bluuebunny/crossref_metadata_2025 huggingface dataset:
Outbreak of Vancomycin-Resistant
Enterococcus faecium
of the Phenotype VanB in a Hospital in Warsaw, Poland: Probable Transmission of the Resistance Determinants into an Endemic Vancomycin-Susceptible Strain
Authors: Kawalec Magdalena , Gniadkowski Marek , Zaleska Maria , Ozorowski Tomasz , Konopka Lech , Hryniewicz Waleria
Published: July 2002

The first outbreak caused by vancomycin-resistant enterococci of the VanB phenotype in Poland was analyzed. It occurred in a single ward of a Warsaw hospital which is a specialized center for the treatment of hematological disorders. Between July 1999 and February 2000, 11 patients in the ward were found to be infected and/or colonized by
Enterococcus faecium
that was resistant in vitro to vancomycin and susceptible to teicoplanin. PCR analysis confirmed that the vancomycin-resistant
E. faecium
(VREM) isolates carried the
vanB
gene, which is responsible for the VanB phenotype. Pulsed-field gel electrophoresis (PFGE) typing revealed that the isolates belonged to four distinct PFGE types and that one of these was clearly predominant, including isolates collected from seven different patients. The isolates contained one or more copies of the
vanB
gene cluster of the identical, unique
Dra
I/
Pag
I (
Bsp
HI) restriction fragment length polymorphism type, which resided in either the same or different plasmid molecules or chromosomal regions. All this data suggested that the outbreak was due to both clonal spread of a single strain and horizontal transfer of resistance genes among nonrelated strains, which could be mediated by plasmids and/or by
vanB
gene cluster-containing transposons. The comparative analysis of vancomycin-susceptible
E. faecium
(VSEM) isolates collected from infections in the same ward at the time of the VREM outbreak has led to identification of a widespread VSEM strain that was possibly related to the major VREM clone. It is very likely that this endemic VSEM strain has acquired vancomycin-resistance determinants and that the acquisition occurred more than once during the outbreak.
Replicate PCR Testing and Probit Analysis for Detection and Quantitation of
Chlamydia pneumoniae
in Clinical Specimens
Authors: Smieja M. , Mahony J. B. , Goldsmith C. H. , Chong S. , Petrich A. , Chernesky M.
Published: July 2002

Nucleic acid amplification of clinical specimens with low target concentration has variable sensitivity. We examined whether testing multiple aliquots of extracted DNA increased the sensitivity and reproducibility of
Chlamydia pneumoniae
detection by PCR. Nested and non-nested
C. pneumoniae
PCR assays were compared using 10 replicates of 16 serial dilutions of
C. pneumoniae
ATCC VR-1310. The proportion positive versus the
C. pneumoniae
concentration was modeled by probit regression analysis. To validate the model, 10 replicates of 26 previously positive patient specimens of peripheral blood mononuclear cells (PBMC), sputum, or nasopharyngeal swabs (NPS) were tested. The proportion of replicates that were positive varied with the concentration of
C. pneumoniae
in the sample. At concentrations above 5 infection-forming units (IFU)/ml, both nested and non-nested PCR assay sensitivities were 90% or greater. The nested PCR was more sensitive (median detection, 0.35 versus 0.61 IFU/ml; relative median detection, 0.58; 95% confidence interval, 0.31 to 0.99;
P
= 0.04). In clinical specimens, replicate PCR detected 15 of 26 (nested) versus 1 of 26 (non-nested,
P
< 0.001). For PBMC specimens, testing 1, 3, or 5 replicates detected 3, 5, or 9 of 10 positive specimens, respectively. Median
C. pneumoniae
concentrations were estimated at 0.07 IFU/ml for PBMC and at <0.03 IFU/ml for NPS specimens. We conclude that performing 5 or 10 replicates considerably increased the sensitivity and reproducibility of
C. pneumoniae
PCR and enabled quantitation for clinical specimens. Due to sampling variability, PCR tests done without replication may miss a large proportion of positive specimens, particularly for specimens with small amounts of target
C. pneumoniae
DNA present.
One-Tube Real-Time Isothermal Amplification Assay To Identify and Distinguish Human Immunodeficiency Virus Type 1 Subtypes A, B, and C and Circulating Recombinant Forms AE and AG
Authors: de Baar Michel P. , Timmermans Eveline C. , Bakker Margreet , de Rooij Esther , van Gemen Bob , Goudsmit Jaap
Published: July 2002

To halt the human immunodeficiency virus type 1 (HIV-1) epidemic requires interventions that can prevent transmission of numerous HIV-1 subtypes. The most frequently transmitted viruses belong to the subtypes A, B, and C and the circulating recombinant forms (CRFs) AE and AG. A fast one-tube assay that identifies and distinguishes among subtypes A, B, and C and CRFs AE and AG of HIV-1 was developed. The assay amplifies a part of the
gag
gene sequence of the genome of all currently known HIV-1 subtypes and can identify and distinguish among the targeted subtypes as the reaction proceeds, because of the addition of subtype-specific molecular beacons with multiple fluorophores. The combination of isothermal nucleic acid sequence-based amplification and molecular beacons is a new approach in the design of real-time assays. To obtain a sufficiently specific assay, we developed a new strategy in the design of molecular beacons, purposely introducing mismatches in the molecular beacons. The subtype A and CRF AG isolates reacted with the same molecular beacon. We tested the specificity and sensitivity of the assay on a panel of the culture supernatant of 34 viruses encompassing all HIV-1 subtypes: subtypes A through G, CRF AE and AG, a group O isolate, and a group N isolate. Assay sensitivity on this panel was 92%, with 89% correct subtype identification relative to sequence analysis. A linear relationship was found between the amount of input RNA in the reaction mixture and the time that the reaction became positive. The lower detection level of the assay was approximately 10
3
copies of HIV-1 RNA per reaction. In 38% of 50 serum samples from HIV-1-infected individuals with a detectable amount of virus, we could identify subtype sequences with a specificity of 94% by using sequencing and phylogenetic analysis as the “gold standard.” In conclusion, we showed the feasibility of the approach of using multiple molecular beacons labeled with different fluorophores in combination with isothermal amplification to identify and distinguish subtypes A, B, and C and CRFs AE and AG of HIV-1. Because of the low sensitivity, the assay in this format would not be suited for clinical use but can possibly be used for epidemiological monitoring as well as vaccine research studies.
Use of Recommended Laboratory Testing Methods among Patients with Tuberculosis in California
Authors: Kellam Steffi , Pascopella Lisa , Desmond Edward , Reingold Arthur , Chin Daniel P.
Published: July 2002

This study assessed the extent to which laboratory methods recommended by the Centers for Disease Control and Prevention were used in tuberculosis testing of patients in California in 1998. While recommended methods were used for most patients, there was room for improvement by hospital and independent non-health maintenance organization laboratories.
Use of the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test for Early Detection of
Mycobacterium tuberculosis
in BACTEC 12B Medium
Authors: Desmond E. P. , Loretz K.
Published: July 2002

To achieve better sensitivity than direct testing and better turnaround time than current culture and identification methods, the Gen-Probe Mycobacterium Tuberculosis Direct method was used to detect
Mycobacterium tuberculosis
in BACTEC 12B medium cultures when they first gave a growth index (GI) of at least 10 (MTD/BACTEC method). Of 179 acid-fast, smear-positive specimens that were culture positive for
M. tuberculosis
, all were positive by the MTD/BACTEC method (sensitivity, 100%). Positive results were obtained only with tuberculosis patients. For diagnostic specimens from untreated patients, the mean time to achieve a GI of 10 was 6 days.
Capnocytophaga cynodegmi
Cellulitis, Bacteremia, and Pneumonitis in a Diabetic Man
Authors: Sarma Podila S. , Mohanty Smruti
Published: July 2002

Capnocytophaga cynodegmi
(formerly “DF-2 like organism”), a commensal organism of the canine oral cavity, is a capnophilic, gram-negative, facultative bacillus.
C. cynodegmi
has rarely been encountered in human diseases. We report the first known case of cellulitis, bacteremia, and pneumonitis caused by
C. cynodegmi
in a diabetic man from central India following a dog bite.
Differentiation of Mycobacterial Species by PCR-Restriction Analysis of DNA (342 Base Pairs) of the RNA Polymerase Gene (
rpoB
)
Authors: Kim Bum-Joon , Lee Keun-Hwa , Park Bo-Na , Kim Seo-Jeong , Bai Gill-Han , Kim Sang-Jae , Kook Yoon-Hoh
Published: July 2002

PCR amplification-restriction analysis (PRA) of
rpoB
DNA (342 bp), which comprises the Rif
r
region, was used for the differential identification of 49 mycobacteria. The DNA had been used previously for the identification of mycobacterial species by comparative sequence analysis (B. J. Kim et al., J. Clin. Microbiol. 37:1714–1720, 1999). Digestion with four restriction enzymes (
Hae
III,
Hin
dII,
Mva
I, and
Acc
II), which were selected on the basis of
rpoB
DNA sequences, generated distinctive PRA patterns that allowed not only the reference strains but also the clinical isolates of mycobacteria to be distinguished. Both rapidly and slowly growing mycobacteria were distinctly differentiated by
Hae
III digestion of the amplified
rpoB
DNA. By
Hin
dII digestion the
Mycobacterium tuberculosis
complex was distinguished from the other mycobacteria. Furthermore, six subspecies of
Mycobacterium kansasii
(subspecies I to VI) as well as the closely related
Mycobacterium gastri
, and other closely related species, were distinguished by simultaneous digestion of
Mva
I and
Acc
II. According to the
rpoB
PRA scheme, 240 strains of clinical isolates could be identified. It was also possible to detect and identify
M. tuberculosis
directly from sputa and bronchoalveolar lavage specimens. These results suggest that PRA of
rpoB
DNA is a simple and feasible method not only for the differentiation of culture isolates but also for the rapid detection and identification of pathogenic mycobacteria in primary clinical specimens.
Prevalence of Iron Transport Gene on Pathogenicity-Associated Island of Uropathogenic
Escherichia coli
in
E. coli
O157:H7 Containing Shiga Toxin Gene
Authors: Ye Changyun , Xu Jianguo
Published: July 2002

Uropathogenc
Escherichia coli
(UPEC) CFT073 has a pathogenicity-associated island (PAI
CFT073
), which causes pyelonephritis and cystitis. Using PCR method, we found the
prrA
gene of PAI
CFT073
in
E. coli
O157:H7 EDL933. Further detailed PCR screening of 38 open reading frames, the right and left junction sequences of PAI
CFT073
, revealed that it is the
prrA-modD-yc73-fepC
gene cluster but not the PAI
CFT073
present in
E. coli
O157:H7 EDL933. A rapid preliminary analysis suggested that the
prrA-modD-yc73-fepC
gene cluster of the PAI
CFT073
, is present in 43 strains of
E. coli
O157:H7 containing Shiga toxin (Stx) gene but absent in 19 strains of
E. coli
O157:H7 without Stx gene. A strict co-occurrence of the
prrA-modD-yc73-fepC
gene cluster and Stx genes was observed, regardless of their origin. The
prrA-modD-yc73-fepC
gene cluster encode proteins probably involved in iron uptake system, which strongly suggests the importance of iron metabolism in the Stx-mediated virulence. In addition, the
prrA-modD-yc73-fepC
gene cluster may be used as a diagnostic marker to distinguish
E. coli
O157:H7 strains containing Stx gene from that without Stx gene, and possibly to quickly detect other pathogenic gram-negative bacteria containing the Stx gene.
Comparison of a Polymer Conjugate-Enhanced Enzyme Immunoassay to Ligase Chain Reaction for Diagnosis of
Chlamydia trachomatis
in Endocervical Swabs
Authors: Chernesky Max , Jang Dan , Copes Debby , Patel Jay , Petrich Astrid , Biers Kathleen , Sproston Arlene , Kapala Julius
Published: July 2002

Two endocervical swabs from each of 1,123 women were collected into manufacturer-supplied transport tubes and tested for
Chlamydia trachomatis
by a polymer conjugate-enhanced (PCE) enzyme immunoassay (EIA) (IDEIA PCE Chlamydia; DAKO) and a ligase chain reaction assay (LCx Chlamydia; Abbott). After confirmation by the EIA blocking test, the sensitivity of the IDEIA PCE remained at 91.8% and the specificity increased from 98.2 to 99.8% compared to LCx.
Liver Abscess Caused by
Klebsiella pneumoniae
in Siblings
Authors: Chiu Cheng-Hsun , Su Lin-Hui , Wu Tsu-Lan , Hung Iou-Jih
Published: July 2002

Klebsiella pneumoniae
has been emerging as the leading cause of liver abscess in diabetic patients. Results of molecular typing of
K. pneumoniae
isolates from two siblings with liver abscess, their family members, and the environment suggest a possibility of cross infection of liver abscess by the fecal-oral route within diabetic patients.
rpoB
Sequence Analysis of Cultured
Tropheryma whippelii
Authors: Drancourt Michel , Carlioz Antoine , Raoult Didier
Published: July 2002

Until recently no isolate of
Tropheryma whippelii
was available, and therefore genetic studies were limited to those based on PCR amplification of conserved genes. In this study we determined the nucleotide sequence of
rpoB
(encoding the β-subunit of RNA polymerase) from a cultured strain of
T. whippelii
using degenerate consensus PCR and genome walking. The
T. whippelii rpoB
consists of 3,657 bp with a 50.4% GC content and encodes 1,218 amino acids with a calculated molecular mass of 138 kDa. Comparison of
T. whippelii
RpoB with other eubacterial RpoB proteins indicated sequence similarity ranging from 57.19 (
Mycoplasma pneumoniae
) to 74.63% (
Mycobacterium tuberculosis
). Phylogenetic analysis of
T. whippelii
based on comparison of its RpoB sequence with sequences available for other bacteria was consistent with that previously derived from the 16S ribosomal DNA (rDNA) sequence, indicating that it belongs to the actinomyces clade. The sequence comparison allowed the design of a primer pair, TwrpoB.F and TwrpoB.R, specific for
T. whippelii rpoB.
When incorporated into a PCR, this primer pair allowed the detection of
T. whippelii rpoB
in three of three 16S rDNA PCR-positive biopsy specimens and zero of seven negative controls.
rpoB
could therefore be targeted in PCR-mediated detection and identification of this emerging bacterial species. This approach has previously been shown useful for the identification of related mycobacteria. This study underscores that a method involving isolation and then propagation of emerging bacteria is a useful way to quickly achieve extensive molecular knowledge of these pathogens.
Pasteurella
multocida
subsp.
multocida
and
P. multocida
subsp.
septica
Differentiation by PCR Fingerprinting and α-Glucosidase Activity
Authors: Hunt Gerardo Sharon , Citron Diane M. , Claros Marina C. , Fernandez Helen T. , Goldstein Ellie J. C.
Published: July 2002

Pasteurella multocida
is composed of three subspecies that are often differentiated by fermentation of sorbitol and dulcitol. We studied 35 dulcitol-negative
P. multocida
isolates from infected dog and cat bite wounds, 16 of which yielded weak and/or conflicting fermentation reactions in Andrades sorbitol, thus making it difficult to distinguish between the two dulcitol-negative subspecies of
P. multocida
, i.e.,
P. multocida
subsp.
multocida
and
P. multocida
subsp.
septica
. All isolates and two control strains were further analyzed using a PCR fingerprinting technique with a single primer (M13 core) and assessed for α-glucosidase (α-Glu) activity. Although the PCR fingerprint patterns and α-Glu activity did not correlate well with the sorbitol fermentation reactions, they did correlate well with each other. All strains identified as
P. multocida
subsp.
septica
were positive for α-Glu activity and exhibited the group I PCR fingerprint profile. All strains categorized as
P. multocida
subsp.
multocida
displayed either the group II or group III PCR fingerprint profile; 9 of 11 of these isolates were α-Glu negative. These data suggest that both PCR fingerprinting and α-Glu activity provide reliable means for differentiating
P. multocida
subsp.
multocida
from
P. multocida
subsp.
septica
, particularly in strains that produce weak and/or discrepant sorbitol fermentation reactions.
Detection of RTX Toxin Gene in
Vibrio cholerae
by PCR
Authors: Chow K. H. , Ng T. K. , Yuen K. Y. , Yam W. C.
Published: July 2002

A PCR that amplifies a recently discovered
Vibrio cholerae
RTX (repeat in toxin) toxin gene was developed. Among 166 clinical and environmental isolates of
V. cholerae
causing epidemics and sporadic cases of cholera in various parts of the world, all were found to be toxigenic by both PCR and HEp-2 cell cytotoxicity assay. Standard strains of the classical biotype containing a deletion within the gene cluster exhibited negative results by both assays. This is the first rapid genotyping method for differentiation of
V. cholerae
O1 classical biotype strains from El Tor biotype strains as well as strains of other non-O1 serogroups including serogroup O139. The PCR assay that was developed also specifically detects RTX toxin genes in
V. cholerae
, as clinical isolates of
Vibrio parahaemolyticus
, diarrheagenic
Escherichia coli,
Aeromonas
species, and
Plesiomonas
species were all negative by the RTX toxin-specific PCR as well as the HEp-2 cytotoxicity assay. These findings highlight the characteristics of the RTX toxins in
V. cholerae
. Their role in the pathogenicity of the bacterium requires further investigation.
Novel Fluorescent Broth Microdilution Method for Fluconazole Susceptibility Testing of
Candida albicans
Authors: Liao Robert S. , Rennie Robert P. , Talbot James A.
Published: July 2002

A comparative evaluation of the reference National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method with a novel fluorescent carboxyfluorescein diacetate (CFDA)-modified microdilution method for the susceptibility testing of fluconazole was conducted with 68
Candida
strains, including 53
Candida albicans
, 5
Candida tropicalis
, 5
Candida glabrata
, and 5
Candida parapsilosis
strains. We found trailing endpoints and discordant fluconazole MICs of <8 μg/ml at 24 h and of ≥64 μg/ml at 48 h for 12 of the
C. albicans
strains. These strains satisfy the definition of the low-high MIC phenotype. All 12 low-high phenotype strains were correctly shown to be susceptible at 48 h with the CFDA-modified microdilution method. For the 41 non-low-high phenotype
C. albicans
strains, the CFDA-modified microdilution method yielded 97.6% (40 of 41 strains) agreement within ±1 dilution at 24 h compared with the reference method and 92.7% (38 of 41 strains) agreement within ±1 dilution at 48 h compared with the reference method. The five strains each from
C. tropicalis
,
C. glabrata,
and
C. parapsilosis
that were tested showed 100% agreement within ±2 dilutions for the two methods being evaluated.
Molecular and Antigenic Comparison of
Ehrlichia canis
Isolates from Dogs, Ticks, and a Human in Venezuela
Authors: Unver Ahmet , Perez Miriam , Orellana Nelson , Huang Haibin , Rikihisa Yasuko
Published: July 2002

We previously culture isolated a strain of
Ehrlichia canis
, the causative agent of canine ehrlichiosis, from a human in Venezuela. In the present study, we examined whether dogs and ticks are infected with
E. canis
in Venezuela and, if so, whether this is the same strain as the human isolate. PCR analysis using
E. canis
-specific primers revealed that 17 of the 55 dog blood samples (31%) and all three pools of four
Rhipicephalus sanguineus
ticks each were positive. An ehrlichial agent (Venezuelan dog
Ehrlichia
[VDE]) was isolated and propagated in cell culture from one dog sample and was further analyzed to determine its molecular and antigenic characteristics. The 16S rRNA 1,408-bp sequence of the new VDE isolate was identical to that of the previously reported Venezuelan human
Ehrlichia
isolate (VHE) and was closely related (99.9%) to that of
E. canis
Oklahoma. The 5′ (333-bp) and 3′ (653-bp) sequences of the variable regions of the 16S rRNA genes from six additional
E. canis
-positive dog blood specimens and from three pooled-tick specimens were also identical to those of VHE. Western blot analysis of serum samples from three dogs infected with VDE by using several ehrlichial antigens revealed that the antigenic profile of the VDE was similar to the profiles of VHE and
E. canis
Oklahoma. Identical 16S rRNA gene sequences among ehrlichial organisms from dogs, ticks, and a human in the same geographic region in Venezuela and similar antigenic profiles between the dog and human isolates suggest that dogs serve as a reservoir of human
E. canis
infection and that
R. sanguineus
, which occasionally bites humans residing or traveling in this region, serves as a vector. This is the first report of culture isolation and antigenic characterization of an ehrlichial agent from a dog in South America, as well as the first molecular characterization of
E. canis
directly from naturally infected ticks.
Quantitation of Varicella-Zoster Virus DNA in Patients with Ramsay Hunt Syndrome and Zoster Sine Herpete
Authors: Furuta Yasushi , Ohtani Fumio , Sawa Hirofumi , Fukuda Satoshi , Inuyama Yukio
Published: July 2002

Varicella-zoster virus (VZV) reactivation causes facial nerve palsy in Ramsay Hunt syndrome (RHS) and zoster sine herpete (ZSH) with and without zoster rash, respectively. In the present study, we analyzed the VZV DNA copy number in saliva samples from 25 patients with RHS and 31 patients with ZSH using a TaqMan PCR assay to determine differences in the viral load between the two diseases. VZV copy number in saliva peaked near the day of the appearance of zoster in patients with RHS. Consequently, VZV DNA was less frequently detected in patients with RHS who exhibited facial palsy several days after the appearance of zoster. These findings suggest that the VZV load in saliva samples reflects the kinetics of viral reactivation in patients with RHS. In addition, VZV DNA was equally detected in saliva from patients with RHS and ZSH, and there was no significant difference in the highest viral copy number between patients with RHS and those with ZSH. The VZV load does not appear to reflect a major difference between RHS and ZSH.
Characterization of Clinical Isolates ofKlebsiella pneumoniaefrom 19 Laboratories Using the National Committee for Clinical Laboratory Standards Extended-Spectrum β-Lactamase Detection Methods
Authors: Steward Christine D. , Rasheed J. Kamile , Hubert Susannah K. , Biddle James W. , Raney Patti M. , Anderson Gregory J. , Williams Portia P. , Brittain Kelley L. , Oliver Antonio , McGowan John E. , Tenover Fred C.
Published: July 2002

Extended-spectrum β-lactamases (ESBLs) are enzymes found in gram-negative bacilli that mediate resistance to extended-spectrum cephalosporins and aztreonam. In 1999, the National Committee for Clinical Laboratory Standards (NCCLS) published methods for screening and confirming the presence of ESBLs inKlebsiella pneumoniae, Klebsiella oxytoca, andEscherichia coli. To evaluate the confirmation protocol, we tested 139 isolates ofK. pneumoniaethat were sent to Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) from 19 hospitals in 11 U.S. states. Each isolate met the NCCLS screening criteria for potential ESBL producers (ceftazidime [CAZ] or cefotaxime [CTX] MICs were ≥2 μg/ml for all isolates). Initially, 117 (84%) isolates demonstrated a clavulanic acid (CA) effect by disk diffusion (i.e., an increase in CAZ or CTX zone diameters of ≥5 mm in the presence of CA), and 114 (82%) demonstrated a CA effect by broth microdilution (reduction of CAZ or CTX MICs by ≥3 dilutions). For five isolates, a CA effect could not be determined initially by broth microdilution because of off-scale CAZ results. However, a CA effect was observed in two of these isolates by testing cefepime and cefepime plus CA. The cefoxitin MICs for 23 isolates that failed to show a CA effect by broth microdilution were ≥32 μg/ml, suggesting either the presence of an AmpC-type β-lactamase or porin changes that could mask a CA effect. By isoelectric focusing (IEF), 7 of the 23 isolates contained a β-lactamase with a pI of ≥8.3 suggestive of an AmpC-type β-lactamase; 6 of the 7 isolates were shown by PCR to contain bothampC-type andblaOXAgenes. The IEF profiles of the remaining 16 isolates showed a variety of β-lactamase bands, all of which had pIs of ≤7.5. All 16 isolates were negative by PCR with multiple primer sets forampC-type,blaOXA, andblaCTX-Mgenes. In summary, 83.5% of theK. pneumoniaeisolates that were identified initially as presumptive ESBL producers were positive for a CA effect, while 5.0% contained β-lactamases that likely masked the CA effect. The remaining 11.5% of the isolates studied contained β-lactamases that did not demonstrate a CA effect. An algorithm based on phenotypic analyses is suggested for evaluation of such isolates.
Detection of Cervical Infections in Colposcopy Clinic Patients
Authors: Lanham Stuart , Herbert Amanda , Basarab Adriana , Watt Peter
Published: July 2002

The purpose of this study was to determine if
Neisseria gonorrhoeae; Chlamydia trachomatis
; herpes simplex virus; cytomegalovirus; Epstein-Barr virus; human herpesviruses 6, 7, and 8; or adeno-associated virus influenced the production of cervical intraepithelial neoplasia. Two hundred thirty-one cervical smear samples were tested for the presence of the organisms by PCR. In addition, human papillomavirus types in the samples were determined by PCR and classified into cancer risk types of high, moderate, and low. There was no link with cervical intraepithelial neoplasia status and detection of herpes simplex virus, cytomegalovirus, Epstein-Barr virus, human herpesviruses 6 and 8, gonorrhea, or chlamydia. However, high-grade cervical intraepithelial neoplasia was found more frequently with mixed infection by moderate-risk human papillomavirus types and human herpesvirus 7 than with these papillomavirus types alone. The presence of human herpesvirus 7 may increase the oncogenic potential of moderate-risk human papillomavirus types.
Automated Ribotyping Using Different Enzymes To Improve Discrimination ofListeria monocytogenesIsolates, with a Particular Focus on Serotype 4b Strains
Authors: De Cesare Alessandra , Bruce James L. , Dambaugh Timothy R. , Guerzoni Maria E. , Wiedmann Martin
Published: July 2002

To develop improved automated subtyping approaches forListeria monocytogenes, we characterized the discriminatory power of different restriction enzymes for ribotyping. When 15 different restriction enzymes were used for automated ribotyping of 16 selectedL. monocytogenesisolates, the restriction enzymesEcoRI,PvuII, andXhoI showed high discriminatory ability (Simpson's index of discrimination > 0.900) and produced complete and reproducible restriction cut patterns. These three enzymes were thus evaluated for their ability to differentiate among isolates representing the two major serotype 4b epidemic clones, those having ribotype reference pattern DUP-1038 (51 isolates) and those having pattern DUP-1042 (20 isolates). Among these isolates,PvuII provided the highest discrimination for a single enzyme (nine different subtypes; index of discrimination = 0.518). A combination ofPvuII andXhoI showed the highest discriminatory ability (index of discrimination = 0.590) for these isolates. A group of 44 DUP-1038 isolates and a group of 12 DUP-1042 isolates were identical to each other even when the combined data for all three enzymes were used. We conclude that automated ribotyping using different enzymes allows improved discrimination ofL. monocytogenesisolates, including epidemic serotype 4b strains. We furthermore confirm that most of the isolates representing the genotypes linked to the two major epidemicL. monocytogenesclonal groups form two genetically homogeneous groups.
Extensive Allelic Variation among
Francisella tularensis
Strains in a Short-Sequence Tandem Repeat Region
Authors: Johansson Anders , Göransson Ingela , Larsson Pär , Sjöstedt Anders
Published: July 2002

Members of the genus
Francisella
and the species
F. tularensis
appear to be genetically very similar despite pronounced differences in virulence and geographic localization, and currently used typing methods do not allow discrimination of individual strains. Here we show that a number of short-sequence tandem repeat (SSTR) loci are present in
F. tularensis
genomes and that two of these loci, SSTR9 and SSTR16, are together highly discriminatory. Labeled PCR amplification products from the loci were identified by an automated DNA sequencer for size determination, and each allelic variant was sequenced. Simpson's index of diversity was 0.97 based on an analysis of 39 nonrelated
F. tularensis
isolates. The locus showing the highest discrimination, SSTR9, gave an index of diversity of 0.95. Thirty-two strains isolated from humans during five outbreaks of tularemia showed much less variation. For example, 11 of 12 strains isolated in the Ljusdal area, Sweden in 1995 and 1998 had identical allelic variants. Phenotypic variants of strains and extensively cultured replicates within strains did not differ, and, for example, the same allelic combination was present in 55 isolates of the live-vaccine strain of
F. tularensis
and another one was present in all 13 isolates of a strain passaged in animals. The analysis of short-sequence repeats of
F. tularensis
strains appears to be a powerful tool for discrimination of individual strains and may be useful for a detailed analysis of the epidemiology of this potent pathogen.
Prevalence of Molecular Types and
mecA
Gene Carriage of Coagulase-Negative Staphylococci in a Neonatal Intensive Care Unit: Relation to Nosocomial Septicemia
Authors: Krediet Tannette G. , Jones Mark E. , Janssen Karin , Gerards Leo J. , Fleer André
Published: July 2002

Molecular typing of isolates revealed that neonatal coagulase-negative staphylococcal (CONS) septicemia is most frequently caused by predominant, antibiotic-resistant CONS types, which are widely distributed among both neonates and staff of the neonatal unit, suggesting cross-contamination. Therefore, infection control measures may be valuable in the prevention of this common nosocomial septicemia.
Heteroresistance to Vancomycin in
Enterococcus faecium
Authors: Alam M. Rabiul , Donabedian Susan , Brown William , Gordon James , Chow Joseph W. , Zervos Marcus J. , Hershberger Ellie
Published: July 2002

This study presents the first report of vancomycin heteroresistance in an
Enterococcus faecium
isolate from a patient. The original isolate was susceptible in vitro to vancomycin. E-tests showed growth of subcolonies in a zone of inhibition with a vancomycin MIC of >256 μg/ml. Both the susceptible and resistant colonies were from the same strain as determined by PFGE, and both contained the
vanA
gene as determined by PCR.
Molecular Typing of the Etiologic Agent of Human Granulocytic Ehrlichiosis
Authors: Carter Sarah E. , Ravyn M. Dana , Xu Yaning , Johnson Russell C.
Published: July 2002

The
p44
gene of the agent of human granulocytic ehrlichiosis (aoHGE) encodes a 44-kDa major outer surface protein. A technique was developed for the typing of the aoHGE based on the PCR amplification of the
p44
gene followed by a multiple restriction digest with
Hin
dIII,
Eco
RV, and
Asp
I to generate restriction fragment length polymorphism patterns. Twenty-four samples of the aoHGE were collected from geographically dispersed sites in the United States and included isolates from humans, equines, canines, small mammals, and ticks. Six granulocytic ehrlichiosis (GE) types were identified. The GE typing method is relatively simple to perform, is reproducible, and is able to differentiate among the various isolates of granulocytic ehrlichiae in the United States. These characteristics suggest that this GE typing method may be an important epizootiological and epidemiological tool.
βlasEN: Microdilution Panel for Identifying β-Lactamases Present in Isolates of
Enterobacteriaceae
Authors: Sanders Christine C. , Ehrhardt Anton F. , Moland Ellen Smith , Thomson Kenneth S. , Zimmer Barbara , Roe Darcie E.
Published: July 2002

A dried investigational use-only microdilution panel named βlasEN (a short named derived from the panel’s purpose, to identify β-lactamases in
Enterobacteriaceae
) containing 10 β-lactam drugs with and without β-lactamase inhibitors was developed to identify β-lactamases among clinical isolates of
Escherichia coli
,
Klebsiella pneumoniae
,
Klebsiella oxytoca
,
Citrobacter koseri
,
Citrobacter freundii
group,
Enterobacter
spp., and
Serratia marcescens
. The MICs obtained with a collection of 383 organisms containing well-characterized β-lactamases were used to develop numeric codes and logic pathways for computerized analysis of results. The resultant logic pathways and βlasEN panel were then used to test and identify β-lactamases among 885 isolates of
Enterobacteriaceae
recovered in cultures obtained at six different hospital laboratories across the United States. β-Lactamases present in 801 (90.5%) of the 885 isolates were identified by βlasEN by using the existing logic pathways and codes or after minor modifications were made to the existing codes. The 84 strains that gave codes that βlasEN could not identify were collected, reidentified, and retested by using βlasEN. Three strains had been misidentified, 54 strains gave different codes upon repeat testing that could be identified by βlasEN, and 27 strains repeated new codes. The β-lactamases in these strains were identified, and the new codes were added to the βlasEN logic pathways. These results indicate that βlasEN can identify clinically important β-lactamases among most isolates of
Enterobacteriaceae
. The results also show that good quality control and attention to proper performance of the tests are essential to the correct performance of βlasEN.
Active Copper- and Zinc-Containing Superoxide Dismutase in the Cryptic Genospecies of
Haemophilus
Causing Urogenital and Neonatal Infections Discriminates Them from
Haemophilus influenzae
Sensu Stricto
Authors: Langford Paul R. , Sheehan Brian J. , Shaikh Taheed , Kroll J. Simon
Published: July 2002

The presence of active copper- and zinc-containing superoxide dismutase in isolates of the cryptic genospecies of
Haemophilus
, responsible for urogenital, neonatal, and mother-infant infections, can be used as a biochemical marker to discriminate them from
H. influenzae
sensu stricto strains.
Identification of Four Distinct Genotypes of
Candida dubliniensis
and Detection of Microevolution In Vitro and In Vivo
Authors: Gee Sarah F. , Joly Sophie , Soll David R. , Meis Jacques F. G. M. , Verweij Paul E. , Polacheck Itzhack , Sullivan Derek J. , Coleman David C.
Published: July 2002

The present study investigates further the population structure of
Candida dubliniensis
and its ability to exhibit microevolution. Using 98 isolates (including 80 oral isolates) from 94 patients in 15 countries, we confirmed the existence of two distinct populations within the species
C. dubliniensis
, designated Cd25 group I and Cd25 group II, respectively, on the basis of DNA fingerprints generated with the
C. dubliniensis
-specific probe Cd25. The majority of Cd25 group I isolates (48 of 71, 67.6%) were from human immunodeficiency virus (HIV)-infected individuals, whereas the majority of Cd25 group II isolates (19 of 27, 70.4%) were from HIV-negative individuals (
P
≤ 0.001). Nucleotide sequence analysis of the internal transcribed spacer (ITS) regions of the rRNA genes from 19 representative isolates revealed the presence of four separate genotypes. All of the Cd25 group I isolates tested belonged to genotype 1, while the Cd25 group II population was comprised of three distinct genotypes (genotypes 2 to 4), which corresponded to distinct clades within the Cd25 group II population. These findings were confirmed using genotype-specific PCR primers with 70 isolates. We also showed that
C. dubliniensis
can exhibit microevolution in vivo and in vitro as occurs in other yeast species. DNA fingerprinting using the
C. dubliniensis
probes Cd25, Cd24, and Cd1 and karyotype analysis of multiple oral isolates recovered from the same specimen from each of eight separate patients revealed microevolution in six of eight of the clonal populations. Similarly, sequential clonal isolates from various anatomical sites in two separate patients exhibited microevolution. Microevolution was also shown to occur when two clinical isolates susceptible to fluconazole were exposed to the drug in vitro. The epidemiological significance of the four
C. dubliniensis
genotypes and the ability of
C. dubliniensis
to undergo microevolution has yet to be established.
Lectin Typing of
Campylobacter concisus
Authors: Aabenhus Rune , Hynes Sean O. , Permin Henrik , Moran Anthony P. , Andersen Leif P.
Published: July 2002

A total of 44 clinical isolates and the type strain of the putative pathogen
Campylobacter concisus
were grouped based on their reactions with plant lectins. The optimized lectin typing system used
C. concisus
strains proteolytically pretreated and subsequently typed by using a panel of four lectins. The system grouped all 45 strains into 13 lectin reaction patterns, leaving no strain untypeable due to autoagglutination. Lectin types were both stable and reproducible.
Culture-Negative Endocarditis Due to
Chlamydia pneumoniae
Authors: Gdoura R. , Pereyre S. , Frikha I. , Hammami N. , Clerc M. , Sahnoun Y. , Bebear C. , Daoud M. , de Barbeyrac B. , Hammami A.
Published: July 2002

We report on the case of a 54-year-old woman diagnosed as having culture-negative endocarditis (clinical and histopathologic evidence compatible with a recent episode of endocarditis). The responsibility of
Chlamydia pneumoniae
in this episode of endocarditis was suggested by a serological study and was then confirmed by the positive results of PCR and in situ hybridization tests with aortic and mitral valves tissues. To our knowledge, this is the first case of endocarditis due to
C. pneumoniae
confirmed by molecular biology-based techniques.
Monoclonal Antibodies Specific for Hippurate Hydrolase of
Campylobacter jejuni
Authors: Steele Marina , Gyles Carlton , Chan Voon Loong , Odumeru Joseph
Published: July 2002

Eleven monoclonal antibodies raised against recombinant
Campylobacter jejuni
hippurate hydrolase were tested for binding to lysates from 19
C. jejuni
strains, 12 other
Campylobacter
strains, and 21 non-
Campylobacter
strains. Several monoclonal antibodies bound to
C. jejuni
but not to other
Campylobacter
species and may be useful in a species-specific immunoassay.
Use of PGMY Primers in L1 Consensus PCR Improves Detection of Human Papillomavirus DNA in Genital Samples
Authors: Coutlée François , Gravitt Patti , Kornegay Janet , Hankins Catherine , Richardson Harriet , Lapointe Normand , Voyer Hélène , Franco Eduardo
Published: July 2002

The novel PGMY L1 consensus primer pair is more sensitive than the MY09 and MY11 primer mix for detection and typing with PCR of human papillomavirus (HPV) DNA in genital specimens. We assessed the diagnostic yield of PGMY primers for the detection and typing of HPV by comparing the results obtained with PGMY09/PGMY11 and MY09/MY11/HMB01 on 299 genital samples. Amplicons generated with PGMY primers were typed with the line blot assay (PGMY-line blot), while HPV amplicons obtained with the degenerate primer pool MY09/MY11/HMB01 were detected with type-specific radiolabeled probes in a dot blot assay (standard consensus PCR test). Cervicovaginal lavage samples (
N
= 272) and cervical scrape samples (
N
= 27) were tested in parallel with both PCR tests. The PGMY-line blot test detected the presence of HPV DNA more frequently than the standard consensus PCR assay. The concordance for HPV typing between the two assays was 84.3% (214 of 255 samples), for a good kappa value of 0.69. Of the 177 samples containing HPV DNA by at least one method, 40 samples contained at least one HPV type detected only with PGMY-line blot, whereas positivity exclusively with the standard consensus PCR test was found for only 7 samples (
P
< 0.001). HPV types 45 and 52 were especially more frequently detected with PGMY than MY primers. However, most HPV types were better amplified with PGMY primers, including HPV-16. Samples with discordant results between the two PCR assays more frequently contained multiple HPV types. Studies using PGMY instead of MY primers have the potential to report higher detection rates of HPV infection not only for newer HPV types but also for well-known genital types.
Genotyping of Human Papillomavirus in Liquid Cytology Cervical Specimens by the PGMY Line Blot Assay and the SPF
10
Line Probe Assay
Authors: van Doorn Leen-Jan , Quint Wim , Kleter Bernhard , Molijn Anco , Colau Brigitte , Martin Marie-Thérèse , Kravang-In  , Torrez-Martinez Norah , Peyton Cheri L. , Wheeler Cosette M.
Published: July 2002

A comparison of two PCR-based human papillomavirus (HPV) DNA detection and genotyping systems (PGMY LBA and SPF
10
LiPA) was conducted in two laboratories. Both systems are based on broad-spectrum PCR for the detection of HPV DNA, followed by reverse hybridization with type-specific probes. A total of 400 selected cervical scrape specimens in PreservCyt solution (55% normal cytology, 18% atypical squamous cells of unknown significance, 14.8% low-grade squamous intraepithelial lesions [SIL], and 12.5% high-grade SIL) were tested for the presence of HPV DNA. In this selected group of specimens, the overall agreement between the two methods for the detection of any HPV DNA was high (κ = 0.859). When the 20 common HPV genotypes identified by both methods were considered (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 56, 58, 59, 66, and 68), compatible genotype-specific results were observed in 96.5% of the samples, even when multiple HPV genotypes were present. However, for some specific HPV genotypes, there were significant differences in HPV detection by the two methods. PGMY LBA detected more HPV type 42 (
P
= 0.002), HPV type 56 (
P
= 0.039), and HPV type 59 (
P
< 0.001), whereas SPF
10
LiPA detected more HPV type 31 (
P
< 0.001) and HPV type 52 (
P
= 0.031). For the remaining genotypes, including HPV types 16 and 18, the results obtained by the two methods were not significantly different. In general, both genotyping methods are highly suitable for clinical and epidemiological studies.
Polymorphic Amplified Typing Sequences Provide a Novel Approach to
Escherichia coli
O157:H7 Strain Typing
Authors: Kudva Indira T. , Evans Peter S. , Perna Nicole T. , Barrett Timothy J. , DeCastro G. Joel , Ausubel Frederick M. , Blattner Frederick R. , Calderwood Stephen B.
Published: July 2002

Escherichia coli
O157:H7 (O157) strains are commonly typed by pulsed-field gel electrophoresis (PFGE) following digestion of genomic DNA with the restriction enzyme
Xba
I. We have shown that O157 strains differ from each other by a series of discrete insertions or deletions, some of which contain recognition sites for
Xba
I, suggesting that these insertions and deletions are responsible for the differences in PFGE patterns. We have devised a new O157 strain typing protocol, polymorphic amplified typing sequences (PATS), based on this information. We designed PCR primer pairs to amplify genomic DNA flanking each of 40 individual
Xba
I sites in the genomes of two O157 reference strains. These primer pairs were tested with 44 O157 isolates, 2 each from 22 different outbreaks of infection. Thirty-two primer pairs amplified identical fragments from all 44 isolates, while eight primer pairs amplified regions that were polymorphic between isolates. The isolates could be differentiated solely on the basis of which of the eight polymorphic amplicons was detected. PATS correctly identified 21 of 22 outbreak pairs as being identical or highly related, whereas PFGE correctly identified 14 of the 22 outbreak pairs as being identical or highly related; PATS was also able to type isolates from three outbreaks that were untypeable by PFGE. However, PATS was less sensitive than PFGE in discriminating between outbreaks. These data suggest that typing by PATS may provide a simple procedure for strain typing of O157 and other bacteria and that further evaluation of the utility of this method for epidemiologic investigations is warranted.
Recombinant BBK32 Protein in Serodiagnosis of Early and Late Lyme Borreliosis
Authors: Heikkilä Tero , Seppälä Ilkka , Saxén Harri , Panelius Jaana , Peltomaa Miikka , Julin Tiina , Carlsson Sten-Anders , Lahdenne Pekka
Published: July 2002

Borrelial protein BBK32 was evaluated as an antigen in the serodiagnosis of early and disseminated Lyme borreliosis (LB).
bbk32
was cloned and sequenced from eight isolates of the three pathogenic
Borrelia
species. The identities between the amino acid sequences of the BBK32 proteins from
Borrelia burgdorferi
sensu stricto,
B. garinii
, and
B. afzelii
isolates were 71 to 100%. By immunoglobulin G (IgG) Western blotting (WB) or enzyme-linked immunosorbent assay (ELISA), up to 74 and 100% of acute- and convalescent-phase samples, respectively, from 23 patients with erythema migrans (EM) were positive for recombinant BBK32 protein from
B. afzelii
. In the serology of disseminated LB, the three variant BBK32 antigens cross-reacted. In total, 14 of 14 samples from patients with neuroborreliosis and 15 of 15 samples from patients with Lyme arthritis were positive. The specificities of the IgG ELISA with the variant BBK32 antigens for EM and disseminated borreliosis were 81 to 92% and 89 to 95%, respectively. Our findings indicate that the BBK32 proteins are promising serodiagnostic antigens for the detection of early and disseminated LB but that variant BBK32 proteins may be needed either in parallel or in combination with an immunoassay for LB to cover all the relevant borrelial species that cause the disease.
Genome Scale Comparison ofMycobacterium aviumsubsp.paratuberculosiswithMycobacterium aviumsubsp.aviumReveals Potential Diagnostic Sequences
Authors: Bannantine John P. , Baechler Emily , Zhang Qing , Li LingLing , Kapur Vivek
Published: July 2002

The genetic similarity betweenMycobacterium aviumsubsp.paratuberculosisand other mycobacterial species has confounded the development ofM. aviumsubsp.paratuberculosis-specific diagnostic reagents. Random shotgun sequencing of theM. aviumsubsp.paratuberculosisgenome in our laboratories has shown >98% sequence identity withMycobacterium aviumsubsp.aviumin some regions. However, an in silico comparison of the largest annotatedM. aviumsubsp.paratuberculosiscontigs, totaling 2,658,271 bp, with the unfinishedM. aviumsubsp.aviumgenome has revealed 27 predictedM. aviumsubsp.paratuberculosiscoding sequences that do not align withM. aviumsubsp.aviumsequences. BLASTP analysis of the 27 predicted coding sequences (genes) shows that 24 do not match sequences in public sequence databases, such as GenBank. These novel sequences were examined by PCR amplification with genomic DNA from eight mycobacterial species and ten independent isolates ofM. aviumsubsp.paratuberculosis. From these analyses, 21 genes were found to be present in allM. aviumsubsp.paratuberculosisisolates and absent from all other mycobacterial species tested. One region of theM. aviumsubsp.paratuberculosisgenome contains a cluster of eight genes, arranged in tandem, that is absent in other mycobacterial species. This region spans 4.4 kb and is separated from other predicted coding regions by 1,408 bp upstream and 1,092 bp downstream. The gene upstream of this eight-gene cluster has strong similarity to mycobacteriophage integrase sequences. The GC content of this 4.4-kb region is 66%, which is similar to the rest of the genome, indicating that this region was not horizontally acquired recently. Southern hybridization analysis confirmed that this gene cluster is present only inM. aviumsubsp.paratuberculosis. Collectively, these studies suggest that a genomics approach will help in identifying novelM. aviumsubsp.paratuberculosisgenes as candidate diagnostic sequences.