bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in enteric neural circuitry and smooth muscle in the inflamed and infected gut
Authors: Mawe G. M. , Collins S. M. , Shea‐Donohue T.
Published: April 2004

Much of the morbidity associated with inflammatory bowel disease (IBD) and infection is caused by disordered gastrointestinal motor and secretory functions. Given that intestinal smooth muscle tone and epithelial cell secretion are regulated by the enteric nervous system (ENS), it is quite likely that inflammation‐induced changes in the enteric neural circuitry contribute to intestinal dysmotility and diarrhoea. Indeed, discoveries over the past decades have demonstrated that gut inflammation and infections are associated with changes in key elements all along the enteric neural circuitry from the sensory transducers, the enterochromaffin (EC) cells, to the terminals of motor neurones.
Use of Quantitative 16S Ribosomal DNA Detection for Diagnosis of Central Vascular Catheter-Associated Bacterial Infection
Authors: Warwick S. , Wilks M. , Hennessy E. , Powell-Tuck J. , Small M. , Sharp J. , Millar M. R.
Published: April 2004

Many
central vascular catheters (CVCs) are removed unnecessarily because
current diagnostic methods for CVC-associated infection are unreliable.
A quantitative PCR assay using primers and probe targeted to bacterial
16S ribosomal DNA was used to measure the levels of bacterial DNA in
blood samples drawn through the CVC in a population of patients
receiving intravenous nutrition. Bacterial DNA concentrations were
raised in 16 of 16 blood samples taken during episodes of probable
bacterial CVC-associated infection. Bacterial DNA concentrations were
raised in 4 of 29 episodes in which bacterial CVC-associated infection
was unlikely. The use of this technique has the potential to
substantially reduce the unnecessary removal of
CVCs.
Simultaneous Detection of Marine Fish Pathogens by Using Multiplex PCR and a DNA Microarray
Authors: González Santiago F. , Krug Melissa J. , Nielsen Michael E. , Santos Ysabel , Call Douglas R.
Published: April 2004

We coupled multiplex PCR and a DNA microarray to construct an assay suitable for the simultaneous detection of five important marine fish pathogens (
Vibrio vulnificus
,
Listonella anguillarum
,
Photobacterium damselae
subsp.
damselae
,
Aeromonas salmonicida
subsp.
salmonicida
, and
Vibrio parahaemolyticus
). The array was composed of nine short oligonucleotide probes (25-mer) complementary to seven chromosomal loci (
cyt
,
rpoN
,
gyrB
,
toxR
,
ureC
,
dly
, and
vapA
) and two plasmid-borne loci (
fatA
and A.sal). Nine primer sets were designed to amplify short fragments of these loci (100 to 177 bp) in a multiplex PCR. PCR products were subsequently labeled by nick translation and hybridized to the microarray. All strains of the five target species (
n
= 1 to 21) hybridized to at least one species-specific probe. Assay sensitivities ranged from 100% for seven probes to 83 and 67% for the two remaining probes. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria (
n
= 40 strains; 100% specificity). Using purified genomic DNA, we were able to detect PCR products with <20 fg of genomic DNA per reaction (equivalent to four or five cells), and the array was at least fourfold more sensitive than agarose gel electrophoresis for detecting PCR products. In addition, our method allowed the tentative identification of virulent strains of
L. anguillarum
serotype O1 based on the presence of the
fatA
gene (67% sensitivity and 100% specificity). This assay is a sensitive and specific tool for the simultaneous detection of multiple pathogenic bacteria that cause disease in fish and humans.
Study of Genotypes andvirB4Secretion Gene ofBartonella henselaeStrains from Patients with Clinically Defined Cat Scratch Disease
Authors: Woestyn Sophie , Olivé Nathalie , Bigaignon Geoffroy , Avesani Véronique , Delmée Michel
Published: April 2004

Bartonella henselaeis the causative agent of cat scratch disease (CSD), which usually presents as a self-limiting lymphadenopathy. Occasionally, the bacteria will spread and be responsible for tissue and visceral involvement. TwoB. henselaegenotypes (genotypes I and II) have been described to be responsible for uncomplicated CSD on the basis of 16S rRNA sequence analysis. A type IV secretion system (T4SS) similar to the virulence-associated VirB system ofAgrobacterium tumefacienswas recently identified in theB. henselaeHouston-1 genotype I strain. We studied the correlations of theB. henselaegenotypes with the clinical presentations and with the presence of T4SS. Isolates originated from CSD patients whose lymph nodes were prospectively analyzed.B. henselaegenotype I was identified in 13 of 42 patients (30%). Among these, two teenage twins presented with hepatosplenic CSD and one immunocompetent adult presented with osteomyelitis. Genotype II was detected in 28 of 42 patients (67%), all of whom presented with uncomplicated CSD. The last patient was infected with both genotypes. T4SS was studied by PCR amplification of thevirB4gene. Amplification ofvirB4codons 146 to 256, 273 to 357, and 480 to 537 enabled us to detect 66, 90, and 100% of theB. henselaeisolates, respectively. Sequence analysis revealed sequence variations that correlated with genotype distribution. Our studies suggest thatB. henselaegenotype I strains harborvirB4genes that are different from those harbored by genotype II strains and that genotype I strains might be more pathogenic.
Use of the BD PHOENIX Automated Microbiology System for Direct Identification and Susceptibility Testing of Gram-Negative Rods from Positive Blood Cultures in a Three-Phase Trial
Authors: Funke Guido , Funke-Kissling Pascale
Published: April 2004

The present study describes the use of the automated BACTEC 9240 blood culture system, the Serum Separator Tube (SST), and the BD PHOENIX Automated Microbiology System in combination for the direct identification and antimicrobial susceptibility testing (AST) of gram-negative rods (GNRs) from positive blood cultures (BCs) without subculture. The study was conducted in three phases: (i) the recovery yield of
Escherichia coli
ATCC 25922 was determined with the SST between 0 and 8 h after spiked BC bottles turned positive; (ii) the identifications and susceptibility testing results obtained with the PHOENIX system for nine American Type Culture Collection strains of GNRs processed by the SST procedure and for colonies from agar medium were compared; and (iii) the procedure with the BACTEC system, SSTs, and the PHOENIX system was applied to positive cultures of blood from 309 patients during a 3-month period. The SST procedure with
E. coli
yielded sufficient numbers of cells to perform direct inoculation at any time between 0 and 8 h after a BC bottle turned positive. By using the identities obtained from pure cultures with the PHOENIX system and other biochemical identification systems as reference methods, the agreement between the reference methods and the PHOENIX system tested directly by using cultures of blood from patients was 92.9%. The 7.1% discrepant results were due to 6.5% incorrect identifications with the PHOENIX system with BC samples and 0.6% incorrect identifications with the PHOENIX system with samples from agar cultures. By AST the overall categorical accuracy was 99.0%, with 0.1% very major errors, 0.1% major errors, and 0.8% minor errors. In conclusion, use of the combination of the BACTEC system, SSTs, and the PHOENIX system has the potential to allow the agar isolation step to be skipped and the procedures for rapid direct identification and susceptibility testing of GNRs from positive BCs to be improved both in hospital-based and in central non-hospital-based laboratories.
Performance and Cost Evaluation of One Commercial andSix In-House Conventional and Real-Time ReverseTranscription-PCR Assays for Detection of Severe AcuteRespiratory SyndromeCoronavirus
Authors: Mahony James B. , Petrich Astrid , Louie Lisa , Song Xinyu , Chong Sylvia , Smieja Marek , Chernesky Max , Loeb Mark , Richardson Susan
Published: April 2004

We
evaluated seven reverse transcription-PCR (RT-PCR) assays, including
six in-house assays and one commercial assay for the detection of
severe acute respiratory syndrome coronavirus (SARS-CoV) RNA in
clinical specimens. RT-PCR assays targeted different genomic regions
and included three conventional assays (one nested and two non-nested)
run on a conventional heat block and four real-time assays performed in
a LightCycler (LC; Roche Diagnostics). All in-house assays were
optimized for assay parameters, including MgCl
2
, primer, and
probe concentrations. The commercial assay was the RealArt HPA CoV
RT-PCR assay (Artus), which was run in the LC. Testing serial dilutions
of cultured SARS-CoV showed that the analytical sensitivity of the
assays ranged from 10
−8
to 10
−6
,
corresponding to 1 and 100 copies of viral RNA, respectively.
Significant differences in analytical sensitivities were observed
between assays (
P
< 0.01, probit regression analysis
for 50% sensitivity levels for the top two assays versus the
others). Testing 68 clinical specimens (including 17 respiratory tract
specimens, 29 urine samples, and 22 stools or rectal swabs)
demonstrated that six of the seven assays detected at least 17 of 18
positives (defined as positive in at least two assays), and two of the
assays had a sensitivity of 100%. There were no significant
differences in sensitivity between the assays (
P
= 0.5
[Cochrance Q test, least sensitive 15 of 18 versus 18 of
18]). The specificities of the assays ranged from 94.0 to
100% without significant differences (
P
= 0.25
to 0.5 [McNemar test]). The reagent and technologist cost of
performing the in-house PCR assays ranged from $5.46 to $9.81 Canadian
dollars (CDN) per test. The commercial assay cost was
considerably higher at $40.37 per test. The results demonstrated good
performance for all assays, providing laboratories that need to do SARS
RNA testing with a choice of assay
formats.
Analysis of Polymorphic Microsatellite Markers for Typing
Penicillium marneffei
Isolates
Authors: Lasker Brent A. , Ran Yuping
Published: April 2004

Penicillium marneffei
is an emerging opportunistic dimorphic fungal pathogen that is endemic in Southeast Asia. A typing method based on the analysis of size polymorphisms in microsatellite loci was investigated. Three loci available from the GenBank database were identified to harbor microsatellites. PCR primers flanking the microsatellite repeats were designed with one primer in the set fluorescently labeled. PCR products were then sized by automated capillary electrophoresis. As expected for a haploid fungus, a single band was observed for each microsatellite locus for all isolates. Polymorphic microsatellite marker (PMM) analysis detected a total of 22 different allelic types for 35 isolates of
P. marneffei
with a high discriminatory power (
D
= 0.956). Microsatellites I, II, and III detected 14, 10, and 7 alleles, respectively. The reproducibility of length polymorphisms was confirmed by using different DNA preparations from the same isolate or by repeated runs from the same DNA preparation. PMM profiles for eight isolates passaged in vitro for 7 to 8 weeks were identical to the original culture, demonstrating short-term stability and reproducibility. PCR products were not observed for other dimorphic fungi or human DNA. Comparison of allelic frequencies in isolates obtained from China and Thailand identified distinct allele combinations, suggesting the potential geographic isolation of populations. Due to the high discriminatory power, reproducibility, and potential for high throughput, PMM analysis may provide a good typing method for epidemiologic and surveillance investigations of
P. marneffei.
Epidemiology and Clinical Course of
Burkholderia cepacia
Complex Infections, Particularly Those Caused by Different
Burkholderia cenocepacia
Strains, among Patients Attending an Italian Cystic Fibrosis Center
Authors: Manno Graziana , Dalmastri Claudia , Tabacchioni Silvia , Vandamme Peter , Lorini Renata , Minicucci Laura , Romano Luca , Giannattasio Alessandro , Chiarini Luigi , Bevivino Annamaria
Published: April 2004

In this study, the epidemiology of
Burkholderia cepacia
complex (Bcc) recovered from the sputum of 75 patients attending the Genoa Cystic Fibrosis (CF) Center at the Gaslini Children's Hospital (Genoa, Italy) was investigated, and the clinical course of the CF patients infected with the different species and genomovars of Bcc was evaluated. All isolates were analyzed for genomovar status by
recA
gene polymorphism and subsequently random amplified polymorphic DNA fingerprinting.
Burkholderia cenocepacia
is the predominant species recovered from the CF patients infected with Bcc at the Genoa CF Center. Of the other eight species comprising the Bcc, only a few isolates belonging to
B. cepacia
genomovar I,
Burkholderia stabilis
, and
Burkholderia pyrrocinia
were found. Of the four
recA
lineages of
B. cenocepacia
, most patients were infected by epidemic strains belonging to lineages IIIA and IIID, whereas only a few patients harbored IIIB strains. Patient-to-patient spread of Bcc among CF patients was mostly associated with
B. cenocepacia
, in particular with strains belonging to
recA
lineages IIIA and IIID. The mortality of CF patients infected with Bcc at the Genoa CF Center was significantly higher than mortality among CF patients not infected with Bcc. All of the deaths were associated with the presence of
B. cenocepacia
, except the case of a patient infected with
B. cepacia
genomovar I. Within
B. cenocepacia
, infection with epidemic strains belonging to lineages IIIA and IIID was associated with higher rates of mortality than was infection with lineage IIIB strains. No significant differences in lung function, body weight, and mortality rate were observed between patients infected with epidemic strains belonging to either
B. cenocepacia
IIIA or
B. cenocepacia
IIID.
SYBR Green-Based Real-Time Quantitative PCR Assay for Detection of West Nile Virus Circumvents False-Negative Results Due to Strain Variability
Authors: Papin James F. , Vahrson Wolfgang , Dittmer Dirk P.
Published: April 2004

Real-time quantitative PCR is used routinely for the high-throughput diagnosis of viral pathogens, such as West Nile virus (WNV). Rapidly evolving RNA viruses present a challenge for diagnosis because they accumulate mutations that may render them undetectable. To explore the effect of sequence variations on assay performance, we generated every possible single point mutation within the target region of the widely used TaqMan assay for WNV and found that the TaqMan assay failed to detect 47% of possible single nucleotide variations in the probe-binding site and was unable to detect any targets with more than two mutations. In response, we developed and validated a less expensive assay with the intercalating dye SYBR green. The SYBR green-based assay was as sensitive as the TaqMan assay for WNV. Importantly, it detected 100% of possible WNV target region variants. The assay developed here adds an additional layer of protection to guard against false-negative results that result from natural variations or drug-directed selection and provides a rapid means to identify such variants for subsequent detailed analysis.
Incidence of Bloodstream Infections Due to
Candida
Species and In Vitro Susceptibilities of Isolates Collected from 1998 to 2000 in a Population-Based Active Surveillance Program
Authors: Hajjeh Rana A. , Sofair Andre N. , Harrison Lee H. , Lyon G. Marshall , Arthington-Skaggs Beth A. , Mirza Sara A. , Phelan Maureen , Morgan Juliette , Lee-Yang Wendy , Ciblak Meral A. , Benjamin Lynette E. , Thomson Sanza Laurie , Huie Sharon , Yeo Siew Fah , Brandt Mary E. , Warnock David W.
Published: April 2004

To determine the incidence of
Candida
bloodstream infections (BSI) and antifungal drug resistance, population-based active laboratory surveillance was conducted from October 1998 through September 2000 in two areas of the United States (Baltimore, Md., and the state of Connecticut; combined population, 4.7 million). A total of 1,143 cases were detected, for an average adjusted annual incidence of 10 per 100,000 population or 1.5 per 10,000 hospital days. In 28% of patients,
Candida
BSI developed prior to or on the day of admission; only 36% of patients were in an intensive care unit at the time of diagnosis. No fewer than 78% of patients had a central catheter in place at the time of diagnosis, and 50% had undergone surgery within the previous 3 months.
Candida albicans
comprised 45% of the isolates, followed by
C. glabrata
(24%),
C. parapsilosis
(13%), and
C. tropicalis
(12%). Only 1.2% of
C. albicans
isolates were resistant to fluconazole (MIC, ≥64 μg/ml), compared to 7% of
C. glabrata
isolates and 6% of
C. tropicalis
isolates. Only 0.9% of
C. albicans
isolates were resistant to itraconazole (MIC, ≥1 μg/ml), compared to 19.5% of
C. glabrata
isolates and 6% of
C. tropicalis
isolates. Only 4.3% of
C. albicans
isolates were resistant to flucytosine (MIC, ≥32 μg/ml), compared to <1% of
C. parapsilosis
and
C. tropicalis
isolates and no
C. glabrata
isolates. As determined by E-test, the MICs of amphotericin B were ≥0.38 μg/ml for 10% of
Candida
isolates, ≥1 μg/ml for 1.7% of isolates, and ≥2 μg/ml for 0.4% of isolates. Our findings highlight changes in the epidemiology of
Candida
BSI in the 1990s and provide a basis upon which to conduct further studies of selected high-risk subpopulations.
Comparison of Multiplex PCR Assay with Culture for Detection of Genital Mycoplasmas
Authors: Stellrecht Kathleen A. , Woron Amy M. , Mishrik Nada G. , Venezia Richard A.
Published: April 2004

Ureaplasma
, spp.
Mycoplasma genitalium
, and
Mycoplasma hominis
are associated with infection of the genitourinary tract, reproductive failure, and neonatal morbidity and mortality. We have developed a multiplex PCR for the detection of these
Mycoplasmatales
in a single amplification reaction. The analytical sensitivities of this assay were 10.8, 10.8, and 8.8 CFU for each organism, respectively. This multiplex PCR was compared to culture on 26 cervical swabs, 2 vaginal swabs, 4 female urine specimens, 49 semen samples, 2 male urine specimens, and 1 nonspecified sample. A total of 21 specimens were culture positive (25%); 17 of these were PCR positive. An additional 11 specimens were PCR positive but culture negative. Of the 21 culture-positive specimens, 17 (81%) grew
Ureaplasma
spp. and 4 (19%) grew
Mycoplasma
spp. Of the 28 PCR-positive specimens,
Ureaplasma
spp. was detected in 23 (82%),
M. hominis
was detected in 3 (11%), and both were detected in 2 (7%). In a confirmatory analysis, all samples were tested by amplification of a second target of the ureaplasma genome. True-positive cases were defined as a positive result by culture or by both amplification assays. The multiplex PCR detected organisms in 26 of the 30 true-positive specimens, as well as in 2 other specimens. Based on a 36% prevalence of infection, the sensitivity, specificity, and positive and negative predictive values of multiplex PCR analyses were 87, 96, 94, and 93%, respectively. Multiplex PCR offers a rapid, sensitive, and easy method to detect genital mycoplasmas.
Development of a Nucleic Acid Sequence-Based Amplification Assay That Uses
gag
-Based Molecular Beacons To Distinguish between Human Immunodeficiency Virus Type 1 Subtype C and C′ Infections in Ethiopia
Authors: Ayele Workenesh , Pollakis Georgios , Abebe Almaz , Fisseha Bitew , Tegbaru Belete , Tesfaye Girma , Mengistu Yohannes , Wolday Dawit , van Gemen Bob , Goudsmit Jaap , Dorigo-Zetsma Wendelien , de Baar Michel P.
Published: April 2004

A
gag
-based molecular beacon assay utilizing real-time nucleic acid sequence-based amplification technology has been developed to differentiate between the two genetic subclusters of human immunodeficiency virus type 1 (HIV-1) subtype C (C and C′) circulating in Ethiopia. Of 41 samples, 36 could be classified as C or C′ by sequencing of the
gag
gene. All 36 isolates were correctly identified by the
gag
beacon test. Three isolates with genomes that were recombinant in
gag
were unambiguously typed as belonging to the C′ subcluster. Further analysis revealed that these contained the most sequence homology with a reference subcluster C′ sequence in the target region of the beacon and hence were correct for the analyzed region. For one sample, sequencing and
gag
molecular beacon results did not match, while another isolate could not be detected at all by the beacon assay. Overall, high levels of sensitivity and specificity were achieved for both beacons (90.5% sensitivity and 100% specificity for the C beacon and 100% sensitivity and 95.2% specificity for the C′ beacon). The availability of a diagnostic test which can quickly and reliably discriminate between C and C′ HIV-1 infections in Ethiopia is an important first step toward studying their respective biological characteristics. As the assay is specific to the Ethiopian HIV-1 subtype C epidemic, it will contribute to characterizing the circulating viruses in this population, thereby generating the information necessary for the development of a potential efficacious HIV-1 vaccine appropriate for the Ethiopian context.
Detection of Multiple Macrolide- and Lincosamide-Resistant Strains of
Streptococcus pyogenes
from Patients in the Boston Area
Authors: Hasenbein Meredith E. , Warner John E. , Lambert Kathleen G. , Cole Sarah E. , Onderdonk Andrew B. , McAdam Alexander J.
Published: April 2004

Macrolide (including erythromycin and azithromycin) and lincosamide (including clindamycin) antibiotics are recommended for treatment of penicillin-allergic patients with
Streptococcus pyogenes
pharyngitis. Resistance to erythromycin in
S. pyogenes
can be as high as 48% in specific populations in the United States. Macrolide and lincosamide resistance in
S. pyogenes
is mediated by several different genes. Expression of the
erm
(A) or
erm
(B) genes causes resistance to erythromycin and inducible or constitutive resistance to clindamycin, respectively, whereas expression of the
mef
(A) gene leads to resistance to erythromycin but not clindamycin. We studied the resistance of
S. pyogenes
to erythromycin and clindamycin at an urban tertiary-care hospital. Of 196 sequential isolates from throat cultures, 15 (7.7%) were resistant to erythromycin. Three of these were also constitutively resistant to clindamycin and had the
erm
(B) gene. Five of the erythromycin-resistant isolates were resistant to clindamycin upon induction with erythromycin and had the
erm
(A) gene. The remaining seven erythromycin-resistant isolates were susceptible to clindamycin even upon induction with erythromycin and had the
mef
(A) gene. Pulsed-field gel electrophoresis analysis and
emm
typing demonstrated that the erythromycin-resistant
S. pyogenes
comprised multiple strains. These results demonstrate that multiple mechanisms of resistance to macrolide and lincosamide antibiotics are present in
S. pyogenes
strains in the United States.
Rapid and Sensitive Method Using Multiplex Real-Time PCR for Diagnosis of Infections by Influenza A and Influenza B Viruses, Respiratory Syncytial Virus, and Parainfluenza Viruses 1, 2, 3, and 4
Authors: Templeton Kate E. , Scheltinga Sitha A. , Beersma Matthias F. C. , Kroes Aloys C. M. , Claas Eric C. J.
Published: April 2004

Laboratory diagnosis of viral respiratory infections is generally performed by virus isolation in cell culture and immunofluorescent assays. Reverse transcriptase PCR is now recognized as a sensitive and specific alternative for detection of respiratory RNA viruses. A rapid real-time multiplex PCR assay was developed for the detection of influenza A and influenza B viruses, human respiratory syncytial virus (RSV), parainfluenza virus 1 (PIV1), PIV2, PIV3, and PIV4 in a two-tube multiplex reaction which used molecular beacons to discriminate the pathogens. A total of 358 respiratory samples taken over a 1-year period were analyzed by the multiplex assay. The incidence of respiratory viruses detected in these samples was 67 of 358 (19%) and 87 of 358 (24%) by culture and real-time PCR, respectively. Culture detected 3 influenza A virus, 2 influenza B virus, 57 RSV, 2 PIV1, and 2 PIV3 infections. All of these culture-positive samples and an additional 5 influenza A virus, 6 RSV, 2 PIV1, 1 PIV2, 1 PIV3, and 3 PIV4 infections were detected by the multiplex real-time PCR. The application of real-time PCR to clinical samples increases the sensitivity for respiratory viral diagnosis. In addition, results can be obtained within 6 h, which increases clinical relevance. Therefore, use of this real-time PCR assay would improve patient management and infection control.
Immunological Characterization of the Spike Protein of the Severe Acute Respiratory Syndrome Coronavirus
Authors: Lu Liqun , Manopo Ivanus , Leung Bernard P. , Chng Hiok Hee , Ling Ai Ee , Chee Li Lian , Ooi Eng Eong , Chan Shzu-Wei , Kwang Jimmy
Published: April 2004

Severe acute respiratory syndrome (SARS) is a novel infectious disease caused by the SARS-associated coronavirus (SARS-CoV). There are four major structural proteins in the SARS-CoV, including the nucleocapsid, spike, membrane, and small envelope proteins. In this study, two sets of truncated fragments of spike protein were generated, the first were approximately 210-bp nonoverlapping fragments and the second were overlapping segments of 750 to 900 bp. From these 23 fragments, we identified a fragment of 259 amino acids (amino acids 441 to 700) that is a major immunodominant epitope. This fragment was highly expressed, and the purified fragment C could detect all 33 SARS patient serum samples tested, collected from 7 to 60 days after the onset of fever, but had no reactivity with all 66 healthy human serum samples tested. Thus, fragment C of spike protein was identified as an immunodominant antigen and could be used for serological detection of SARS-CoV infection.
Molecular Diagnosis of Human Adenoviruses D and E by a Phylogeny-Based Classification Method Using a Partial Hexon Sequence
Authors: Shimada Yasushi , Ariga Toshihide , Tagawa Yoshitsugu , Aoki Koki , Ohno Shigeaki , Ishiko Hiroaki
Published: April 2004

Human adenoviruses (HAdVs) are the major causes of a variety of acute illnesses. Virus isolation and neutralization tests are usually done to identify the causative virus, but these tests are labor-intensive and time-consuming, and standardized antisera are in limited supply. This study investigated a rapid and reliable method of virus identification based on PCR and phylogenetic analysis. The phylogenetic tree constructed by neighbor joining on the basis of the newly determined partial hexon sequences from 33 prototypes of HAdV-D and -E, along with 11 available prototypes of HAdV-A to -C and -F from GenBank, allowed HAdVs to be grouped into six distinct clusters. These clusters correspond closely to the six newly designated species, HAdV-A to -F. The partial hexon sequences of 57 isolates from patients with acute conjunctivitis obtained over 20 years plus those of 44 prototype strains were analyzed. Each isolate formed a monophyletic cluster along with its respective prototype strain, allowing serotype identification. Partial-hexon-based classification appears to be an effective tool for studying the molecular epidemiology of HAdVs.
Immunoblot Method To Detect
Streptococcus pneumoniae
and Identify Multiple Serotypes from Nasopharyngeal Secretions
Authors: Bronsdon Melinda A. , O'Brien Katherine L. , Facklam Richard R. , Whitney Cynthia G. , Schwartz Benjamin , Carlone George M.
Published: April 2004

Conventional culture techniques are limited in the ability to detect multiple
Streptococcus pneumoniae
serotypes in nasopharyngeal (NP) secretions. We developed an immunoblot (IB) method with monoclonal antibodies (MAbs) to detect
S. pneumoniae
and to identify serotypes. NP specimens stored in skim milk-tryptone-glucose-glycerol medium were assessed by the IB method and the reference culture method (RM). In the RM, four optochin-sensitive alpha-hemolytic colonies resembling pneumococci were typed by the Quellung reaction. In the IB method, a nitrocellulose membrane blot of surface growth was reacted with a pneumococcal surface adhesion (PsaA) MAb and visualized. Of 47 NP specimens, 32 (68%) were found to be positive and 13 (28%) were found to be negative for pneumococci by both methods; each method alone yielded one positive result. The sensitivity and specificity of the IB method for the detection of pneumococci were 97 and 93%, respectively. To identify serotypes, blots were tested with serotype-specific MAbs (4, 6A, 6B, 9V, 14, 18C, 19F, and 23F). To detect the remaining serotypes, positive serotype-specific replicate blots were compared visually to an original anti-PsaA-positive blot; four unidentified colonies were subcultured and serotyped by the Quellung reaction. Fifty-eight
S. pneumoniae
-positive NP specimens containing 69 pneumococcal strains (23 serotypes) were tested; 68 (98.6%) of the strains were detected by the IB method, and 66 (95.6%) were detected by the RM. For 11 specimens found to contain two serotypes, both methods detected both serotypes in 7 (63.6%), the IB method alone detected the two serotypes in 3 (27.3%), and the RM alone detected both serotypes in 1 (9%). The IB method identified multiple clones and minor populations of pneumococci in NP secretions. This method is useful for detecting specific serotypes and carriage of multiple serotypes in epidemiologic surveillance and carriage studies.
Improved Enzyme-Linked Immunosorbent Assay Using C-Terminal Truncated Recombinant Antigens of
Babesia bovis
Rhoptry-Associated Protein-1 for Detection of Specific Antibodies
Authors: Boonchit Suthisak , Xuan Xuenan , Yokoyama Naoaki , Goff Will L. , Waghela Suryakant D. , Wagner Gale , Igarashi Ikuo
Published: April 2004

An enzyme-linked immunosorbent assay (ELISA) based on a recombinant rhoptry-associated protein-1 (RAP-1) of
Babesia bovis
has been previously developed, but it was imperfect because some cross-reactions were still present in
Babesia bigemina
-infected bovine sera. To improve its accuracy for the specific detection of the antibodies to
B. bovis
, we constructed three C-terminal truncated recombinant antigens of the RAP-1—rCT1 (amino acids [aa] 301 to 408), rCT2 (aa 388 to 490), and rCT3 (aa 466 to 565)—by using a baculovirus expression system and evaluated their diagnostic potentials using ELISA. rCT1 and rCT2 were better diagnostic antigens in their sensitivities and diagnostic efficiencies than rCT3, although none of the recombinant antigens showed any cross-reactivity to
B. bigemina
-infected bovine sera. These results confirmed that the N-terminal 300-aa region caused cross-reactivity of the entire RAP-1 antigen, and the C-terminal truncated recombinant antigens were shown to be useful reagents for species-specific serodiagnosis.
Reassessment of Sequence-Based Targets for Identification ofBacillusSpecies
Authors: Blackwood K. S. , Turenne C. Y. , Harmsen D. , Kabani A. M.
Published: April 2004

TheBacillusgenus is a large heterogeneous group in need of an efficient method for species differentiation. To determine the current validity of a sequence-based method for identification and provide contemporary data, PCR and sequencing of a 500-bp product encompassing the V1 to V3 regions of the 16S rRNA gene were undertaken using 65 of the 83 type strains of this genus. This region proved discriminatory between most species (70.0 to 100% similarity), the exceptions being clinically relevantB. cereusandB. anthracisas well as nonpathogenicB. psychrotoleransandB. psychrodurans.Consequently, 27 type and clinical strains from theB. cereusgroup were used to test alternate targets (rpoB,vrrA, and the 16S-23S spacer region) for identification. TherpoBgene proved the best alternate target, with a conserved 4-nucleotide difference betweenB. cereusandB. anthracis. The high 16S rRNA gene sequence similarities between some strains demonstrated the need for a polyphasic approach to the systematics of this genus. This approach is one focus of the Ribosomal Differentiation of Medical Microorganisms mandate. Accordingly, the 16S rRNA gene sequences generated in this study have been submitted for inclusion into its publicly accessible, quality-controlled database athttp://www.ridom_rdna.de/.
Hepatitis C Virus (HCV) Core Antigen Assay To Detect Ongoing HCV Infection in Thai Injection Drug Users
Authors: Netski Dale M. , Wang Xiao-Hong , Mehta Shruti H. , Nelson Kenrad , Celentano David , Thongsawat Satawat , Maneekarn Niwat , Suriyanon Vinai , Jittiwutikorn Jaroon , Thomas David L. , Ticehurst John R.
Published: April 2004

We evaluated a quantitative enzyme immunoassay (trak-C) for hepatitis C virus core antigen (HCV core Ag) by testing serum specimens from 820 injection drug users in Thailand with anti-HCV antibodies. The HCV genotypes in this population include genotypes 3 and 6, which have not been extensively tested with this assay. Among these specimens, 629 (76.7%) yielded positive results, with HCV core Ag concentrations predominantly spanning (35.7%) or above (58.2%) the measurable range of 1.5 to 100 pg/ml. To assess reproducibility, we retested 30 specimens representing six core Ag ranges; the mean coefficient of variation for each range was ≤9.7% (highest for 1.5 to 25 pg/ml). We also tested 204 specimens of the 820-specimen set for HCV RNA: while 146 (71.6%) were core Ag positive, 168 (82.4%) had detectable HCV RNA, of which 96% were typeable as genotype 3 (39%), 1 (31%), or 6 (26%) by nested reverse transcription-PCR. Among RNA-positive specimens, 86.9% had core Ag; 94% of the RNA negatives were core Ag negative. While there was no apparent bias for detecting core Ag representing the tested genotypes, median quantified results were higher for types 1a and 6 than for genotype 3 (P= 0.01); similarly, the median core Ag concentration was higher in HCV-human immunodeficiency virus-coinfected subjects than in HCV-monoinfected subjects. Our results demonstrated a good correlation between core Ag and HCV RNA in this population with high frequencies of genotypes 3 and 6. Because most core Ag concentrations were greater than those in the measurable range, we recommend a 10-fold dilution of the specimen before quantification. Reproducibility, low technical requirements, and high throughput should make this assay useful for clinical or research monitoring of HCV levels during active infection.
Convenient Selective Differential Broth for Isolation of Vancomycin-Resistant
Enterococcus
from Fecal Material
Authors: Novicki Thomas J. , Schapiro Jeffrey M. , Ulness Bruce K. , Sebeste Ann , Busse-Johnston Laurel , Swanson Kristine M. , Swanzy Susan R. , Leisenring Wendy , Limaye Ajit P.
Published: April 2004

Studies have shown that vancomycin broth enrichment is superior to direct plating for the detection of vancomycin-resistant enterococcus (VRE), but vancomycin selective broth is not generally commercially available. We developed an easy-to-prepare VRE selective differential broth and compared it to direct plating on bile esculin azide (BEA) agar for the isolation of VRE from fecal samples. A total of 528 consecutive rectal swabs and stools were inoculated onto BEA agar and into BEA broth with vancomycin at a concentration of 15 μg/ml (BEA VAN
15μg/ml
broth). After 1 to 2 days of incubation, broths were subcultured to BEA VAN
6μg/ml
agar. Bile esculin-positive colonies from the direct and broth subculture plates were evaluated for the presence of VRE by standard microbiological techniques. Addition of the broth enrichment step led to the detection of significantly more VRE isolates than did direct plating alone (28 versus 18 VRE isolates, respectively). In all, 30 VRE strains were isolated from 29 cultures, all of which were
Enterococcus faecium
. MICs of vancomycin ranged from 32 μg/ml (
n
= 2) to > 256 μg/ml (
n
= 28). Twenty-two VRE isolates were available for further testing: sixteen exhibited a VanA phenotype and six were of the VanB phenotype.
van
genotypes were in agreement with phenotypes for all VRE isolates except one, which could not be genotyped. The broth method also resulted in significantly fewer bile esculin-positive, non-VRE isolates requiring further workup. We have thus developed an easily prepared vancomycin selective differential broth that is significantly more sensitive and specific in the detection of VRE than is direct fecal plating to BEA agar.
Characterization of
ompA
Genotypes by Sequence Analysis of DNA from All Detected Cases of
Chlamydia trachomatis
Infections during 1 Year of Contact Tracing in a Swedish County
Authors: Lysén Maria , Österlund Anders , Rubin Carl-Johan , Persson Tina , Persson Ingrid , Herrmann Björn
Published: April 2004

In this study we aimed to characterize the
ompA
gene by sequencing DNA from all detected cases of
Chlamydia trachomatis
infection in a Swedish county during 2001, in order to improve the efficiency of contact tracing. Approximately 990 bp of the
ompA
gene was amplified, and sequence analysis was achieved in 678 (94%) of 725
C. trachomatis
-positive cases in this unselected population. The most prevalent genotype was serotype E (39%), followed by F (21%), G (11%), D (9%), K (9%), J (7%), H (2%), B (1%), and Ia (1%). Serotype E was found in five genotype variants, with the reference sequence comprising 96% of all E cases. Serotype D was the most variable, and of seven sequence variants, three were identified as recombinants with serotype E. Altogether 29 genetic variants were detected, and mutations and recombination events are discussed. Clinical manifestations were not associated with genotypes. Sequence variation was linked to sexual networks identified by contact tracing and improved epidemiological knowledge but was of limited benefit.
Identification of a Protein Biomarker Unique to the Pandemic O3:K6 Clone of
Vibrio parahaemolyticus
Authors: Williams Tracie L. , Musser Steven M. , Nordstrom Jessica L. , DePaola Angelo , Monday Steven R.
Published: April 2004

The present method of characterizing
Vibrio parahaemolyticus
strains involves serotyping or detection methods based on assessment of the presence or absence of genes thought to be markers of an organism's pathogenicity. It is unclear whether these assays detect all pathogenic
V. parahaemolyticus
strains since a clear correlation between the presence of a particular gene and the organism's pathogenicity has not yet been observed. We have described a proteomics-based method to distinguish individual
V. parahaemolyticus
strains on the basis of their protein profiles and identified a specific protein that is characteristic of the pandemic O3:K6 strain and its clonal derivatives. In the pandemic clone of
V. parahaemolyticus
, a histone-like DNA-binding protein, HU-α, has a C-terminal amino acid sequence different from those of other strains of
V. parahaemolyticus
. Upon further study, it was discovered that the gene encoding this protein has a 16-kbp insert at the 3′ terminus of the open reading frame for this protein. By using the protein sequence of the unique biomarker for the pandemic clone of
V. parahaemolyticus
, it was possible to rationally design specific PCR-based probes and assays that permit the rapid and precise identification of pandemic strains of
V. parahaemolyticus
.
Significance of Amplified Fragment Length Polymorphism in Identification and Epidemiological Examination of
Candida
Species Colonization in Children Undergoing Allogeneic Stem Cell Transplantation
Authors: Ball L. M. , Bes M. A. , Theelen B. , Boekhout T. , Egeler R. M. , Kuijper E. J.
Published: April 2004

Candida albicans
and non-
C. albicans Candida
species are increasingly being isolated from patients in high-risk categories, most notably, those who have undergone stem cell transplantation (SCT). Identification of the presence of non-
C. albicans Candida
species early in the course of the transplant procedure is important, as these species exhibit different sensitivities to the available antifungal treatments and cause mortality at rates that vary from those for
C. albicans
. Amplified fragment length polymorphism (AFLP) analysis has been shown to be a reliable method of reproducibly identifying medically important
Candida
species. We investigated the use of serial AFLP analysis of 54 routine surveillance cultures for the identification and epidemiological examination of
Candida
sp. colonization in five consecutive children undergoing allogeneic SCT. One child became colonized with a
C. albicans
strain and remained colonized with this strain during the whole admission period. Another child had persistent colonization with a
C. albicans
strain with striking variations in its AFLP patterns over time, which was considered indicative of microevolution.
Candida dubliniensis
,
Candida lusitaniae
, and
Saccharomyces cerevisiae
were identified in the three remaining patients, with two children being simultaneously and transiently colonized with different species. These findings show that colonization with yeasts during transplantation is a complex and dynamic interaction between the host and the organism(s). In our study three strains from eight separate time points were incorrectly identified as
C. albicans
by a rapid enzyme test. AFLP analysis of surveillance cultures allowed more accurate and informative epidemiological evaluations of pathogenic yeasts in children during transplantation.
Multilocus Short Sequence Repeat Sequencing Approach for Differentiating among
Mycobacterium avium
subsp.
paratuberculosis
Strains
Authors: Amonsin Alongkorn , Li Ling Ling , Zhang Qing , Bannantine John P. , Motiwala Alifiya S. , Sreevatsan Srinand , Kapur Vivek
Published: April 2004

We
describe a multilocus short sequence repeat (MLSSR) sequencing approach
for the genotyping of
Mycobacterium avium
subsp.
paratuberculosis
(
M. paratuberculosis
) strains.
Preliminary analysis identified 185 mono-, di-, and trinucleotide
repeat sequences dispersed throughout the
M. paratuberculosis
genome, of which 78 were perfect repeats. Comparative nucleotide
sequencing of the 78 loci of six
M. paratuberculosis
isolates
from different host species and geographic locations identified a
subset of 11 polymorphic short sequence repeats (SSRs), with an average
of 3.2 alleles per locus. Comparative sequencing of these 11 loci was
used to genotype a collection of 33
M. paratuberculosis
isolates representing different multiplex PCR for IS
900
loci
(MPIL) or amplified fragment length polymorphism (AFLP) types. The
analysis differentiated the 33
M. paratuberculosis
isolates
into 20 distinct MLSSR types, consistent with geographic and
epidemiologic correlates and with an index of discrimination of 0.96.
MLSSR analysis was also clearly able to distinguish between sheep and
cattle isolates of
M. paratuberculosis
and easily and
reproducibly differentiated strains representing the predominant MPIL
genotype (genotype A18) and AFLP genotypes (genotypes Z1 and Z2) of
M. paratuberculosis
described previously. Taken together, the
results of our studies suggest that MLSSR sequencing enables facile and
reproducible high-resolution subtyping of
M. paratuberculosis
isolates for molecular epidemiologic and population genetic
analyses.
Identification of
Bacteroides thetaiotaomicron
on the Basis of an Unexpected Specific Amplicon of Universal 16S Ribosomal DNA PCR
Authors: Teng Lee-Jene , Hsueh Po-Ren , Huang Yu-Hsuan , Tsai Jui-Chang
Published: April 2004

We applied a set of commonly used universal primers (primers RW01 and DG74) to amplify partial fragments of 16S ribosomal DNA for bacterial identification and found an unexpected amplicon (547 bp), in addition to the expected 362-bp product, in samples containing
Bacteroides thetaiotaomicron
. It was demonstrated that the internal sequence (508 bp, excluding the primers) of the 547-bp amplicon was identical to the genomic sequence from nucleotide positions 165800 to 166307 of
B. thetaiotaomicron
type strain VPI-5482 by a BLAST search of the sequences in the GenBank database. The existence of this unexpected yet specific amplicon strongly indicated the presence of
B. thetaiotaomicron
in the sample, and it was found that it could be used to discriminate
B. thetaiotaomicron
from closely related species. Another set of PCR primers specific for
B. thetaiotaomicron
was developed on the basis of the sequence of this 547-bp genomic fragment. Both PCR-based assays showed the same sensitivity (88%) and specificity (100%).
Evaluation of the Velogene Genomic Assay for Detection of
vanA
and
vanB
Genes in Vancomycin-Resistant
Enterococcus
Species
Authors: Appleman Maria D. , Citron Diane M. , Kwok Richard
Published: April 2004

The Velogene (VEL) genomic assay is a qualitative DNA probe for
vanA
and
vanB
in enterococci. 150 clinical isolates were tested with the VEL assay and characterized by pigment production, catalase levels, motility, growth in 6 μg of vancomycin/ml, vancomycin and teicoplanin susceptibility, API 20S assay, and genotyping by multiplex PCR. The VEL assay identified all enterococcal strains with
vanA
and
vanB
genes.
Reactivation of Porcine Cytomegalovirus through Allogeneic Stimulation
Authors: Guedes Maria Isabel M. C. , Risdahl Jack M. , Wiseman Barry , Molitor Thomas W.
Published: April 2004

Reactivation of latent porcine cytomegalovirus after coculture of peripheral blood mononuclear cells (PBMCs) from pigs with different genetic backgrounds was investigated. Nine of 10 allogeneic coculture pairs were PCR (DNA) positive, whereas 7 coculture pairs had porcine cytomegalovirus (PCMV) RNA, an indication of virus replication. The cell subpopulations harboring PCMV were monocytes and CD8
+
T cells.
Nested PCR Assay for Detection of
Leishmania donovani
in Slit Aspirates from Post-Kala-Azar Dermal Leishmaniasis Lesions
Authors: Sreenivas Gannavaram , Ansari N. A. , Kataria Joginder , Salotra Poonam
Published: April 2004

A nested PCR assay to detect parasite DNA in slit aspirates from skin lesions of patients with post-kala-azar dermal lesihmaniasis (PKDL) is described. PCR results were positive in 27 of 29 (93%) samples by nested PCR assay, while only 20 of 29 (69%) were positive in a primary PCR assay. The nested PCR assay allowed reliable diagnosis of PKDL in a noninvasive manner.
Performance of a Commercial, Type-Specific Enzyme-Linked Immunosorbent Assay for Detection of Herpes Simplex Virus Type 2-Specific Antibodies in Ugandans
Authors: Laeyendecker Oliver , Henson Charla , Gray Ronald H. , Nguyen Ruby H.-N. , Horne Bobbi Jo , Wawer Maria J. , Serwadda David , Kiwanuka Noah , Morrow Rhoda Ashley , Hogrefe Wayne , Quinn Thomas C.
Published: April 2004

Two hundred forty-eight human immunodeficiency virus (HIV)-positive and 496 HIV-negative subjects in Uganda were tested by HerpeSelect herpes simplex virus type 2 enzyme-linked immunosorbent assay (ELISA) and Western blotting to optimize the ELISA for use in this population. A higher index cutoff value was required for optimal sensitivity and specificity, and overall performance of the assay was not affected by HIV status.
Two Genotypes of Canine Coronavirus Simultaneously Detected in the Fecal Samples of Dogs with Diarrhea
Authors: Pratelli Annamaria , Decaro Nicola , Tinelli Antonella , Martella Vito , Elia Gabriella , Tempesta Maria , Cirone Francesco , Buonavoglia Canio
Published: April 2004

Sixty-nine fecal samples from diarrheic puppies were examined by reverse transcription-PCR assays for the M and the S genes of canine coronaviruses (CCoVs). The isolates in 10 samples were recognized as CCoV type I, and the isolates in 6 samples were recognized as CCoV type II, while isolates of both genotypes were simultaneously detected in 53 samples.
Molecular Characterization of a Prophage of
Salmonella enterica
Serotype Typhimurium DT104
Authors: Tanaka Kiyoshi , Nishimori Kei , Makino Sou-Ichi , Nishimori Tomoko , Kanno Toru , Ishihara Ryoko , Sameshima Toshiya , Akiba Masato , Nakazawa Muneo , Yokomizo Yuichi , Uchida Ikuo
Published: April 2004

Isolates of the
Salmonella enterica
serotype Typhimurium definitive phage type (DT104) were found to contain the same prophage (designated phage ST104). The complete sequence of the DNA genome of prophage ST104 was determined. The entire DNA sequence consisted of 41,391 bp, including 64 open reading frames, and exhibited high similarity to P22 and to phage type conversion phage ST64T.