis
. Overall agreement between Etest and BMD MICs was 96% for fluconazole and 95% for voriconazole. Where a discrepancy was observed between Etest and the reference method, the Etest tended to give lower values with both fluconazole and voriconazole. The Etest method using RPMI agar appears to be a useful method for determining fluconazole and voriconazole susceptibilities of uncommon species of
Candida
.
Hepatitis C Virus Subtyping by a Core-Envelope 1-Based Reverse Transcriptase PCR Assay with Sequencing and Its Use in Determining Subtype Distribution among Danish Patients
Authors: Corbet Sylvie , Bukh Jens , Heinsen Anja , Fomsgaard Anders 
Published: March 2003

ABSTRACT
A reverse transcriptase PCR (RT-PCR) assay using conserved primers deduced from the core-envelope 1 (C-E1) region of the hepatitis C virus (HCV) genome was developed for subtyping purposes. The sensitivity and specificity of this assay tested against two HCV reference panels containing genotype 1 through 5 subtypes were similar to those of an RT-PCR assay from the 5′-untranslated region (5′-UTR). The sensitivity of the RT-PCR typing assay in the more variable C-E1 region was, however, lower than that of the RT-PCR in the highly conserved 5′-UTR when testing multiple clinical samples. Thus, 71 (88%) of 81 consecutive samples from hospitalized Danish patients positive for HCV antibodies and RNA (5′-UTR) were positive also in the C-E1 RT-PCR assay. Phylogenetic analysis of the E1 sequences obtained by direct sequencing of HCV from two reference panels and 71 Danish patients allowed us to readily distinguish the subtypes. In contrast, phylogenetic analysis of their corresponding 5′-UTR sequences was able to predict only major genotypes. Three different genotypes and four subtypes were identified among Danish samples: 1a (43%), 1b (11%), 2b (6%), and 3a (39%). An isolate from a Somalian refugee was identified as a new HCV type related to Somalian isolates described as subtype 3h. The most common genotype in Denmark is genotype 1 (53%), which is the most difficult to treat. However, Denmark had the highest prevalence in Europe of subtype 3a, which responds more favorably to treatment. The described C-E1 RT-PCR with sequencing is suggested as an easy routine assay for definitive genotyping and subtyping of HCV.
Use of DNA Extracts from Ziehl-Neelsen-Stained Slides for Molecular Detection of Rifampin Resistance and Spoligotyping of
Mycobacterium tuberculosis
Authors: Van Der Zanden A. G. M. , Te Koppele-Vije E. M. , Vijaya Bhanu N. , Van Soolingen D. , Schouls L. M. 
Published: March 2003

ABSTRACT

Multidrug resistance among new cases of tuberculosis (TB) is increasingly becoming a significant problem in countries with a high prevalence of TB and with inadequate therapies for TB. Rifampin resistance is widely used as a marker for multidrug-resistant (MDR) TB; therefore, a new approach to the retrospective measurement of rifampin resistance without the need of a viable culture has been introduced. In many developing countries culture is unavailable and diagnosis relies on clinical manifestations and the results of Ziehl-Neelsen staining of sputum smears. We determined rifampin resistance directly with DNA extracts from Ziehl-Neelsen-stained slides by identification of mutations in the
rpoB
gene using reverse line blot hybridization and DNA sequencing. Analysis of the
rpoB
gene revealed that samples containing rifampin-resistant
Mycobacterium tuberculosis
carried altered codons representing amino acid positions 516, 526, and 531 of the RNA polymerase. Although the sensitivities of both methods were equal (84%), sequencing of the
rpoB
gene was more accurate in identifying mutations in the core region of the
rpoB
gene. Sequence analysis of the
rpoB
gene in extracts from Ziehl-Neelsen-stained slides may be used to quantify more precisely the magnitude of MDR TB and, more importantly, provide information on trends in the development of resistance on a global scale. The nature of rifampin resistance and the genotype can be determined by analysis of Ziehl-Neelsen-stained slides in a laboratory equipped for sequencing and spoligotyping without the need to ship biohazardous materials.
Molecular Characterization of Gentamicin-Resistant
Enterococci
in the United States: Evidence of Spread from Animals to Humans through Food
Authors: Donabedian S. M. , Thal L. A. , Hershberger E. , Perri M. B. , Chow J. W. , Bartlett P. , Jones R. , Joyce K. , Rossiter S. , Gay K. , Johnson J. , Mackinson C. , Debess E. , Madden J. , Angulo F. , Zervos M. J. 
Published: March 2003

ABSTRACT

We evaluated the molecular mechanism for resistance of 360 enterococci for which the gentamicin MICs were ≥128 μg/ml. The
aac(6′)-Ie-aph(2")-Ia
,
aph(2")-Ic
, and
aph(2")-Id
genes were identified by PCR in isolates from animals, food, and humans. The
aph(2")-Ib
gene was not identified in any of the isolates. Two
Enterococcus faecalis
isolates (MICs > 1,024 μg/ml) from animals failed to generate a PCR product for any of the genes tested and likely contain a new unidentified aminoglycoside resistance gene. Pulsed-field gel electrophoresis (PFGE) analysis showed a diversity of strains. However, 1 human and 18 pork
E. faecalis
isolates from Michigan with the
aac(6′)-Ie-aph(2")-Ia
gene had related PFGE patterns and 2
E. faecalis
isolates from Oregon (1 human and 1 grocery store chicken isolate) had indistinguishable PFGE patterns. We found that when a gentamicin-resistant gene was present in resistant enterococci from animals, that gene was also present in enterococci isolated from food products of the same animal species. Although these data indicate much diversity among gentamicin-resistant enterococci, the data also suggest similarities in gentamicin resistance among enterococci isolated from humans, retail food, and farm animals from geographically diverse areas and provide evidence of the spread of gentamicin-resistant enterococci from animals to humans through the food supply.
Influence of Culture Conditions on the Fatty Acid Profiles of Laboratory-Adapted and Freshly Isolated Strains of
Helicobacter pylori
Authors: Scherer Christiane , Müller Karl-D. , Rath Peter-M. , Ansorg Rainer A. M. 
Published: March 2003

ABSTRACT

Cellular fatty acids of
Helicobacter pylori
have taxonomic, physiological, and pathogenic implications. However, little is known about the fatty acid composition under various culture conditions.
H. pylori
is usually grown on blood-supplemented complex media, and the fatty acids in the blood may affect the fatty acids in the cells. In addition, frequently subcultivated laboratory-adapted strains may have properties different from those of fresh clinical isolates, which are culturable only for a limited number of passages. Therefore, the cellular fatty acid profiles of laboratory-adapted strains (LAS) and freshly isolated strains (FIS) were compared after growth on agar that was fatty acid free and growth on blood agar that contained fatty acids. LAS ATCC 43504, 51932, and 700392 and the FIS IMMi 88, 89, and 92, each with <10 subcultures, were cultured in parallel on a fatty acid-free agar (ISAF) and on 5% sheep blood agar (SBA), which contained oleic acid (18:1 9
c
), hexadecanoic acid (16:0), and octadecanoic acid (18:0). ISAF-grown cultures showed no 18:1 9
c
and no appreciable differences between the profiles of FIS and LAS. After culture on SBA, the strains showed 18:1 9
c
and increased 16:0 and 18:0 content combined with decreased tetradecanoic acid (14:0) content compared to ISAF-grown cells. The changes in the fatty acid profiles were much more pronounced in FIS than in LAS. LAS are obviously characterized by a lower uptake of the fatty acids from the growth medium than FIS. Furthermore, it could be shown that this LAS behavior is most likely a primary strain attribute that is favored under laboratory conditions. The pronounced uptake of fatty acids by strains with FIS behavior may be associated with the expression of virulence properties.
New Types of Toxin A-Negative, Toxin B-Positive Strains among
Clostridium difficile
Isolates from Asia
Authors: Rupnik Maja , Kato Naoki , Grabnar Miklavz , Kato Haru 
Published: March 2003

ABSTRACT

A total of 56
C. difficile
strains were selected from 310 isolates obtained from different hospitals in Japan and Korea and from healthy infants from Indonesia. Strains that had been previously typed by pulsed-field gel electrophoresis and PCR ribotyping, were characterized by toxinotyping and binary toxin gene detection. When toxinotyped, 35 strains were determined to be toxinotype 0, whereas 21 strains showed variations in toxin genes and could be grouped into 11 variant toxinotypes. Six of the toxinotypes had been described before (I, III, IV, VIII, IX, and XII). In addition, five new toxinotypes were defined (XVI to XX). Three of the new toxinotypes (XVIII, XIX, and XX) vary only in repetitive regions of
tcdA
and produce both toxins. In two strains from toxinotypes XVI and XVII, the production of TcdA could not be detected with commercial immunological kits. Strain J9965 (toxinotype XVII) was in PaLoc similar but not identical to another known A
−
B
+
strain,
C. difficile
8864. Strain SUC 36 (toxinotype XVI), on the other hand, was similar to well-defined group consisting of toxinotypes V, VI, and VII, which thus far includes only A
+
B
+
strains. Toxinotypes XVI and XVII represent two new groups of A
−
B
+
strains. Strains of the well-known A
−
B
+
group from toxinotype VIII have a nonsense mutation at the beginning of
tcdA
gene, and the introduction of a stop codon at amino acid position 47 results in nonproduction of TcdA. The 5′-end sequence of
tcdA
in two newly described A
−
B
+
strains does not contain an identical mutation. The prevalence of variant
C. difficile
strains varied greatly among nine hospitals. Only five strains from four different hospitals were positive in PCR for amplification of the binary toxin gene.
In Vitro Susceptibility Testing of
Aspergillus
spp.: Comparison of Etest and Reference Microdilution Methods for Determining Voriconazole and Itraconazole MICs
Authors: Pfaller J. B. , Messer S. A. , Hollis R. J. , Diekema D. J. , Pfaller M. A. 
Published: March 2003

ABSTRACT

The performance of the Etest for voriconazole and for itraconazole susceptibility testing of 376 isolates of
Aspergillus
spp. was assessed in comparison with the National Committee for Clinical Laboratory Standards (NCCLS) proposed standard microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. Etest MICs were determined with RPMI agar containing 2% glucose and were read after incubation for 48 h at 35°C. The isolates included
A. fumigatus
,
A. flavus
,
A. niger
,
A. terreus
,
A. versicolor
,
A. glaucus
,
A. nidulans
,
A. ustus
, and
A. sydowii
. Overall agreement percentages between the Etest and microdilution MICs were 97.6% for voriconazole and 95.8% for itraconazole. Where a discrepancy was observed between Etest and the reference method, the Etest tended to give lower values with voriconazole and higher values with itraconazole. The Etest method using RPMI agar appears to be a useful method for determining the voriconazole and itraconazole susceptibilities of
Aspergillus
spp.
Comparison of Four Chromogenic Media and Hektoen Agar for Detection and Presumptive Identification of
Salmonella
Strains in Human Stools
Authors: Perez J. M. , Cavalli P. , Roure C. , Renac R. , Gille Y. , Freydiere A. M. 
Published: March 2003

ABSTRACT

Several chromogenic media have been developed to enhance the specificity of
Salmonella
detection. We compared the performance of four commercial chromogenic media—namely, ABC medium (Lab M. Ltd., Bury, United Kingdom), COMPASS Salmonella agar (Biokar Diagnostics, Beauvais, France), CHROMagar Salmonella agar (CHROMagar Company, Paris, France), and SM ID agar (bioMerieux, Marcy l'Etoile, France)—with conventional Hektoen medium. Nine hundred sixteen stool samples from inpatients at three hospitals were cultured, in parallel, on the five media, both by direct inoculation and after selective enrichment in selenite broth. Sixty-four
Salmonella
strains with 12 serotypes were isolated on at least one medium. After 48 h of incubation, sensitivity before and after enrichment was 62.5 and 89.1% with ABC medium, 77.1 and 93.8% with COMPASS agar, 66.7 and 89.1% with CHROMagar, 68.8 and 85.9% with SM ID agar, and 85.4 and 98.4% with Hektoen agar, respectively. Broth enrichment and prolonged incubation (48 versus 24 h) increased the sensitivity of all five media. Only one strain was not isolated on Hektoen agar. The number of false-positive isolates was higher with all five media after enrichment in selenite broth and after incubation for 48 h compared to 24 h. The specificity of the four chromogenic media was better than 91% after incubation for 24 h (77.7% with Hektoen agar) and better than 84% after incubation for 48 h (74.8% with Hektoen agar). This higher specificity reduces the need for confirmatory tests, thereby cutting technical time and reagent requirements. Both COMPASS agar and CHROMagar Salmonella, which after simple additional tests showed close efficiencies (96 and 97%, respectively), can be recommended as single-plate media of choice for the detection and presumptive identification of salmonellae in stools.
Two-Center Collaborative Evaluation of the Performance of the BD Phoenix Automated Microbiology System for Identification and Antimicrobial Susceptibility Testing of
Enterococcus
spp. and
Staphylococcus
spp
Authors: Fahr Anne-Marie , Eigner Ulrich , Armbrust Martina , Caganic Alexandra , Dettori Giuseppe , Chezzi Carlo , Bertoncini Luca , Benecchi Magda , Menozzi Maria Grazia 
Published: March 2003

ABSTRACT

The performance of the BD Phoenix Automated Microbiology System (BD Diagnostic Systems, Sparks, Md.) was assessed for identification (ID) and antimicrobial susceptibility testing (AST) for the majority of clinically encountered bacterial isolates in a European collaborative two-center trial. A total of 469 bacterial isolates of the genera
Staphylococcus
(275 isolates),
Enterococcus
(179 isolates), and
Streptococcus
(15 isolates, for ID only) were investigated; of these, 367 were single patient isolates, and 102 were challenge strains tested at one center. Sixty-four antimicrobial drugs were tested, including the following drug classes: aminoglycosides, beta-lactam antibiotics, beta-lactam-beta-lactamase inhibitors, carbapenems, cephems, folate antagonists, quinolones, glycopeptides, macrolides-lincosamides-streptogramin B (MLS), and others. Phoenix ID results were compared to those of the laboratories' routine ID systems (API 32 Staph, API 32 Strep, and VITEK 2 [bioMérieux, Marcy l'Etoile, France]); Phoenix AST results were compared to those of frozen standard broth microdilution (SBM) panels according to NCCLS guidelines (NCCLS document M 100-S 9, approved standard M 7-A 4). Discrepant results were repeated in duplicate. Concordant IDs of 97.1, 98.9, and 100% were observed for staphylococci, enterococci, and streptococci, respectively. For AST results the overall essential agreement was 93.3%; the category agreement was 97.3%; and the very major error rate, major error rate, and minor error rate were 1.2, 1.9, and 1.3%, respectively. In conclusion, the Phoenix ID results showed high agreement with results of the systems to which they were being compared; the AST performance was highly equivalent to that of the SBM reference method.
Proficiency Testing Program for Clinical Laboratories Performing Antifungal Susceptibility Testing of Pathogenic Yeast Species
Authors: Ramani Rama , Chaturvedi Vishnu 
Published: March 2003

ABSTRACT

Antifungal susceptibility testing is expected to facilitate the selection of adequate therapy for fungal infections. The general availability of antifungal susceptibility testing in clinical laboratories is low, even though a number of standard methods are now available. The objective of the present study was to develop and evaluate a proficiency testing program (PTP) for the antifungal susceptibility testing of pathogenic yeasts in laboratories licensed by the New York State Department of Health. A number of quality control standards, and methods for documenting laboratory performance, were developed in consultation with the laboratory directors. The participating laboratories were provided with five American Type Culture Collection strains of pathogenic yeasts for which the minimum inhibitory concentrations (MICs) of amphotericin B and fluconazole were well defined. A majority of laboratories (14 of 17) used broth microdilution, and these were evenly split between the NCCLS M-27A protocol and the Sensititre YeastOne method. The other three laboratories performed susceptibility testing with Etest. Overall, the levels of agreement between MIC reference ranges and the reported MICs were 85 and 74% for amphotericin B and for fluconazole, respectively. All laboratories except one successfully detected fluconazole resistance in a
Candida krusei
strain. However, amphotericin B resistance in a
Candida lusitaniae
strain was not detected by any of the participating labs. It is concluded that a suitably designed PTP could adequately monitor the competence of clinical laboratories performing antifungal susceptibility testing.
High-Level Expression and Purification of a Truncated Merozoite Antigen-2 ofBabesia equiinEscherichia coliand Its Potential for Immunodiagnosis
Authors: Huang Xiaohong , Xuan Xuenan , Yokoyama Naoaki , Xu Longshan , Suzuki Hiroshi , Sugimoto Chihiro , Nagasawa Hideyuki , Fujisaki Kozo , Igarashi Ikuo 
Published: March 2003

ABSTRACTThe gene encoding a truncated merozoite antigen-2 (EMA-2t) ofBabesia equiwas cloned and highly expressed inEscherichia colias a glutathioneS-transferase fusion protein (G-rEMA-2t). Both G-rEMA-2t and rEMA-2t (after the removal of glutathioneS-transferase) had good antigenicity. Either Western blot analysis with rEMA-2t or enzyme-linked immunosorbent assay (ELISA) with G-rEMA-2t clearly discriminated the sera of horses experimentally infected withB. equifrom sera of horses infected withBabesia caballiand healthy horses, although rEMA-2t was not suitable for ELISA, probably owing to its poor absorbability to the plates. The specific antibodies inB. equi-infected horses were detectable during both acute and latent infection (6 to 244 days postinfection). Horse sera from Jilin Province, China, were examined by the two tests. The seroprevalence ofB. equiwas 49.2% (31 of 63 sera) by Western blot analysis with rEMA-2t and 47.6% (30 of 63 sera) by ELISA with G-rEMA-2t. The correspondence was 98.4% (62 of 63 sera) between the two tests. The results indicate that G-rEMA-2t and rEMA-2t proteins should be suitable antigens for the development of an effective immunodiagnostic assay due to their high sensitivity, specificity, and great yield.
Differentiation of
Candida albicans
and
Candida dubliniensis
by Using Recombinant Human Antibody Single-Chain Variable Fragments Specific for Hyphae
Authors: Bliss Joseph M. , Sullivan Mark A. , Malone Jane , Haidaris Constantine G. 
Published: March 2003

ABSTRACT

To identify antigens specific for the filamentous form of
Candida albicans
, a combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to select phage clones capable of binding to the surfaces of viable
C. albicans
filaments. Eight distinct phage clones that bound specifically to filament surface antigens not expressed on blastoconidia were identified. Single-chain antibody variable fragments (scFv) derived from two of these phage clones (scFv5 and scFv12) were characterized in detail. Filament-specific antigen expression was detected by an indirect immunofluorescence assay. ScFv5 reacted with
C. dubliniensis
filaments, while scFv12 did not. Neither scFv reacted with
C. glabrata
,
C. parapsilosis
,
C. rugosa
,
C. tropicalis
, or
Saccharomyces cerevisiae
grown under conditions that stimulated filament formation in
C. albicans
and
C. dubliniensis
. Epitope detection by the two scFv was sensitive to proteinase K treatment but not to periodate treatment, indicating that the cognate epitopes were composed of protein. The antigens reactive with scFv5 and scFv12 were extractable from the cell surface with Zymolyase, but not with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol, and migrated as polydisperse, high-molecular-weight bands on SDS-polyacrylamide gel electrophoresis gels. The epitopes were detected on clinical specimens obtained from infants with thrush and urinary candidiasis without passage of the organisms on laboratory media, confirming epitope expression in human infection. The availability of a monoclonal immunologic reagent that recognizes filaments from both
C. albicans
and
C. dubliniensis
and another specific only to
C. albicans
adds to the repertoire of potential diagnostic reagents for differentiation between these closely related species.