bluuebunny/crossref_metadata_2025_split huggingface dataset:
Production of Cytolethal Distending Toxins by Pathogenic
Escherichia coli
Strains Isolated from Human and Animal Sources: Establishment of the Existence of a New
cdt
Variant (Type IV)
Authors: Tóth István , Hérault Fréderique , Beutin Lothar , Oswald Eric 
Published: September 2003

ABSTRACT

Three types of cytolethal distending toxin (CDT), namely, CDT-I, CDT-II, and CDT-III, have been described in
Escherichia coli
. Using primers designed for the detection of sequences common to the
cdtB
genes, we analyzed by PCR a set of 21 CDT-producing
E. coli
strains of intestinal and extraintestinal origins isolated from human and different animal species in several European countries and in the United States. On the basis of the existing differences in the
cdtB
genes,
cdt
-I-,
cdt
-II-, and
cdt
-III-specific primer pairs were designed and used for
cdt
typing. These new primers successfully differentiated all of the previously described
cdt
genes. Six strains proved to be
cdt-
I; eight strains proved to be
cdt
-III. However, none of the type I-, II-, and III-specific primers generated amplicons from six CDT
+
strains, suggesting the existence of a new
cdt
variant. Sequence analysis of the amplicons from two untypeable genes confirmed the existence of a new
cdt
variant that we called
cdt
-IV. Using the new specific primers,
cdt
-IV was detected in human, porcine, and poultry strains of intestinal and extraintestinal origins. To validate all sets of
cdt
specific primers, a group of 353 human
E. coli
strains isolated in Hungary was then investigated for the presence of
cdt
genes. This included 190 strains isolated from patients with urinary tract infections (UTI), 51 strains isolated from other (nonurinary) extraintestinal infections, and 112 intestinal strains isolated from healthy individuals. Of 190 UTI strains, 15 (7.9%) had
cdt
genes. Of 51 non-UTI extraintestinal strains 3 (5.9%) contained the
cdt
gene, and 1 (0.9%) of 112 healthy intestinal strains was PCR positive. Five strains proved to be
cdt
-I, and fourteen strains proved to be
cdt
-IV. The CDT-producing extraintestinal strains belonged to a wide variety of serogroups, including O2, O6, O75, and O170. In conclusion, we have developed a new PCR typing system for CDT able to detect a new CDT variant present in pathogenic
E. coli
strains obtained from animals and humans.
Discrimination of
d
-Tartrate-Fermenting and -Nonfermenting
Salmonella enterica
subsp.
enterica
Isolates by Genotypic and Phenotypic Methods
Authors: Malorny Burkhard , Bunge Cornelia , Helmuth Reiner 
Published: September 2003

ABSTRACT

A multiplex PCR and an improved lead acetate test were developed to discriminate
d
-tartrate-fermenting and -nonfermenting
Salmonella enterica
subsp.
enterica
strains. Both methods showed an accuracy of 100% when 125
Salmonella
strains belonging to 15 serovars were tested. Special emphasis was given to
S. enterica
subsp.
enterica
serovar Paratyphi B isolates because of the clinical importance of its
d
-tartrate-nonfermenting variant and the recently increasing numbers of cases of human outbreaks caused by its fermenting variant (formerly
Salmonella
serovar Java). The lead acetate test described previously (G. A. Alfredsson, R. M. Barker, D. C. Old, and J. P. Duguid, J. Hyg. 70:651-666, 1972) was modified in the inoculation and incubation procedure. The PCR assay was based on the genotypic difference of the presence (
d
-tartrate-fermenting strains) or absence (
d
-tartrate-nonfermenting strains) of the ATG start codon for the gene STM 3356, which encodes a putative cation transporter. Sequence data revealed a nucleotide exchange from G to A within the ATG start codon of gene STM 3356 in the
d
-tartrate-nonfermenting strains. In order to increase the reliability of the PCR assay, a positive control based on a
Salmonella
genus-specific primer set for the detection of
Salmonella
DNA was included. The PCR-based discrimination needs only several hours compared to 6 days needed by the improved lead acetate test to obtain reliable results. Consequently, the PCR
d
-tartrate assay should be the method of choice for the discrimination of
d
-tartrate-fermenting and -nonfermenting
Salmonella
strains in the future.
GeneScan Reverse Transcription-PCR Assay for Detection of Six Common Respiratory Viruses in Young Children Hospitalized with Acute Respiratory Illness
Authors: Erdman Dean D. , Weinberg Geoffrey A. , Edwards Kathryn M. , Walker Frances J. , Anderson Barbara C. , Winter Jörn , González Monica , Anderson Larry J. 
Published: September 2003

ABSTRACT
A reverse transcription-PCR (RT-PCR) assay based on automated fluorescent capillary electrophoresis and GeneScan software analysis was developed to detect six common respiratory viruses in clinical specimens from young children. Assays for human respiratory syncytial virus (HRSV); human parainfluenza viruses 1, 2, and 3 (HPIV1, -2, and -3, respectively); and influenza A and B viruses were incorporated into a single standard assay format. The optimized assay panel was used to test 470 respiratory specimens obtained from 462 children hospitalized with acute respiratory illness that had been previously tested by viral culture (405 specimens) or direct immunofluorescence staining (DIF) (65 specimens). Of 93 specimens positive for respiratory viruses by culture or DIF, 86 (92%) were positive by RT-PCR, including 66 HRSV, 2 HPIV2, 5 HPIV3, 3 influenza A virus, and 10 influenza B virus specimens. An additional 119 respiratory viruses were identified by RT-PCR in 116 patients for whom results were negative by viral isolation or DIF. We conclude that the GeneScan RT-PCR panel can markedly improve detection of acute respiratory virus infections in young children.
Semiquantitative Detection by Real-Time PCR ofAspergillus fumigatusin Bronchoalveolar Lavage Fluids and Tissue Biopsy Specimens from Patients with Invasive Aspergillosis
Authors: Rantakokko-Jalava Kaisu , Laaksonen Sanna , Issakainen Jouni , Vauras Jukka , Nikoskelainen Jukka , Viljanen Matti K. , Salonen Juha 
Published: September 2003

ABSTRACTA real-time PCR method was developed and used to detectAspergillus fumigatusmitochondrial DNA (mtDNA) in bronchoalveolar lavage (BAL) fluids and tissue biopsy specimens. The analytical sensitivity of the assay was oneA. fumigatusconidium per reaction, and the assay was linear at least over 4 orders of magnitude above the detection limit. BAL fluids from 66 immunocompromised patients at risk of invasive pulmonary aspergillosis (IPA) and 33 immunocompetent controls and tissue biopsy specimens from 10 immunocompromised patients were analyzed. The results were related to the clinical diagnosis established according to recently published consensus criteria.A. fumigatusmtDNA positivity was encountered in 16 of 81 (20%) BAL fluid specimens from patients at risk and 1 of 33 (3%) specimens from immunocompetent controls. PCRs were positive in six of seven, two of four, and four of five of the patients with proven, probable, and possible IPA, respectively, as well as in four patients at risk but without any other evidence of IPA. With qualitative detection, the diagnostic sensitivity of PCR was 73%, specificity was 93%, and predictive values of positive (PPV) and negative (NPV) results were 73 and 95%, respectively. Using a threshold cycle of <35 as a limit for positive PCR, the specificity and PPV of PCR in the diagnosis of invasive aspergillosis were 100%, but its sensitivity was only 45% and NPV was 92%. PCR was positive in tissue biopsy specimens from all patients with invasive aspergillosis caused byA. fumigatus. Semiquantitative detection ofA. fumigatusmtDNA in BAL fluid may be helpful in the diagnosis of IPA. PCR is well suited for the verification of the presence ofA. fumigatusin tissue biopsy specimens.
Use of Real-Time PCR with Multiple Targets To Identify
Pseudomonas aeruginosa
and Other Nonfermenting Gram-Negative Bacilli from Patients with Cystic Fibrosis
Authors: Qin Xuan , Emerson Julia , Stapp Jenny , Stapp Lynn , Abe Patrick , Burns Jane L. 
Published: September 2003

ABSTRACT

Pseudomonas aeruginosa
and other gram-negative isolates from patients with cystic fibrosis (CF) may be difficult to identify because of their marked phenotypic diversity. We examined 200 gram-negative clinical isolates from CF respiratory tract specimens and compared identification by biochemical testing and real-time PCR with multiple different target sequences using a standardized combination of biochemical testing and molecular identification, including 16S rRNA partial sequencing and
gyrB
PCR and sequencing as a “gold standard.” Of 50 isolates easily identified phenotypically as
P. aeruginosa
, all were positive with PCR primers for
gyrB
or
oprI
, 98% were positive with exotoxin A primers, and 90% were positive with
algD
primers. Of 50
P. aeruginosa
isolates that could be identified by basic biochemical testing, 100% were positive by real-time PCR with
gyrB
or
oprI
primers, 96% were positive with exotoxin A primers, and 92% were positive with
algD
primers. For isolates requiring more-extensive biochemical evaluation, 13 isolates were identified as
P. aeruginosa
; all 13 were positive with
gyrB
primers, 12 of 13 were positive with
oprI
primers, 11 of 13 were positive with exotoxin A primers, and 10 of 13 were positive with
algD
primers. A single false-positive
P. aeruginosa
result was seen with
oprI
primers. The best-performing commercial biochemical testing was in exact agreement with molecular identification only 60% of the time for this most difficult group. Real-time PCR had costs similar to those of commercial biochemical testing but a much shorter turnaround time. Given the diversity of these CF isolates, real-time PCR with a combination of two target sequences appears to be the optimum choice for identification of atypical
P. aeruginosa
and for non-
P. aeruginosa
gram-negative isolates.
Antimicrobial Susceptibility Testing of Aquatic Bacteria: Quality Control Disk Diffusion Ranges for
Escherichia coli
ATCC 25922 and
Aeromonas salmonicida
subsp.
salmonicida
ATCC 33658 at 22 and 28°C
Authors: Miller R. A. , Walker R. D. , Baya A. , Clemens K. , Coles M. , Hawke J. P. , Henricson B. E. , Hsu H. M. , Mathers J. J. , Oaks J. L. , Papapetropoulou M. , Reimschuessel R. 
Published: September 2003

ABSTRACT

Quality control (QC) ranges for disk diffusion susceptibility testing of aquatic bacterial isolates were proposed as a result of a multilaboratory study conducted according to procedures established by the National Committee for Clinical Laboratory Standards (NCCLS). Ranges were proposed for
Escherichia coli
ATCC 25922 and
Aeromonas salmonicida
subsp.
salmonicida
ATCC 33658 at 22 and 28°C for nine different antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, gentamicin, oxolinic acid, oxytetracycline, ormetoprim-sulfadimethoxine, and trimethoprim-sulfamethoxazole). All tests were conducted on standard Mueller-Hinton agar. With ≥95% of all data points fitting within the proposed QC ranges, the results from this study comply with NCCLS guidelines and have been accepted by the NCCLS Subcommittee for Veterinary Antimicrobial Susceptibility Testing. These QC guidelines will permit greater accuracy in interpreting results and, for the first time, the ability to reliably compare susceptibility test data between aquatic animal disease diagnostic laboratories.
Evaluation of Three Rapid Methods for the Direct Identification of
Staphylococcus aureus
from Positive Blood Cultures
Authors: Chapin Kimberle , Musgnug Michael 
Published: September 2003

ABSTRACT

Staphylococci represent the most commonly encountered blood culture isolates. Differentiating
Staphylococcus aureus
from coagulase-negative staphylococci (CoNS) is important in guiding empirical therapy, especially since the majority of CoNS are contaminants. This study evaluated three rapid methods for the direct identification of
S. aureus
from blood cultures. A total of 157 patient blood cultures with gram stains showing gram-positive cocci in clusters were included. The following assays were evaluated: API RAPIDEC staph (API) (bioMerieux, Durham, N.C.), the tube coagulase test (TCT) read at 4 h, and peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) (AdvanDx, Woburn, Mass.). All assays yielded results of
S. aureus
or non-
S. aureus.
The direct rapid results were compared to results obtained with isolated colonies using the AccuProbe
Staphylococcus aureus
Culture Identification Test (Gen-Probe, San Diego, Calif.). API, TCT, and PNA FISH exhibited sensitivities of 96, 84, and 99% and specificities of 99, 100, and 100%, respectively. Direct identification testing by any of these three assays yielded acceptable performance and timely results. This ability to accurately detect
S. aureus
in blood cultures gives the physician information with which to initiate or tailor antimicrobial therapy. Coupled with direct susceptibility testing of positive blood culture broths, the patient and institution may experience improved outcomes.
Isolation of anAnaplasmasp. Organism from White-Tailed Deer by Tick Cell Culture
Authors: Munderloh Ulrike G. , Tate Cynthia M. , Lynch Meghan J. , Howerth Elizabeth W. , Kurtti Timothy J. , Davidson William R. 
Published: September 2003

ABSTRACTWe used tick cell culture to isolate a bacterium previously referred to as the “white-tailed deer (WTD) agent” from two captive fawns inoculated with blood from wild WTD (Odocoileus virginianus). Buffy coat cells were added to ISE6 tick cell cultures and incubated at 34°C, and 8 days later,Anaplasma-like inclusions were demonstrated in Giemsa-stained culture samples. The microbes became established and could be continuously passaged in tick cells. The identity of a culture isolate designated WTD76 was verified as the WTD agent by using specific PCR primers and by DNA sequencing. Comparison with sequences available in GenBank indicated that the isolate was most closely related first toAnaplasma platysand second toAnaplasma phagocytophilum, supporting its placement in the genusAnaplasma. Transmission electron microscopy of thisAnaplasmasp. organism in tick cell cultures revealed large inclusions filled with pleomorphic and rod-shaped bacteria. Tick cells infected with theAnaplasmasp. organism were used to successfully infect a naive deer, thereby proving the infectivity of the isolate for deer.
Diagnosis of Tick-Borne Encephalitis by a μ-Capture Immunoglobulin M-Enzyme Immunoassay Based on Secreted Recombinant Antigen Produced in Insect Cells
Authors: Jääskeläinen Anu , Han Xiuqi , Niedrig Matthias , Vaheri Antti , Vapalahti Olli 
Published: September 2003

ABSTRACT

Acute tick-borne encephalitis is diagnosed by detection of IgM antibodies to tick-borne encephalitis virus (TBEV) (genus
Flavivirus
) in patient serum. TBEV membrane (M) and envelope (E) proteins have previously been shown to form virus-like particles when expressed in mammalian cells. We expressed the prM/M and E proteins in insect cells with a recombinant baculovirus system and obtained antigenic protein secreted into the cell culture medium, as evidenced by detection by a panel of five monoclonal antibodies to TBEV E protein. According to the sedimentation pattern in sucrose gradient centrifugation, the proteins were most likely secreted as virus-like particles. A μ-capture immunoglobulin M-enzyme immunoassay (IgM-EIA) test was developed and compared to a commercially available TBEV-IgM test (Progen) based on inactivated purified TBEVs. With a panel of 100 TBEV-IgM-negative, 50 TBEV-IgM-positive, and seven dengue virus-IgM-positive serum samples from our diagnostic laboratory, a sensitivity of 100% and specificity of 99% were obtained, and the correlation coefficient of EIA absorbances with the reference test was 0.93. The antigen was also suitable for IgG antibody detection in an immunofluorescent assay format. This is the first time that secreted, fully antigenic E protein has been produced in insect cells for this arthropod-borne flavivirus.
Use of Discriminatory Probes for Strain Typing of Formalin-Fixed, Rabies Virus-Infected Tissues by In Situ Hybridization
Authors: Nadin-Davis Susan A. , Sheen Mary , Wandeler Alexander I. 
Published: September 2003

ABSTRACT
An in situ hybridization (ISH) method has been developed to overcome difficulties encountered in the viral typing of formalin-fixed rabies virus-infected brain tissue. Rabies viruses representative of all strains normally encountered in diagnostic submissions throughout Canada, including 3 strains of terrestrial hosts (arctic fox, western skunk, mid-Atlantic raccoon), 10 strains circulating in several bat reservoirs (BBCAN1 to BBCAN7, LACAN, SHCAN, and MYCAN), and the Evelyn-Rokitniki-Abelseth (ERA) strain, used as an oral vaccine for fox rabies control in Ontario, were targeted. Partial phosphoprotein gene fragments generated from reverse transcription (RT)-PCR products of specimens of each viral type were molecularly cloned and used to produce negative-sense digoxigenin-labeled RNA transcripts. Conditions permitting the use of these transcripts as strain-specific probes were optimized by blotting analyses with RT-PCR amplicons generated with representative rabies viruses and by ISH applied to mouse brains inoculated with these strains. The successful application of this methodology to two rabies virus-positive specimens that were also identified by traditional methods and the retrospective typing of two archival rabies virus-positive equine specimens is described. This technique provides a typing regimen for rabies virus isolates submitted in a form that is normally recalcitrant to alternate typing strategies.
Corynebacterium nigricans
sp. nov.: Proposed Name for a Black-Pigmented
Corynebacterium
Species Recovered from the Human Female Urogenital Tract
Authors: Shukla Sanjay K. , Bernard Kathryn A. , Harney Mary , Frank Daniel N. , Reed Kurt D. 
Published: September 2003

ABSTRACT

Six independent isolates of an unusual black-pigmented
Corynebacterium
species (strains CN-1, CN-2, CN-3415, W70124, 91-0032, and 92-0360) were recovered from the human female urogenital tract. Four of the six source patients had complications of pregnancy, including spontaneous abortion, preterm labor, and low amniotic fluid volume at the time of the pathogen isolation. One isolate was recovered from a vaginal ulcer. All six strains yielded black-pigmented colonies on sheep blood agar, chocolate agar, and colistin-nalidixic acid agar after 24 to 48 h of incubation at 35°C. The dry, adherent colonies pitted the agar surface. The cells were coccobacillary to rod-shaped, catalase positive, nonmotile, and nonlipophilic. Only five of six isolates were available for characterization. Biochemical and chemotaxonomic studies revealed that the strains belong to the genus
Corynebacterium
but differ from known corynebacterial species. Comparative 16S rRNA gene sequence analysis showed that the strains are closely related and form a new subline within the genus
Corynebacterium
. We propose the name
Corynebacterium nigricans
sp. nov. for this group of coryneforms. The type strain of
Corynebacterium nigricans
is CN-1. It is deposited in the American Type Culture Collection (assigned strain number ATCC 700975) and in the Institute Pasteur collection (assigned strain number CIP 107346).
Use of Loop-Mediated Isothermal Amplification of the IS900Sequence for Rapid Detection of CulturedMycobacterium aviumsubsp.paratuberculosis
Authors: Enosawa M. , Kageyama S. , Sawai K. , Watanabe K. , Notomi T. , Onoe S. , Mori Y. , Yokomizo Y. 
Published: September 2003

ABSTRACTWe evaluated the usefulness of loop-mediated isothermal amplification (LAMP) in detecting specific gene sequences ofMycobacterium aviumsubsp.paratuberculosis(MAP). A total of 102 primer sets for LAMP was designed to amplify the IS900, HspX, and F57 gene sequences of MAP. Using each of two primer sets (P-1 and P-2) derived from the IS900fragment, it was possible to detect MAP in a manner similar to that used with nested PCR. The sensitivity of LAMP with P-1 was 0.5 pg/tube, which was more sensitive than nested PCR. When P-2 was used, 5 pg/tube could be detected, which was the same level of sensitivity as that for nested PCR. LAMP with P-1 was specific. Although only 2Mycobacterium scrofulaceumstrains out of 43 non-MAP mycobacterial strains were amplified, the amplification reaction for these strains was less efficient than for MAP strains, and their products could be distinguished from MAP products by restriction digestion. LAMP with P-2 resulted in very specific amplification only from MAP, the same result obtained with nested PCR. Our LAMP method was highly specific, and the white turbidity of magnesium pyrophosphate, a by-product of the LAMP reaction, allowed simple visual detection. Our method is rapid, taking only 2 h, compared with 4 h for nested PCR. In addition, the LAMP method is performed under isothermal conditions and no special apparatus is needed, which makes it more economical and practical than nested PCR or real-time PCR. These results indicate that LAMP can provide a rapid yet simple test for the detection of MAP.
Comparison and Evaluation of Real-Time PCR, Real-Time Nucleic Acid Sequence-Based Amplification, Conventional PCR, and Serology for Diagnosis of
Mycoplasma pneumoniae
Authors: Templeton Kate E. , Scheltinga Sitha A. , Graffelman A. Willy , van Schie Jolanda M. , Crielaard Jantine W. , Sillekens Peter , van den Broek Peterhans J. , Goossens Herman , Beersma Matthias F. C. , Claas Eric C. J. 
Published: September 2003

ABSTRACT

Mycoplasma pneumoniae
is a common cause of community-acquired pneumonia and lower-respiratory-tract infections. Diagnosis has traditionally been obtained by serological diagnosis, but increasingly, molecular techniques have been applied. However, the number of studies actually comparing these assays is limited. The development of a novel duplex real-time PCR assay for detection of
M. pneumoniae
in the presence of an internal control real-time PCR is described. In addition, real-time nucleic acid sequence-based amplification (NASBA) on an iCycler apparatus is evaluated. These assays were compared to serology and a conventional PCR assay for 106 clinical samples from patients with lower-respiratory-tract infection. Of the 106 samples, 12 (11.3%) were positive by all the molecular methods whereas serology with acute sample and convalescent samples detected 6 (5.6%) and 9 (8.5%), respectively. Clinical symptoms of the patients with
Mycoplasma
-positive results were compared to those of the other patients with lower-respiratory-tract infections, and it was found that the results for mean lower age numbers as well as the presence of chills, increased erythrocyte sedimentation rate, and raised C-reactive protein levels showed significant differences. Molecular methods are superior for diagnosis of
M. pneumoniae
, providing more timely diagnosis. In addition, using real-time methods involves less hands-on time and affords the ability to monitor the reaction in the same tube.