bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evidence That the Enterotoxin Gene Can Be Episomal inClostridium perfringensIsolates Associated with Non-Food-Borne Human Gastrointestinal Diseases
Authors: Collie Renee E. , McClane Bruce A. 
Published: December 2019

ABSTRACTClostridium perfringensenterotoxin (CPE) is responsible for the diarrheal and cramping symptoms of humanC. perfringenstype A food poisoning. CPE-producingC. perfringensisolates have also recently been associated with several non-food-borne human gastrointestinal (GI) illnesses, including antibiotic-associated diarrhea and sporadic diarrhea. The current study has used restriction fragment length polymorphism (RFLP) and pulsed-field gel electrophoresis (PFGE) analyses to compare the genotypes of 43cpe-positiveC. perfringensisolates obtained from diverse sources. All North American and European food-poisoning isolates examined in this study were found to carry a chromosomalcpe, while all non-food-borne human GI disease isolates characterized in this study were determined to carry theircpeon an episome. Collectively, these results provide the first evidence that distinct subpopulations ofcpe-positiveC. perfringensisolates may be responsible forC. perfringenstype A food poisoning versus CPE-associated non-food-borne human GI diseases. If these putative associations are confirmed in additional surveys,cpeRFLP and PFGE genotyping assays may facilitate the differential diagnosis of food-borne versus non-food-borne CPE-associated human GI illnesses and may also be useful epidemiologic tools for identifying reservoirs or transmission mechanisms for the subpopulations ofcpe-positive isolates specifically responsible for CPE-associated food-borne versus non-food-borne human GI diseases.
Case of Triple Endocarditis Caused by
Rothia dentocariosa
and Results of a Survey in France
Authors: Kong Robert , Mebazaa Alexandre , Heitz Bertrand , De Briel Dominique André , Kiredjian Martine , Raskine Laurent , Payen Didier 
Published: December 2019

ABSTRACT

This is the first case of endocarditis in which
Rothia dentocariosa
was cultured from three affected valves. In addition, the records of two microbiology laboratories in France showed that
R. dentocariosa
was rarely involved in severe infection and that positive blood cultures were not associated with endocarditis.
Identification of Streptococci to Species Level by Sequencing the Gene Encoding the Manganese-Dependent Superoxide Dismutase
Authors: Poyart Claire , Quesne Gilles , Coulon Stephane , Berche Patrick , Trieu-Cuot Patrick 
Published: December 2019

ABSTRACT

We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (

sodA
int

) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (
S. acidominimus
,
S. agalactiae
,
S. alactolyticus
,
S. anginosus
,
S. bovis
,
S. constellatus
,
S. canis
,
S. cricetus
,
S. downei
,
S. dysgalactiae
,
S. equi
subsp.
equi
,
S. equi
subsp.
zooepidemicus
,
S. equinus
,
S. gordonii
,
S. iniae
,
S. intermedius
,
S. mitis
,
S. mutans
,
S. oralis
,
S. parasanguis
,
S. pneumoniae
,
S. porcinus
,
S. pyogenes
,
S. salivarius
,
S. sanguis
,
S. sobrinus
,
S. suis
,
S. thermophilus
, and
S. vestibularis
). Phylogenetic analysis of these

sodA
int

fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of their

sodA
int

fragments on the phylogenetic tree of the

sodA
int

fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.
Detection of Human Herpesvirus 6 DNA in Serum by a Microplate PCR-Hybridization Assay
Authors: Osiowy Carla , Prud’homme Isabelle , Monette Michael , Zou Shimian 
Published: December 2019

ABSTRACT
PCR was performed on DNA extracts derived from clinical serum samples submitted for human herpesvirus 6 (HHV-6) serological examination. To detect amplified HHV-6 products, a hybridization-based microtiter plate assay (PCR ELISA; Boehringer Mannheim) was used. The assay system was found to be rapid, specific, and sensitive. Approximately three copies of a plasmid-based HHV-6 sequence could be detected, and no cross amplification was observed with HHV-7 genomic DNA. There was no correlation found between HHV-6 DNA detection and serological status in clinical serum samples from individuals more than 2 years old. On the other hand, in serum samples from infants less than 2 years old, a high rate of detection of HHV-6 DNA was observed in those who lacked immunoglobulin G and M antibodies to HHV-6 (55%). In this regard, PCR of serum DNA extracts may be used as a sensitive indicator of active HHV-6 infection in infants prior to their seroconversion.
Pre-AIDS Era Isolates ofPneumocystis cariniif. sp.hominis: High Genotypic Similarity with Contemporary Isolates
Authors: Tsolaki Anthony G. , Beckers Pieter , Wakefield Ann E. 
Published: December 2019

ABSTRACTIsolates ofPneumocystis cariniif. sp.hominiswere examined from six individuals who died ofP. cariniipneumonia between 1968 and 1981 and who had underlying immunodeficiencies which were not due to human immunodeficiency virus infection. DNA sequence variation was analyzed in the genes encoding the mitochondrial large subunit rRNA (mt LSU rRNA), the internal transcribed spacer (ITS) regions of the nuclear rRNA, thearomlocus, and the mitochondrial small subunit rRNA. No major variations were observed when these isolates were compared to isolates from HIV-infected individuals. A small number of minor differences were detected. A new position at which variation occurred in the mt LSU rRNA was observed in one sample. Three new ITS sequence types were identified. A total of nine different ITS sequence types were found in the six samples. Mixed infection with different ITS sequence types ofP. cariniif. sp.hominiswas observed in four of the six samples. The ITS locus was the most informative of the four loci for distinguishing among the isolates ofP. cariniif. sp.hominis. The data suggest that isolates ofP. cariniif. sp.hominisfrom before the AIDS pandemic are genetically very similar to those currently found in HIV-infected individuals.
Improved Amplification of Microbial DNA from Blood Cultures by Removal of the PCR Inhibitor Sodium Polyanetholesulfonate
Authors: Fredricks David N. , Relman David A. 
Published: December 2019

ABSTRACT

Molecular methods are increasingly used to identify microbes in clinical samples. A common technical problem with PCR is failed amplification due to the presence of PCR inhibitors. Initial attempts at amplification of the bacterial 16S rRNA gene from inoculated blood culture media failed for this reason. The inhibitor persisted, despite numerous attempts to purify the DNA, and was identified as sodium polyanetholesulfonate (SPS), a common additive to blood culture media. Like DNA, SPS is a high-molecular-weight polyanion that is soluble in water but insoluble in alcohol. Accordingly, SPS tends to copurify with DNA. An extraction method was designed for purification of DNA from blood culture media and removal of SPS. Blood culture media containing human blood and spiked with
Escherichia coli
was subjected to an organic extraction procedure with benzyl alcohol, and removal of SPS was documented spectrophotometrically. Successful amplification of the extracted
E. coli
16S rRNA gene was achieved by adding 5 μl of undiluted processed sample DNA to a 50-μl PCR mixture. When using other purification methods, the inhibitory effect of SPS could be overcome only by dilution of these samples. By our extraction technique, even uninoculated blood culture media were found to contain bacterial DNA when they were subjected to broad-range 16S rRNA gene consensus PCR. We conclude that the blood culture additive SPS is a potent inhibitor of PCR, is resistant to removal by traditional DNA purification methods, but can be removed by a benzyl alcohol extraction protocol that results in improved PCR performance.
Human Exposure to a Granulocytic Ehrlichia and Other Tick-Borne Agents in Connecticut
Authors: Magnarelli Louis A. , Ijdo Jacob W. , Anderson John F. , Padula Steven J. , Flavell Richard A. , Fikrig Erol 
Published: December 2019

ABSTRACT

Indirect fluorescent-antibody (IFA) staining methods with
Ehrlichia equi
(MRK or BDS strains) and Western blot analyses containing a human granulocytic ehrlichiosis (HGE) agent (NCH-1 strain) were used to confirm probable human cases of infection in Connecticut during 1995 and 1996. Also included were other tests for
Ehrlichia chaffeensis
, the agent of human monocytic ehrlichiosis (HME),
Babesia microti
, and
Borrelia burgdorferi
. Thirty-three (8.8%) of 375 patients who had fever accompanied by marked leukopenia or thrombocytopenia were serologically confirmed as having HGE. Western blot analyses of a subset of positive sera confirmed the results of the IFA staining methods for 15 (78.9%) of 19 seropositive specimens obtained from different persons. There was frequent detection of antibodies to a 44-kDa protein of the HGE agent. Serologic testing also revealed possible cases of Lyme borreliosis (
n
= 142), babesiosis (
n
= 41), and HME (
n
= 21). Forty-seven (26.1%) of 180 patients had antibodies to two or more tick-borne agents. Therefore, when one of these diseases is clinically suspected or diagnosed, clinicians should consider the possibility of other current or past tick-borne infections.
PCR for Detection and Identification of
Abiotrophia
spp
Authors: Roggenkamp Andreas , Leitritz Lorenz , Baus Kerstin , Falsen Enevold , Heesemann Jürgen 
Published: December 2019

ABSTRACT

Members of the genus
Abiotrophia
, formerly known as nutritionally variant streptococci, are important pathogens causing septicemia and endocarditis. Cultivation and biochemical differentiation of
Abiotrophia
spp. are often difficult. Based on 16S rRNA sequences, two PCR assays for detection and identification of
Abiotrophia
spp. were developed. The first PCR assay was positive for all
Abiotrophia
spp. Subsequently performed restriction fragment length polymorphism analysis allowed the verification of the PCR amplicons and the differentiation of the three species. The second PCR assay was positive only for
A. elegans
, the most fastidious species of
Abiotrophia
. Both PCR assays were shown to be specific and sensitive and should facilitate the identification of
Abiotrophia
spp.
Prolonged Replication of a Type 1 Vaccine-Derived Poliovirus in an Immunodeficient Patient
Authors: Kew Olen M. , Sutter Roland W. , Nottay Baldev K. , McDonough Michael J. , Prevots D. Rebecca , Quick Linda , Pallansch Mark A. 
Published: December 2019

ABSTRACT
VP1 sequences were determined for poliovirus type 1 isolates obtained over a 189-day period from a poliomyelitis patient with common variable immunodeficiency syndrome (a defect in antibody formation). The isolate from the first sample, taken 11 days after onset of paralysis, contained two poliovirus populations, differing from the Sabin 1 vaccine strain by ∼10%, differing from diverse type 1 wild polioviruses by 19 to 24%, and differing from each other by 5.5% of nucleotides. Specimens taken after day 11 appeared to contain only one major poliovirus population. Evolution of VP1 sequences at synonymous third-codon positions occurred at an overall rate of ∼3.4% per year over the 189-day period. Assuming this rate to be constant throughout the period of infection, the infection was calculated to have started ∼9.3 years earlier. This estimate is about the time (6.9 years earlier) the patient received his last oral poliovirus vaccine dose, approximately 2 years before the diagnosis of immunodeficiency. These findings may have important implications for the strategy to eliminate poliovirus immunization after global polio eradication.
Prospective Evaluation of Criteria for Microbiological Diagnosis of Prosthetic-Joint Infection at Revision Arthroplasty
Authors: Atkins Bridget L. , Athanasou Nicholas , Deeks Jonathan J. , Crook Derrick W. M. , Simpson Hamish , Peto Timothy E. A. , McLardy-Smith Peter , Berendt Anthony R. , Group The Osiris Collaborative Study 
Published: December 2019

ABSTRACT
A prospective study was performed to establish criteria for the microbiological diagnosis of prosthetic joint infection at elective revision arthroplasty. Patients were treated in a multidisciplinary unit dedicated to the management and study of musculoskeletal infection. Standard multiple samples of periprosthetic tissue were obtained at surgery, Gram stained, and cultured by direct and enrichment methods. With reference to histology as the criterion standard, sensitivities, specificities, and likelihood ratios (LRs) were calculated by using different cutoffs for the diagnosis of infection. We performed revisions on 334 patients over a 17-month period, of whom 297 were evaluable. The remaining 37 were excluded because histology results were unavailable or could not be interpreted due to underlying inflammatory joint disease. There were 41 infections, with only 65% of all samples sent from infected patients being culture positive, suggesting low numbers of bacteria in the samples taken. The isolation of an indistinguishable microorganism from three or more independent specimens was highly predictive of infection (sensitivity, 65%; specificity, 99.6%; LR, 168.6), while Gram staining was less useful (sensitivity, 12%; specificity, 98%; LR, 10). A simple mathematical model was developed to predict the performance of the diagnostic test. We recommend that five or six specimens be sent, that the cutoff for a definite diagnosis of infection be three or more operative specimens that yield an indistinguishable organism, and that because of its low level of sensitivity, Gram staining should be abandoned as a diagnostic tool at elective revision arthroplasty.
Multiplex PCR for Typing and Subtyping Influenza and Respiratory Syncytial Viruses
Authors: Stockton J. , Ellis J. S. , Saville M. , Clewley J. P. , Zambon M. C. 
Published: December 2019

ABSTRACT
A multiplex reverse transcription (RT)-PCR method that has been developed is capable of detecting and subtyping influenza A (H1N1 and H3N2) and B viruses as well as respiratory syncytial virus (RSV) types A and B in respiratory clinical samples taken as part of a national community-based surveillance program of influenza-like illness in England and Wales. The detection of each different pathogen depended on distinguishing five amplification products of different sizes on agarose gels following RT-PCR with multiple primer sets. The multiplex RT-PCR was tested with 65 nasopharyngeal apirates from which RSV had been isolated and 237 combined nose and throat swabs from which influenza A (H1N1 and H3N2) or B virus had been detected by virus isolation, as well as 40 respiratory samples from which other viruses including cytomegalovirus, herpes simplex virus, enteroviruses, and parainfluenza viruses had been grown. For the typing and subtyping of influenza A and B viruses and RSV types A and B, the multiplex RT-PCR gave an excellent (100%) correlation with the results of conventional typing and subtyping with specific antisera. Multiplex RT-PCR can also be used to accurately detect more than one viral template in the same reaction mixture, allowing viral coinfections to be identified with the same respiratory specimen.
Use of Molecular and Reference Susceptibility Testing Methods in a Multicenter Evaluation of MicroScan Dried Overnight Gram-Positive MIC Panels for Detection of Vancomycin and High-Level Aminoglycoside Resistances in Enterococci
Authors: Chen Yao-Shen , Marshall S. A. , Winokur P. L. , Coffman S. L. , Wilke W. W. , Murray P. R. , Spiegel C. A. , Pfaller M. A. , Doern G. V. , Jones R. N. 
Published: December 2019

ABSTRACT

Modified MicroScan gram-positive MIC no. 8 panels (PM-8) were analyzed for their improved ability to detect vancomycin resistance (VR) and high-level aminoglycoside resistance (HLAR) in enterococci. A validation study design that utilized selected challenge strains, recent clinical isolates, and reproducibility experiments in a multicenter format was selected. Three independent medical centers compared the commercial panels to reference broth microdilution panels (RBM) and Synergy Quad Agar (QA). Resistance was verified by demonstration of VR and HLAR genes by PCR tests. The study was conducted in three phases. (i) In the challenge phase (CP), two well-characterized sets of enterococci were obtained from the Centers for Disease Control and Prevention; one set contained 50 isolates for VR testing and one contained 48 isolates for HLAR testing. In addition, a set of 47 well-characterized isolates representing diverse geographic areas, obtained from earlier national surveillance studies, was tested at the University of Iowa College of Medicine (UICM). (ii) In the efficacy phase (EP), each laboratory tested 50 recent, unique clinical isolates by all methods. (iii) In the reproducibility Phase (RP), each laboratory tested the same 10 strains by all methods in triplicate on three separate days. All isolates from the EP were sent to the UICM for molecular characterization of
vanA
, -
B
, -

C
1

, -

C
2–3

, and HLAR genes. In the CP, the ranking of test methods by error rates (in parentheses; very major and major errors combined, versus PCR results) were as follows: for high-level streptomycin resistance (HLSR), QA (12.0%) > PM-8 (5.2%) > RBM (1.6%); for high-level gentamicin resistance (HLGR), RBM (3.7%) > PM-8 (3.1%) > QA (2.6%); and for VR, RBM = QA (3.0%) > PM-8 (1.2%). In the EP, agreement between all methods and the reference PCR result was 98.0% for HLSR, 99.3% for HLGR, and 98.6% for VR. In the RP, the percentages of results ± 1 log
2
dilution of the all-participant mode were as follows: for VR, 100% (PM-8), 98.9% (QA), and 90.0% (RBM); for HLSR, 99.6% (RBM), 98.5% (PM-8), and 82.2% (QA); and for HLGR, 99.6% (RBM), 99.3% (PM-8), and 98.1% (QA). The ability of the PM-8 to detect VR and HLAR in enterococci was comparable to those for reference susceptibility and molecular PCR methods and was considered acceptable for routine clinical laboratory use.
Detection of
Candida dubliniensis
in Oropharyngeal Samples from Human Immunodeficiency Virus-Infected Patients in North America by Primary CHROMagar Candida Screening and Susceptibility Testing of Isolates
Authors: Kirkpatrick William R. , Revankar Sanjay G. , Mcatee Robert K. , Lopez-Ribot Jose L. , Fothergill Annette W. , McCarthy Dora I. , Sanche Stephen E. , Cantu Rebecca A. , Rinaldi Michael G. , Patterson Thomas F. 
Published: December 2019

ABSTRACT

Candida dubliniensis
has been associated with oropharyngeal candidiasis in patients infected with human immunodeficiency virus (HIV).
C. dubliniensis
isolates may have been improperly characterized as atypical
Candida albicans
due to the phenotypic similarity between the two species. Prospective screening of oral rinses from 63 HIV-infected patients detected atypical dark green isolates on CHROMagar Candida compared to typical
C. albicans
isolates, which are light green. Forty-eight atypical isolates and three control strains were characterized by germ tube formation, differential growth at 37, 42, and 45°C, identification by API 20C, fluorescence, chlamydoconidium production, and fingerprinting by Ca3 probe DNA hybridization patterns. All isolates were germ tube positive. Very poor or no growth occurred at 42°C with 22 of 51 isolates. All 22 poorly growing isolates at 42°C and one isolate with growth at 42°C showed weak hybridization of the Ca3 probe with genomic DNA, consistent with
C. dubliniensis
identification. No
C. dubliniensis
isolate but only 18 of 28
C. albicans
isolates grew at 45°C. Other phenotypic or morphologic tests were less reliable in differentiating
C. dubliniensis
from
C. albicans
. Antifungal susceptibility testing showed fluconazole MICs ranging from ≤0.125 to 64 μg/ml. Two isolates were resistant to fluconazole (MIC, 64 μg/ml) and one strain was dose dependent susceptible (MIC, 16 μg/ml). MICs of other azoles, including voriconazole, itraconazole, and SCH 56592, for these isolates were lower.
C. dubliniensis
was identified in 11 of 63 (17%) serially evaluated patients. Variability in phenotypic characteristics dictates the use of molecular and biochemical techniques to identify
C. dubliniensis
. This study identifies
C. dubliniensis
in HIV-infected patients from San Antonio, Tex., and shows that
C. dubliniensis
is frequently detected in those patients by using a primary CHROMagar screen.
Direct Detection of Respiratory Syncytial Virus, Parainfluenza Virus, and Adenovirus in Clinical Respiratory Specimens by a Multiplex Reverse Transcription-PCR Assay
Authors: Osiowy Carla 
Published: December 2019

ABSTRACT

Diagnosis of respiratory virus infections currently involves detection by isolation or antigen detection, which usually identifies only a single suspected agent. To permit identification of more than one respiratory virus in clinical specimens, a rapid detection method involving a single-step, multiplex reverse transcription-PCR (RT-PCR) assay was developed. The assay included five primer sets that amplified the RNA of respiratory syncytial virus subtypes A and B, parainfluenza virus types 1, 2, and 3, and adenovirus types 1 to 7. Initially the assay was tested on tissue culture-grown virus and was found to be specific for all 12 prototype viruses tested, with no interassay cross amplification or amplification of other respiratory viruses. Assay sensitivity allowed a detection range of 0.2 50% tissue culture infectious dose (TCID
50
) for adenovirus to 250 TCID
50
for parainfluenza virus type 1. The multiplex RT-PCR assay was also able to directly detect viruses in respiratory specimens, with virus being detected in 41 of 112 samples as compared to 34 of 112 samples detected by direct immunofluorescence or antigen detection following specimen culture. This suggests that the multiplex RT-PCR assay can be used as a rapid and sensitive diagnostic method for major respiratory viruses.
Comparison of Phenotypic and Genotypic Methods for Detection of Diphtheria Toxin among Isolates of Pathogenic Corynebacteria
Authors: Efstratiou Androulla , Engler Kathryn H. , Dawes Charlotte S. , Sesardic Dorothea 
Published: December 2019

ABSTRACT

We have compared molecular, immunochemical, and cytotoxic assays for the detection of diphtheria toxin from 55 isolates of
Corynebacterium diphtheriae
and
Corynebacterium ulcerans
originally isolated in five different countries. The suitabilities and accuracies of these assays for the laboratory diagnosis of diphtheria were compared and evaluated against the “gold standard” in vivo methods. The in vivo and Vero cell cytotoxicity assays were accurate in their abilities to detect diphtheria toxin but were time-consuming; however, the cytotoxicity assay is a suitable in vitro alternative to the in vivo virulence test. There was complete concordance between all the phenotypic methods. Genotypic tests based upon PCR were rapid; however, PCR must be used with caution because some isolates of
C. diphtheriae
possessed toxin genes but failed to express a biologically active toxin. Therefore, phenotypic confirmation of toxigenicity for the microbiological diagnosis of diphtheria is recommended.