bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid Identification and Differentiation ofBartonellaSpecies Using a Single-Step PCR Assay
Authors: Jensen Wayne A. , Fall Majilinde Z. , Rooney Jane , Kordick Dorsey L. , Breitschwerdt Edward B.
Published: December 2019

Five species ofBartonellahave been reported to infect humans and cause a variety of diseases that can be difficult to diagnose. Four species ofBartonellahave been reported to infect cats and dogs, and two of these species are considered to be zoonotic pathogens. Diagnosis ofBartonellainfections is hampered by the slow, fastidious growth characteristics ofBartonellaspecies. We report on the development of a single-step PCR-based assay for the detection and differentiation of medically relevantBartonellaspecies. PCR-mediated amplification of the 16S-23S rRNA intergenic region resulted in a product of a unique size for eachBartonellaspecies, thereby allowing differentiation without the necessity of restriction fragment length polymorphism analysis or sequencing of the amplified product. The ability of the single-step PCR assay to differentiate betweenBartonellaspecies was determined with characterized isolates and blood samples from animals known to be infected with eitherBartonella henselae,B. clarridgeiae, orB. vinsoniisubsp.berkhoffii. The sensitivity of the single-step PCR assay relative to that of in vitro culture was determined with blood samples fromB. henselae-infected cats.B. henselaetarget DNA was amplified from 100% of samples with greater than 50 CFU/ml and 80% of samples with 10 to 30 CFU/ml. The single-step assay described in the report expedites PCR-based detection and differentiation of medically relevantBartonellaspecies.
Identification of a Novel DNA Probe for Strain Typing
Mycobacterium bovis
by Restriction Fragment Length Polymorphism Analysis
Authors: O'Brien Rory , Flynn Orla , Costello Eamon , O'Grady Don , Rogers Mark
Published: December 2019

Bovine tuberculosis caused by
Mycobacterium bovis
remains a significant disease of farmed cattle in many countries despite ongoing tuberculosis eradication programs. Molecular typing methods such as restriction fragment length polymorphism (RFLP) analysis and spoligotyping have been used to identify related herd breakdowns in an attempt to identify more precisely the route of infection into cattle herds and to trace the transmission of bovine tuberculosis. A recent geographical survey of Irish
M. bovis
isolates demonstrated that a significant proportion of isolates (∼20%) exhibit a common strain type, limiting the value of current strain typing methods as an epidemiological tool. We have identified and cloned a region of the
M. bovis
genome, pUCD, which generates a clear, highly polymorphic banding pattern when used as an RFLP probe on
Alu
I restriction-digested
M. bovis
genomic DNA and which effectively subdivides this common strain type. When used to type 60 Irish
M. bovis
isolates, pUCD exhibited greater discriminatory power than the commonly used mycobacterial RFLP probes IS
6110
, PGRS, and DR and detected an equivalent number of strain types to a combination of these three probes. pUCD also detected significantly more strain types than the spoligotyping technique, while maintaining a high level of concordance between epidemiologically related and unrelated herd breakdowns. The polymorphic element within pUCD remains to be fully characterized, however the potential for this probe to greatly decrease the workload necessary to genotype
M. bovis
by RFLP analysis is compelling.
Oxygen and Carbon Dioxide Regulation of Toxic Shock Syndrome Toxin 1 Production by
Staphylococcus aureus
MN8
Authors: Yarwood Jeremy M. , Schlievert Patrick M.
Published: December 2019

The production of toxic shock syndrome toxin 1 (TSST-1) by
Staphylococcus aureus
MN8 exposed to a range of oxygen concentrations (0 to 21% [vol/vol]) was examined in batch and thin-film cultures. The response of
S. aureus
to this range of oxygen concentrations was studied in the absence and in the presence of 7% (vol/vol) carbon dioxide. In the absence of carbon dioxide, TSST-1 production in batch cultures increased from negligible levels in the presence of oxygen concentrations of 1% or less to 500 ng/ml in the presence of 2% oxygen and then decreased to 70 ng/ml or less in the presence of oxygen concentrations of 6% and higher. In the presence of carbon dioxide, however, toxin production increased from negligible levels in the presence of 1% oxygen to 1,900 ng/ml in the presence of 21% oxygen. In thin-film cultures, TSST-1 production increased from nearly undetectable levels under anaerobic conditions to 1 and 10 μg/ml under 21% oxygen in the absence and presence of carbon dioxide, respectively. This study demonstrates the controlling effects of both oxygen and carbon dioxide on TSST-1 production.
Molecular Differentiation of Seven
Malassezia
Species
Authors: Gupta Aditya K. , Kohli Yatika , Summerbell Richard C.
Published: December 2019

A system based on PCR and restriction endonuclease analysis was developed to distinguish the seven currently recognized
Malassezia
species. Seventy-eight strains, including authentic culture collection strains and routine clinical isolates, were investigated for variation in the ribosomal DNA repeat units. Two genomic regions, namely, the large subunit of the ribosomal gene and the internal transcribed spacer (ITS) region, were amplified by PCR, and products were digested with restriction endonucleases. The patterns generated were useful in identification of five out of seven
Malassezia
species.
M. sympodialis
was readily distinguishable in that its ITS region yielded a 700-bp amplified fragment, whereas the other six species yielded an 800-bp fragment.
M. globosa
and
M. restricta
were very similar in the regions studied and could be distinguished only by performing a hot start-touchdown PCR on primers for the β-tubulin gene. Primers based on the conserved areas of the
Candida cylindracea
lipase gene, which were used in an attempt to amplify
Malassezia
lipases, yielded an amplification product after annealing at 55°C only with
M. pachydermatis
. This specific amplification may facilitate the rapid identification of this organism.
Computer-Assisted Analysis and Epidemiological Value of Genotyping Methods for
Campylobacter jejuni
and
Campylobacter coli
Authors: de Boer Paulo , Duim Birgitta , Rigter Alan , van der Plas Jan , Jacobs-Reitsma Wilma F. , Wagenaar Jaap A.
Published: December 2019

For epidemiological tracing of the thermotolerant
Campylobacter
species
C. jejuni
and
C. coli
, reliable and highly discriminatory typing techniques are necessary. In this study the genotyping techniques of flagellin typing (
flaA
typing), pulsed-field gel electrophoresis (PFGE), automated ribotyping, and amplified fragment length polymorphism (AFLP) fingerprinting were compared. The following aspects were compared: computer-assisted analysis, discriminatory power, and use for epidemiological typing of campylobacters. A set of 50 campylobacter poultry isolates from The Netherlands and neighboring countries was analyzed. Computer-assisted analysis made cluster analysis possible and eased the designation of different genotypes. AFLP fingerprinting was the most discriminatory technique, identifying 41 distinct genotypes, while PFGE identified 38 different types,
flaA
typing discriminated 31 different types, and ribotyping discriminated 26 different types. Furthermore, AFLP analysis was the most suitable method for computer-assisted data analysis. In some cases combining the results of AFLP fingerprinting, PFGE, and
flaA
typing increased our ability to differentiate strains that appeared genetically related. We conclude that AFLP is a highly discriminatory typing method and well suited for computer-assisted data analysis; however, for optimal typing of campylobacters, a combination of multiple typing methods is needed.
Sensitive Method for Detection of Human Herpesviruses 6 and 7 in Saliva Collected in Field Studies
Authors: Zerr Danielle M. , Huang Meei-Li , Corey Lawrence , Erickson Matthew , Parker Heather L. , Frenkel Lisa M.
Published: December 2019

To facilitate studies of the epidemiology and natural history of human herpesviruses 6 and 7 in infants, a practical method for collecting and quantifying the DNA of these viruses was developed. Saliva was collected using small strips of filter paper, and virus was detected using a real-time quantitative fluorescent-probe PCR assay. The sensitivity and specificity of this method even after prolonged drying of the specimens compared favorably to those of our traditional method of collecting and assaying saliva.
Growth Supplements for
Helicobacter pylori
Authors: Jiang Xiuping , Doyle Michael P.
Published: December 2019

The growth response of
Helicobacter pylori
in broth was determined in the presence of ferrous sulfate, sodium pyruvate, and mucin (porcine stomach). The addition of either ferrous sulfate and sodium pyruvate or mucin to brain heart infusion broth with 7% horse serum (BHI-HS) enhanced the growth of
H. pylori
. The best growth of strain NB2-1, which was the slowest growing of 10
H. pylori
strains evaluated, occurred in the presence of 0.05% ferrous sulfate and 0.05% sodium pyruvate. The addition of 0.3% mucin to BHI-HS reduced the lag time of
H. pylori
by 48 h and enhanced the growth. On the basis of the results for 10
H. pylori
strains, the combination of ferrous sulfate (0.025%), sodium pyruvate (0.025%), and mucin (0.15%) in BHI-HS counteracted the inhibitory effects of the antibiotics used in culture media for selective growth of
H. pylori
. Results suggest that these supplements may be useful for enhancement of the growth of
H. pylori
in enrichment media.
A Three-Year Study of
Campylobacter jejuni
Genotypes in Humans with Domestically Acquired Infections and in Chicken Samples from the Helsinki Area
Authors: Hänninen Marja-Liisa , Perko-Mäkelä Päivikki , Pitkälä Anna , Rautelin Hilpi
Published: December 2019

Campylobacter jejuni
isolates from stool samples of patients with domestically acquired sporadic infections and from chicken from retail shops were studied during seasonal peaks from June to September over a 3-year period from 1996 to 1998. A large number of pulsed-field gel electrophoresis (PFGE) genotypes (a combined
Sma
I-
Sac
II pattern) were identified each year. Certain genotypes persisted for the whole study period, and predominant genotypes represented 28 to 52% of the strains during a restricted period of time. The peak level of positive chicken samples was between July and August of each study year, when 10 to 33% of the samples were positive for campylobacter. The same PFGE genotypes found in humans were also detected in the chicken samples. This suggests that common genotypes were circulating in the area.
Brucella abortus
Infection Acquired in Microbiology Laboratories
Authors: Fiori Pier Luigi , Mastrandrea Scilla , Rappelli Paola , Cappuccinelli Piero
Published: December 2019

We report an outbreak of laboratory-acquired
Brucella abortus
infection originating in the accidental breakage of a centrifuge tube. A total of 12 laboratory workers were infected (attack rate of 31%), with an incubation time ranging from 6 weeks to 5 months. Antibody titers were evaluated weekly in all personnel exposed, allowing the diagnosis of the infection in most cases before the onset of clinical symptoms, so that specific therapy could be administrated.
Infective Endocarditis Complicated with Progressive Heart Failure due to β-Lactamase-Producing
Cardiobacterium hominis
Authors: Lu Po-Liang , Hsueh Po-Ren , Hung Chien-Ching , Teng Lee-Jene , Jang Tsrang-Neng , Luh Kwen-Tay
Published: December 2019

We describe a 66-year-old woman with infective endocarditis due to
Cardiobacterium hominis
whose condition, complicated by severe aortic regurgitation and congestive heart failure, necessitated aortic valve replacement despite treatment with ceftriaxone followed by ciprofloxacin. The blood isolate of
C. hominis
produced β-lactamase and exhibited high-level resistance to penicillin (MIC, ≧256 μg/ml) and reduced susceptibility to vancomycin (MIC, 8 μg/ml).
Genetic Characterization of Type 2 Porcine Circovirus (PCV-2) from Pigs with Postweaning Multisystemic Wasting Syndrome in Different Geographic Regions of North America and Development of a Differential PCR-Restriction Fragment Length Polymorphism Assay To Detect and Differentiate between Infections with PCV-1 and PCV-2
Authors: Fenaux Martijn , Halbur Patrick G. , Gill Mike , Toth Thomas E. , Meng Xiang-Jin
Published: December 2019

Postweaning multisystemic wasting syndrome (PMWS) is an emerging disease in swine. Increasing evidence indicates that a variant strain of porcine circovirus (PCV), designated type 2 PCV (PCV-2), is responsible for PMWS. To determine the extent of genetic heterogeneity of PCV-2 isolates, the complete genomes of six PCV-2 isolates from different regions of North America were amplified by PCR and sequenced. Sequence and phylogenetic analyses confirmed that two distinct genotypes of PCV exist: nonpathogenic genotype PCV-1 and PMWS-associated genotype PCV-2. However, within the PCV-2 genotype, several minor branches that have been identified appear to be associated with geographic origins. The genomic sequences of two French PCV-2 isolates diverge the most from those of other PCV-2 isolates and form a distinct branch. Other minor but distinguishable branches have also been identified for a Taiwan PCV-2 isolate and two of the Canadian PCV-2 isolates. All the U.S. PCV-2 isolates are closely related, but the Canadian isolates vary, to some extent, in their genomic sequences. The data from this study indicate that although the genome of PCV-2 is generally stable among different isolates, PCV-2 isolates from different geographic regions vary in their genomic sequences. This variation may have important implications for PCV-2 diagnosis and research. On the basis of genetic analyses of available PCV strains, a universal PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed to detect and differentiate between infections with PCV-1 and PCV-2. This PCR-RFLP assay should be useful for studying the pathogenesis of PCV-2, for detecting PCV-2 infection in pigs from different geographic regions, and for screening donor pigs for use in xenotransplantation.
Helicobacter canadensis
sp. nov. Isolated from Humans with Diarrhea as an Example of an Emerging Pathogen
Authors: Fox James G. , Chien Chih Ching , Dewhirst Floyd E. , Paster Bruce J. , Shen Zeli , Melito Pasquale L. , Woodward David L. , Rodgers Frank G.
Published: December 2019

We recently analyzed 11 helicobacter isolates cultured from diarrhea patients in Canada. These isolates had been characterized biochemically by restriction fragment length polymorphism (RFLP;
Alu
I,
Hha
I) analysis and by fatty-acid analysis as
Helicobacter pullorum
. However, four of the isolates differed biochemically from
H. pullorum
by their inability to hydrolyze indoxyl acetate and their resistance to nalidixic acid. Using complete 16S rRNA analysis, we determined that these four strains clustered near
H. pullorum
but had a sequence difference of 2% and therefore represent a novel helicobacter,
Helicobacter canadensis
. This novel helicobacter could also be distinguished from
H. pullorum
by RFLP analysis using
Apa
LI. The number of novel
Helicobacter
spp. associated with gastrointestinal disease in humans and animals is rapidly increasing. There are now six
Helicobacter
spp. isolated from diarrheic humans, the other five being
H. pullorum
,
H. canis
, “
H. rappini
,”
H. fennelliae
, and
H. cinaedi
. This finding highlights the importance of careful molecular analysis in addition to standard biochemical tests in identifying the increasing number of
Helicobacter
spp. isolated from humans and animals.
Failure To Detect
Chlamydia pneumoniae
in the Late-Onset Alzheimer's Brain
Authors: Ring Robert H. , Lyons Joseph M.
Published: December 2019

Epidemiological studies have yet to identify a single cause for the most common late-onset form of Alzheimer's disease. The common respiratory pathogen
Chlamydia pneumoniae
recently has been implicated as a risk factor for this form of Alzheimer's disease. Were this true, there would be a dramatic shift in current paradigms of Alzheimer's disease research and treatment. In the absence of published confirmation, we obtained postmortem brain tissue from late-onset Alzheimer's disease patients (
n
= 15) and representative controls (
n
= 5) and extracted DNA from up to six separate brain regions in each instance, including those areas particularly relevant to Alzheimer's disease neuropathology. Each sample of DNA (
n
= 101) was assayed five times or more for the presence of
C. pneumoniae
DNA using a nested-PCR protocol targeting a species-specific gene sequence coding for the major outer membrane protein of this organism. We were unable unequivocally to detect
C. pneumoniae
in any of the 101 samples tested by PCR and failed to culture the organism from tissue samples. We conclude that
C. pneumoniae
is neither strongly nor uniquely associated with the neuropathology seen in late-onset Alzheimer's disease.
Detection of Herpes Simplex Virus DNA by Real-Time PCR
Authors: Kessler Harald H. , Mühlbauer Gerhard , Rinner Beate , Stelzl Evelyn , Berger Annemarie , Dörr Hans-Wilhelm , Santner Brigitte , Marth Egon , Rabenau Holger
Published: December 2019

Molecular detection of herpes simplex virus (HSV) DNA is recognized as the reference standard assay method for the sensitive and specific diagnosis of central nervous system infections caused by HSV. In this study, a molecular assay based on real-time PCR on the LightCycler (LC) instrument was evaluated and compared with a home-brew molecular assay. The detection limit of the LC assay was determined with 10-fold dilutions of plasmid pS4 with the
Sal
I restriction fragment of the DNA polymerase gene and with the First European Union Concerted Action HSV Proficiency Panel. A total of 59 cerebrospinal fluid (CSF) specimens were investigated for the comparative study. With plasmid pS4, the detection limit of the LC assay was found to be 10
4
copies per ml, i.e., 12.5 copies per run. When samples of the First European Union Concerted Action HSV Proficiency Panel were tested, 2 × 10
3
to 5 × 10
3
HSV type 1 genome equivalents (GE) per ml, i.e., 2.5 to 6.3 GE per run, could consistently be detected. There was a correlation between the LC assay and the home-brew assay in 55 of 59 specimens. In conclusion, the LC assay allows very rapid detection of HSV DNA in CSF. It was found to be laborsaving and showed sufficient sensitivity.
Comparison of Two Amplification Technologies for Detection and Quantitation of Human Immunodeficiency Virus Type 1 RNA in the Female Genital Tract
Authors: Bremer James , Nowicki Marek , Beckner Suzanne , Brambilla Donald , Cronin Mike , Herman Steven , Kovacs Andrea , Reichelderfer Patricia
Published: December 2019

Human immunodeficiency virus type 1 (HIV-1) RNA levels in female genital tract and peripheral blood samples were compared using two commercial amplification technologies: the Roche AMPLICOR HIV-1 MONITOR test and either the Organon Teknika nucleic acid sequence-based amplification (NASBA-QT) assay or the NucliSens assay. Estimates of HIV-1 RNA copy number were derived from internal kit standards and analyzed unadjusted and adjusted to a common set of external standards. We found a discordance rate of approximately 18% between the two technologies for the detection of HIV-1 in either the genital tract or peripheral blood samples. Detection discordance was not consistent among specimens or among women. There were no significant differences in adjusted or unadjusted estimates of HIV-1 RNA copy number in the genital tract samples using the AMPLICOR HIV-1 MONITOR test and either the NASBA-QT assay or the NucliSens assay. In addition, the estimated HIV-1 RNA copy number in peripheral blood samples did not differ when tested with the NucliSens assay and the AMPLICOR HIV-1 MONITOR test using kit standards. However, there was a significant difference in estimated RNA copy number between the NASBA-QT assay and the AMPLICOR HIV-1 MONITOR test for internal kit standards, which, as we have previously shown, was eliminated after adjustment with the external standards. Our results suggest that the Roche and Organon Teknika assays are equivalent for quantifying HIV-1 RNA in female genital tract specimens, although variation in detection does exist.
Genetic Characteristics ofBorrelia coriaceaeIsolates from the Soft TickOrnithodoros coriaceus(Acari: Argasidae)
Authors: Hendson Mavis , Lane Robert S.
Published: December 2019

TwoBorreliaisolates (CA434 and CA435) cultured from the soft tickOrnithodoros coriaceuswere analyzed by contour-clamped homogeneous electric field gel electrophoresis of unrestricted andApaI-restricted DNA, standard electrophoresis ofBamHI- andHindIII-restricted DNA, Southern hybridization, restriction fragment length polymorphism and sequencing of the 16S rRNA gene, and amplification of the 5S-23S intergenic spacer region. These isolates were compared withBorrelia coriaceaetype strain Co53,B. burgdorferisensu stricto strain CA4, and the relapsing-fever spirocheteB. parkeri(undesignated). The 16S rRNA region of CA434 and CA435 differed from that ofB. coriaceaetype strain Co53 by the presence of 1 base (C) at position 367 (GenBank accession no.U42286). The linear plasmid profile of CA434 was similar to that of Co53, and theApaI,BamHI, andHindIII restriction fingerprints of the total cellular DNA of CA434 and Co53 were similar. In contrast, CA435 differed somewhat from CA434 and Co53, which demonstrates thatB. coriaceaeis genetically diverse. Southern hybridization showed that the DNAs of CA434 and CA435 hybridized strongly with the digoxigenin-labeled DNA of Co53. Low homology was found between the DNA of Co53 and that ofB. parkeri. The 16S rRNA sequence ofB. parkeriwas identical to previously published results forB. parkeristrain M3001 (GenBank accession numberU42296). CA434 and CA435 represent only the second and third isolates ofB. coriaceaeobtained from any source since its initial isolation from anO. coriaceustick in 1985. All threeB. coriaceaeisolates were derived from adult ticks collected from the same locality in northwestern California. Difficulties encountered in detectingB. coriaceaein, and isolating this spirochete from, the tissues ofO. coriaceusare discussed. The lack of concordance between different detection or isolation methods suggests that reliance upon a single technique may grossly underestimate the true prevalence of spirochetal infection in wild-caughtO. coriaceusticks.
PCR Amplification and Phylogenetic Analysis of
groESL
Operon Sequences from
Ehrlichia ewingii
and
Ehrlichia muris
Authors: Sumner John W. , Storch Gregory A. , Buller Richard S. , Liddell Allison M. , Stockham Steven L. , Rikihisa Yasuko , Messenger Sharon , Paddock Christopher D.
Published: December 2019

Broad-range PCR primers were used to amplify part of the
groESL
operon of the canine pathogen
Ehrlichia ewingii
, recently recognized as a human pathogen, and the murine pathogen
Ehrlichia muris
. Phylogenetic analysis supported the relationships among
Ehrlichia
species previously determined by comparison of 16S rRNA gene sequences. These sequences provide additional PCR targets for species for which few gene sequences have been determined.
Comparison of Polymorphism of Thymidine Kinase Gene and Restriction Fragment Length Polymorphism of Genomic DNA in Herpes Simplex Virus Type 1
Authors: Nagamine Masayoshi , Suzutani Tatsuo , Saijo Masayuki , Hayashi Kozaburo , Azuma Masanobu
Published: December 2019

The polymorphism of the thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1) was analyzed and was compared with the restriction fragment length polymorphism (RFLP) of the whole genome to evaluate the relative efficiency of the TK gene as a potential probe for identification and discrimination of HSV-1. The effectiveness of using the polymorphism of the TK gene in classifying HSV-1 strains was comparable to that of RFLP analysis of 66 sites, suggesting that TK gene sequencing may have important applications in epidemiological studies of HSV-1.
Evaluation of the Binax and Biotest Urinary Antigen Kits for Detection of Legionnaires' Disease Due to Multiple Serogroups and Species of
Legionella
Authors: Benson Robert F. , Tang Patrick W. , Fields Barry S.
Published: December 2019

The Binax and the Biotest urinary antigen kits for the detection of Legionnaires' disease caused by organisms other than
Legionella pneumophila
were compared by testing 45 urine samples from non-
Legionella pneumophila
serogroup 1 patients previously positive in a broad-spectrum enzyme-linked immunosorbent assay (ELISA). Eighteen were positive with the Binax kit, and 13 were positive with the Biotest. Although neither kit is as sensitive as ELISA, these results extend the number of serogroups and species of
Legionella
that can be diagnosed with the Binax or Biotest kit.
Recurrent Infections and Chronic Colonization by an
Escherichia coli
Clone in the Respiratory Tract of a Patient with Severe Cystic Bronchiectasis
Authors: Wang Jann-Yuan , Hsueh Po-Ren , Wang Jann-Tay , Lee Li-Na , Yang Pan-Chyr , Luh Kwen-Tay
Published: December 2019

A 39-year-old woman with cystic bronchiectasis had repeated pulmonary infections from 1996 to 1999, and 6 of a total of 28 isolates of
Escherichia coli
from sputum specimens were studied. Their identical antibiotype and randomly amplified polymorphic DNA patterns indicated a single clone of
E. coli
, which persistently colonized the respiratory tract, causing recurrent infections.
Characterization of Glycopeptide-Resistant
Enterococcus faecium
(GRE) from Broilers and Pigs in Denmark: Genetic Evidence that Persistence of GRE in Pig Herds Is Associated with Coselection by Resistance to Macrolides
Authors: Aarestrup Frank Møller
Published: December 2019

Glycopeptide-resistant enterococci (GRE) from broilers and pigs were characterized to investigate the background for the persistence of GRE in pig herds. All porcine isolates belonged to closely related pulsed-field gel electrophoretic (PFGE) types, with the
ermB
and
vanA
genes located on the same transferable genetic element. Broiler isolates belonged to different PFGE types. The persistence of GRE in Danish pig herds after the ban of glycopeptides may be explained by the genetic link between
ermB
and
vanA
and coselection by use of macrolides for treatment and growth promotion.
Performance of Three Enzyme Immunoassays and Two Direct Fluorescence Assays for Detection of
Giardia lamblia
in Stool Specimens Preserved in ECOFIX
Authors: Fedorko Daniel P. , Williams Esther C. , Nelson Nancy A. , Calhoun Leslie B. , Yan Sizhuang S.
Published: December 2019

ECOFIX is a single-vial stool preservative that is both formalin- and mercury-free. We evaluated the abilities of three commercial
Giardia lamblia
-specific enzyme immunoassays (EIAs) (ProSpecT
Giardia
Microplate Assay [Alexon-Trend Inc.], Giardia Test [Techlab], and Premier
Giardia lamblia
[Meridian Diagnostics, Inc.]) and two commercial direct fluorescent-antibody (FA) assays for
G. lamblia
(Crypto/Giardia IF Test [Techlab] and Merifluor Cryptosporidium/Giardia [Meridian Diagnostics, Inc.]) to detect
G. lamblia
in 34
G. lamblia
-positive and 44
G. lamblia
-negative stool specimens (determined by traditional examination for ova and parasites) preserved in ECOFIX compared to their abilities to detect
G. lamblia
in the same specimens preserved in formalin as the “gold standard” for each assay. Of the 34 formalin-fixed positive specimens, the number detected by each assay was as follows:, Alexon EIA, 34; Meridian EIA, 27; Techlab EIA, 29; Meridian FA assay, 31; and Techlab FA assay, 28. Both FA tests and the Alexon EIA performed well with ECOFIX, but the other two EIAs detected fewer positive specimens (the difference was statistically significant with the Techlab EIA) when ECOFIX was the preservative. Use of
G. lamblia
cyst antigen from cultured organisms preserved in formalin and ECOFIX demonstrated that the Alexon EIA could detect smaller amounts of antigen in ECOFIX than the other two EIAs could and suggested that cyst antigen is more stable in formalin. We recommend that laboratories use an FA assay or the Alexon EIA if they plan to use ECOFIX as their stool preservative.
Clinical and Financial Benefits of Rapid Detection of Respiratory Viruses: an Outcomes Study
Authors: Barenfanger Joan , Drake Cheryl , Leon Nidia , Mueller Tina , Troutt Tammy
Published: December 2019

To assess the expected benefits of rapid reporting of respiratory viruses, we compared patients whose samples were processed using standard techniques such as enzyme immunoassays, shell vial assays, and culture tube assays (year 1) to patients whose samples were processed with the same standard techniques in addition to immunofluorescent testing (FA) directly on cytocentrifuged samples (year 2). The cytospin FA screened for influenza A and B viruses, respiratory syncytial virus (RSV), parainfluenza viruses 1 to 3, and adenovirus (DAKO Diagnostics Ltd.). The specificity of the cytospin FA for all viruses was 100%. The sensitivities for influenza A virus and RSV were 90 and 98%, respectively, but the sensitivities for influenza B virus and adenovirus were unacceptable (14.3 and 0%, respectively). However, since the former viruses account for >85% of our isolates from clinical specimens, the cytospin FA is an excellent screening test since the positive result was available within hours. The mean turnaround time for all positive viruses was 4.5 days in year 1 and 0.9 day in year 2 (
P
= 0.001). This rapid reporting resulted in physicians having access to information sooner, enabling more appropriate treatment. The mean length of stay in the hospital for inpatients with respiratory viral isolates was 10.6 days for year 1 versus 5.3 days for year 2. Mean variable costs for these patients was $7,893 in year 1 and $2,177 in year 2. After subtracting reagent costs and technological time, the savings in variable costs was $144,332/year. Summarizing, the cytospin FA markedly decreased turnaround time and was associated with decreased mortality, length of stay, and costs and with better antibiotic stewardship.
Detection of Significant Bacteriuria by Automated Urinalysis Using Flow Cytometry
Authors: Okada Hiroshi , Sakai Yutaka , Miyazaki Shigenori , Arakawa Soichi , Hamaguchi Yukio , Kamidono Sadao
Published: December 2019

A new flow cytometry-based automated urine analyzer, the UF-50, was evaluated for its ability to screen urine samples for significant bacteriuria. One hundred eighty-six urine specimens from patients attending an outpatient clinic of a university-based hospital were examined. The results obtained with the UF-50 were compared with those obtained by conventional quantitative urine culture. The UF-50 detected significant bacteriuria with a sensitivity of 83.1%, a specificity of 76.4%, a positive predictive value of 62.0%, a negative predictive value of 90.7%, and an accuracy of 78.5%. These results are comparable to those obtained by previously reported screening procedures. Besides detecting significant bacteriuria, the UF-50 can also perform routine urinalysis, including measurement of concentrations of red blood cells, white blood cells, epithelial cells, and casts, within 70 s. This capability renders this new flow cytometry-based urine analyzer superior to previously reported rapid screening methods.
Enteric Virus Infections and Diarrhea in Healthy and Human Immunodeficiency Virus-Infected Children
Authors: Liste Mary B. , Natera Ivelisse , Suarez José A. , Pujol Flor H. , Liprandi Ferdinando , Ludert Juan E.
Published: December 2019

Forty-three stool samples from 27 human immunodeficiency virus (HIV)-seropositive children and 38 samples from 38 HIV-negative children, collected during a 15-month period, were examined for enteric viruses. Diagnostic assays included enzyme immunoassays for rotavirus, adenovirus, and Norwalk virus; polyacrylamide gel electrophoresis for picobirnavirus and atypical rotavirus; and PCR for astrovirus and enterovirus. Specimens from HIV-positive children were more likely than those of HIV-negative children to have enterovirus (56 versus 21%;
P
< 0.0002) and astrovirus (12 versus 0%;
P
< 0.02), but not rotavirus (5 versus 8%;
P
> 0.5). No adenoviruses, picobirnaviruses, or Norwalk viruses were found. The rates of virus-associated diarrhea were similar among HIV-positive and HIV-negative children. Enteroviruses were excreted for up to 6 months in HIV-positive children; however, no evidence for prolonged excretion of poliovirus vaccine was observed. These results suggest that although infection with enterovirus and astrovirus may be frequent in HIV-infected children, enteric viruses are not associated with the diarrhea frequently suffered by these children.
Use of the Neisserial Lipoprotein (Lip) for Subtyping
Neisseria gonorrhoeae
Authors: Trees David L. , Schultz Abbigail J. , Knapp Joan S.
Published: December 2019

The pathogenic
Neisseria
species
N. meningitidis
and
N. gonorrhoeae
possess an outer membrane lipoprotein, designated Lip, which is present in all strains tested. The predicted protein sequence of Lip consists of a consensus AAEAP amino acid repeat. The objective of this study was to determine the feasibility of using the Lip repeat number and sequence for subtyping of
Neisseria gonorrhoeae
. The
lip
genes of each isolate were amplified by PCR and sequenced to determine the repeat number and sequence. Among the 46 strains we examined, eight different Lip repeat numbers were identified, with lengths of 11 (1 strain), 12 (14 strains), 13 (2 strains), 14 (10 strains), 15 (5 strains), 16 (10 strains), 17 (3 strains), and 20 (1 strain) repeats. Analysis indicated differences in the sequences within the repeats that resulted in amino acid alterations in repeat classes that contained multiple strains. Among the 46 isolates examined, we were able to identify 17 unique Lip subtyping patterns.
Real-Time PCR for Diagnosis and Follow-Up of Toxoplasma Reactivation after Allogeneic Stem Cell Transplantation Using Fluorescence Resonance Energy Transfer Hybridization Probes
Authors: Costa J.-M. , Pautas C. , Ernault P. , Foulet F. , Cordonnier C. , Bretagne S.
Published: December 2019

Toxoplasma reactivation is a life-threatening complication of allogeneic stem cell transplantation. A poor prognosis is probably linked to a difficult diagnosis, based on the detection of evidence of parasites in tissue. We developed a real-time PCR test using fluorescence resonance energy transfer hybridization probes to detect and quantify
Toxoplasma gondii
DNA in serum. This PCR test gave reproducible quantitative results over a dynamic range of from 0.75 × 10
6
to 0.75 parasites per PCR mixture. Serial samples from four patients with toxoplasma reactivation were evaluated. Three patients had several consecutive PCR-positive samples which corresponded to ≤0.75 parasites. These three patients became PCR negative during trimethoprim-sulfamethoxazole therapy but never developed clinically apparent toxoplasmosis. In contrast, one patient had an increasing PCR signal, from 1 to 396 parasites in 12 days, and developed cerebral symptoms. The parasite count decreased to 5 parasites in 3 days after pyrimethamine-clindamycin treatment. Real-time quantitative PCR is useful for diagnosis and follow-up of toxoplasma reactivation.
Detection of
Paracoccidioides brasiliensis
in Tissue Samples by a Nested PCR Assay
Authors: Bialek Ralf , Ibricevic Aida , Aepinus Christian , Najvar Laura K. , Fothergill Annette W. , Knobloch Jürgen , Graybill John R.
Published: December 2019

A nested PCR assay for the detection of
Paracoccidioides brasiliensis
DNA was evaluated, using a sequence of the immunogenic
gp43
gene as a target. This gene encodes an outer membrane protein unique to this dimorphic fungus. DNA from six clinical isolates and the ATCC strain 60885 of
P. brasiliensis
, as well as DNA from closely related fungi, was examined to determine detection limits and cross-reactions. PCR was done on DNA extracts of lung homogenates from 23 experimentally
P. brasiliensis
-infected and two uninfected BALB/c mice and from 20
Histoplasma capsulatum
-infected ICR mice. The results were compared to quantitative cultures. A detection limit of 0.5 fg of specific DNA was determined using cloned plasmid DNA. In all seven
P. brasiliensis
isolates, the expected 196-bp nested PCR product was found. Their sequences were 100% identical to the
gp43
gene sequence in GenBank. DNA extracts of all other, related fungi were negative. The PCR assay was positive in 21 out of 23 culture-positive lung homogenates with concentrations of 1 × 10
3
to 1.3 × 10
7
CFU of
P. brasiliensis
per g of lung. Uninfected BALB/c mice and
H. capsulatum
-infected mice samples gave negative results. The high sensitivity and specificity of this new nested PCR assay for the detection of
P. brasiliensis
in tissue samples were demonstrated. The assay may be useful for diagnosis in human tissue samples.
Species Identification of Mycobacteria by PCR-Restriction Fragment Length Polymorphism of the
rpoB
Gene
Authors: Lee Hyeyoung , Park Hee-Jung , Cho Sang-Nae , Bai Gill-Han , Kim Sang-Jae
Published: December 2019

PCR-restriction fragment length polymorphism analysis (PRA) using the novel region of the
rpoB
gene was developed for rapid and precise identification of mycobacteria to the species level. A total of 50 mycobacterial reference strains and 3 related bacterial strains were used to amplify the 360-bp region of
rpoB
, and the amplified DNAs were subsequently digested with restriction enzymes such as
Msp
I and
Hae
III. The results from this study clearly show that most of the mycobacterial species were easily differentiated at the species level by this PRA method. In addition, species with several subtypes, such as
Mycobacterium gordonae
,
M. kansasii
,
M. celatum
, and
M. fortuitum
, were also differentiated by this PRA method. Subsequently, an algorithm was constructed based on the results, and a blinded test was carried out with more than 260 clinical isolates that had been identified on the basis of conventional tests. Comparison of these two sets of results clearly indicates that this new PRA method based on the
rpoB
gene is more simple, more rapid, and more accurate than conventional procedures for differentiating mycobacterial species.
Genetic Characterization of Toxin A-Negative, Toxin B-Positive
Clostridium difficile
Isolates by PCR
Authors: Moncrief J. Scott , Zheng Limin , Neville Laurie M. , Lyerly David M.
Published: December 2019

Toxin-specific enzyme immunoassays, cytotoxicity assays, and PCR were used to analyze 48 toxin A-negative, toxin B-positive
Clostridium difficile
isolates from various geographical sites around the world. All the isolates were negative by the TOX-A TEST and positive by the TOX A/B TEST. A deletion of approximately 1.7 kb was found at the 3′ end of the
toxA
gene for all the isolates, similar to the deletion in toxinotype VIII strains (e.g.,
C. difficile
serotype F 1470). Additional PCR analysis indicated that the toxin B encoded by these isolates contains sequence variations downstream of the active site compared to the sequence of reference strain VPI 10463. This variation may extend the glucosylation spectrum to Ras proteins, as observed previously for closely related lethal toxin from
Clostridium sordellii
and toxin B from toxin A-negative, toxin B-positive strain F 1470. Toxin A-negative, toxin B-positive isolates have recently been associated with disease in humans, and they may be more common than was previously supposed.
A Molecular Epidemiological Study of
Mycobacterium simiae
Isolated from AIDS Patients in Guadeloupe
Authors: Legrand Eric , Devallois Anne , Horgen Lionel , Rastogi Nalin
Published: December 2019

A molecular epidemiological study of
Mycobacterium simiae
strains isolated from AIDS patients in Guadeloupe was performed by the random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) of
Dra
I- or
Xba
I-digested bacterial DNAs. A comparison of RAPD profiles suggested a similarity of banding patterns within a group of patients (two clusters of two and three patients), but the available epidemiological and clinical information did not support this finding. PFGE, on the other hand, showed that all the patients were contaminated with individual isolates. Combined numerical analysis performed by compiling the PFGE patterns obtained after
Xba
I and
Dra
I digestions of bacterial DNAs suggested the occurrence of polyclonal infection in three of nine patients. Our results do not support a common source of
M. simiae
infection in Guadeloupe.
Simple and Reliable Multiplex PCR Assay for Surveillance Isolates of Vancomycin-Resistant Enterococci
Authors: Kariyama Reiko , Mitsuhata Ritsuko , Chow Joseph W. , Clewell Don B. , Kumon Hiromi
Published: December 2019

Conditions have been optimized for the use of a multiplex PCR for the detection of vancomycin-resistant enterococci in nosocomial surveillance specimens. Seven primer sets targeting the genes
vanA
,
vanB
,
vanC1
,
vanC2/C3 Enterococcus faecalis
-specific,
Enterococcus faecium
-specific, and
rrs
(16S rRNA) were used in one reaction tube. The PCR method developed in the present study is simple and reliable for the rapid characterization of vancomycin-resistant enterococci.
Use of 5-Bromo-4-Chloro-3-Indolyl-α-
d
-
N
-Acetylneuraminic Acid in a Novel Spot Test To Identify Sialidase Activity in Vaginal Swabs from Women with Bacterial Vaginosis
Authors: Wiggins R. , Crowley T. , Horner P. J. , Soothill P. W. , Millar M. R. , Corfield A. P.
Published: December 2019

The validity of measuring vaginal sialidase activity to identify bacterial vaginosis (BV) was determined by using 5-bromo-4-chloro-3-indolyl-α-
d
-
N
-acetylneuraminic acid in a near-patient test. The sensitivity and specificity of the test for prediction of BV were 95.6 and 96.3%, respectively. Positive and negative predictive values were 95.6 and 96.3%, respectively. This test may be an alternative to Gram staining.
DNA-Based Diagnostic Approaches for Identification of Burkholderia cepacia Complex, Burkholderia vietnamiensis, Burkholderia multivorans,Burkholderia stabilis, and Burkholderia cepacia Genomovars I and III
Authors: Mahenthiralingam Eshwar , Bischof Jocelyn , Byrne Sean K. , Radomski Christopher , Davies Julian E. , Av-Gay Yossef , Vandamme Peter
Published: December 2019

Bacteria of the Burkholderia cepacia complex consist of five discrete genomic species, including genomovars I and III and three new species: Burkholderia multivorans (formerly genomovar II), Burkholderia stabilis (formerly genomovar IV), andBurkholderia vietnamiensis (formerly genomovar V). Strains of all five genomovars are capable of causing opportunistic human infection, and microbiological identification of these closely related species is difficult. The 16S rRNA gene (16S rDNA) and recAgene of these bacteria were examined in order to develop rapid tests for genomovar identification. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rDNA revealed sequence polymorphisms capable of identifying B. multivorans andB. vietnamiensis but insufficient to discriminate strains of B. cepacia genomovars I and III and B. stabilis. RFLP analysis of PCR-amplified recAdemonstrated sufficient nucleotide sequence variation to enable separation of strains of all five B. cepacia complex genomovars. Complete recA nucleotide sequences were obtained for 20 strains representative of the diversity of the B. cepacia complex. Construction of a recA phylogenetic tree identified six distinct clusters (recA groups):B. multivorans, B. vietnamiensis, B. stabilis, genomovar I, and the subdivision of genomovar III isolates into two recA groups, III-A and III-B. Alignment of recA sequences enabled the design of PCR primers for the specific detection of each of the six latter recA groups. The recA gene was found on the largest chromosome within the genome of B. cepacia complex strains and, in contrast to the findings of a previous study, only a single copy of the gene was present. In conclusion, analysis of the recA gene of theB. cepacia complex provides a rapid and robust nucleotide sequence-based approach to identify and classify this taxonomically complex group of opportunistic pathogens.
Cutaneous Microenvironment of Human Immunodeficiency Virus (HIV)-Seropositive and HIV-Seronegative Individuals, with Special Reference to Staphylococcus aureus Colonization
Authors: Shapiro Michael , Smith Kathleen J. , James William D. , Giblin Walter J. , Margolis David J. , Foglia Arlene N. , McGinley Kenneth , Leyden James J.
Published: December 2019

A cross-sectional quantitative study of cutaneous bacterial and yeast flora at seven body sites in 99 human immunodeficiency virus-seropositive and 50 seronegative military personnel was performed. Statistically significant differences in carriage rates were only observed for Staphylococcus aureus on the foreheads of seropositive individuals. Seronegative individuals demonstrated staphylococcal carriage rates 1.3 to 2 times as great as those of historical controls (defined as healthy individuals not receiving any medications) at five of six body sites. We conclude that seropositive military personnel do not exhibit statistically significant elevations in densities and carriage rates of the microorganisms examined (exceptStaphylococcus aureus), relative to seronegative individuals. Seropositive individuals may be predisposed to staphylococcal carriage. The elevated staphylococcal carriage rates of military personnel undergoing basic training warrants a formal evaluation of the impact of training exercises on cutaneous flora. The information gained may serve to limit the spread of infection during training exercises and battlefield conditions.
Phenotypic and Genotypic Diversity of the Flagellin Gene (fliC) among Clostridium difficile Isolates from Different Serogroups
Authors: Tasteyre Albert , Karjalainen Tuomo , Avesani Véronique , Delmée Michel , Collignon Anne , Bourlioux Pierre , Barc Marie-Claude
Published: December 2019

Phenotypic and genotypic diversity of the flagellin gene (fliC) of Clostridium difficile was studied in 47 isolates from various origins belonging to the serogroups A, B, C, D, F, G, H, I, K, X, and S3. Electron microscopy revealed 17 nonflagellated strains and 30 flagellated strains. PCR and reverse transcription-PCR demonstrated that the flagellin gene was present in all strains and that the fliC gene was expressed in both flagellated and nonflagellated strains. Southern blotting showed the presence of only one copy of the gene and three different hybridization patterns. DNA sequence analysis of fliC from the strains belonging to serogroups C, D, and X, representative of each profile, disclosed great variability in the central domain, whereas the N- and C-terminal domains were conserved. The variability of the flagellin gene fliC was further studied in the isolates by PCR-restriction fragment length polymorphism (RFLP) analysis. Nine different RFLP groups were identified (I to IX), among which three (I, VII, and VIII) corresponded to numerous serogroups whereas the six others (II, III, IV, V, VI, and IX) belonged to a single serogroup. Flagellin gene RFLP analysis could constitute an additional typing method employable in conjunction with other typing methods currently available.