bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative Analysis of Immunoglobulin M (IgM) Capture Enzyme-Linked Immunosorbent Assay Using Virus-Like Particles or Virus-Infected Mouse Brain Antigens To Detect IgM Antibody in Sera from Patients with Evident Flaviviral Infections
Authors: Holmes Derek A. , Purdy David E. , Chao Day-Yu , Noga Amanda J. , Chang Gwong-Jen J.
Published: July 2005

The use of immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) serves as a valuable tool for the diagnosis of acute flaviviral infections, since IgM antibody titers are detectable early, peak at about 2 weeks postinfection, and subsequently decline to lower levels over the next few months. Traditionally, virus-infected tissue culture or suckling mouse brain (SMB) has been the source of viral antigens used in the assay. In an effort to provide a reliable source of standardized viral antigens for serodiagnosis of the medically important flaviviruses, we have developed a eukaryotic plasmid vector to express the premembrane/membrane and envelope proteins which self-assemble into noninfectious virus-like particles (VLPs). In addition to the plasmids for Japanese encephalitis virus, West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and dengue virus type 2 (DENV-2) reported earlier, we recently constructed the DENV-1, -3, and -4 VLP expression plasmids. Three blind-coded human serum panels were assembled from patients having recent DENV, SLEV, and WNV infections to assess the sensitivity and specificity of the MAC-ELISA using VLPs or SMB antigens. In addition, serum specimens from patients infected with either Powassan virus or La Crosse encephalitis virus were used to evaluate the cross-reactivity of seven mosquito-borne viral antigens. The results of the present studies showed higher sensitivity when using SLEV and WNV VLPs and higher specificity when using SLEV, WNV, and the mixture of DENV-1 to -4 VLPs in the MAC-ELISA than when using corresponding SMB antigens. Receiver operating characteristic (ROC) curve analysis, a plot of the sensitivity versus false positive rate (100 − specificity), was applied to discriminate the accuracy of tests comparing the use of VLPs and SMB antigen. The measurement of assay performance by the ROC analysis indicated that there were statistically significant differences in assay performance between DENV and WNV VLPs and the respective SMB antigens. Additionally, VLPs had a lower cutoff positive/negative ratio than corresponding SMB antigens when employed for the confirmation of current infections. The VLPs also performed better than SMB antigens in the MAC-ELISA, as indicated by a higher positive prediction value and positive likelihood ratio test. Cell lines continuously secreting these VLPs are therefore a significantly improved source of serodiagnostic antigens compared to the traditional sources of virus-infected tissue culture or suckling mouse brain.
Ceftriaxone Resistance of NontyphoidalSalmonella entericaIsolates in Northern Taiwan Attributable to Production of CTX-M-14 and CMY-2 β-Lactamases
Authors: Li Wen-Chen , Huang Fu-Yuan , Liu Chang-Pan , Weng Li-Chuan , Wang Nai-Yu , Chiu Nan-Chang , Chiang Chuen-Sheue
Published: July 2005

Among 3,027 nontyphoidalSalmonella entericaisolates identified between January 1999 and December 2002 in a medical center in northern Taiwan, 31 were resistant to the extended-spectrum cephalosporin ceftriaxone (1.02% [31/3,027]), including 2 in 1999 (0.36% [2/549]), 13 in 2000 (1.49% [13/870]), 7 in 2001 (0.78% [7/893]), and 9 in 2002 (1.26% [9/715]). Sixteen of these isolates belonged toSalmonellaserogroup B, nine belonged to serogroup C, four belonged to serogroup D, and two belonged to serogroup E. The majority were from stool cultures. The mechanism of resistance was investigated for eight isolates, including threeS. entericaserovar Typhimurium, oneS. entericaserovar Wagenia, oneS. entericaserovar Senftenberg, oneS. entericaserovar Derby, oneS. entericaserovar Panama, and oneS. entericaserovar Duesseldorf isolate. All eight patients from whom these isolates were recovered had community-acquired infections. All eight isolates were resistant to ampicillin, ceftriaxone, and cefotaxime but susceptible to imipenem and ciprofloxacin. Ceftriaxone resistance was due to the production of the CMY-2 AmpC β-lactamase by seven isolates and the CTX-M-14 β-lactamase by the remaining isolate. Both β-lactamase genes were carried on conjugative plasmids. In a 2.5-kb region encompassing theblaCMY-2gene, at nucleotide 49 upstream of the start codon ofblaCMY-2, three of the sevenblaCMY-2-positive isolates had an A nucleotide and four had a G nucleotide. In conclusion, the ceftriaxone resistance of nontyphoidalSalmonellaisolates in our hospital was attributed to the CTX-M-14 and CMY-2 β-lactamases.
Development and Evaluation of Chlamylege, a New Commercial Test Allowing Simultaneous Detection and Identification of
Legionella
,
Chlamydophila pneumoniae
, and
Mycoplasma pneumoniae
in Clinical Respiratory Specimens by Multiplex PCR
Authors: Ginevra C. , Barranger C. , Ros A. , Mory O. , Stephan J.-L. , Freymuth F. , Joannès M. , Pozzetto B. , Grattard F.
Published: July 2005

This study describes the development and evaluation of a new commercial test, Chlamylege (Argene Inc.), which allows the simultaneous detection in respiratory samples of
Chlamydophila pneumoniae
,
Mycoplasma pneumoniae
, and most
Legionella
species, as well as PCR inhibitors, by using a multiplex PCR and microplate hybridization. The sensitivities of Chlamylege were 1 × 10
−3
IFU, 5 × 10
−2
color-changing units, and 1 CFU per reaction tube for
C. pneumoniae
,
M. pneumoniae
, and
Legionella pneumophila
, respectively. A cohort of 154 clinical samples from patients with documented respiratory infections was analyzed by the kit, including 2 samples from patients with
C. pneumoniae
infection, 9 samples from patients with
M. pneumoniae
infection, 19 samples from patients with
Legionella
species infection, and 114 samples that tested negative for the three pathogens. All the positive specimens were correctly detected and identified by the Chlamylege kit, and no false-positive result was observed with the negative samples. The kit was then evaluated in a pediatric prospective study that included 220 endotracheal aspirates, and the results were compared with those obtained by three single in-house PCR assays. Four specimens were found to be positive for
C. pneumoniae
and six were found to be positive for
M. pneumoniae
by using both strategies. The Chlamylege kit detected two additional samples positive for
M. pneumoniae
and one additional sample positive for a
Legionella
species other than
L. pneumophila
; these three samples were shown to be true positive by other techniques. These overall results demonstrate that the Chlamylege assay is sensitive, specific, and convenient for the rapid detection and identification of atypical pathogens in clinical samples from patients with respiratory infections.
Suspension Microarray with Dendrimer Signal Amplification Allows Direct and High-Throughput Subtyping of
Listeria monocytogenes
from Genomic DNA
Authors: Borucki Monica K. , Reynolds James , Call Douglas R. , Ward Todd J. , Page Brent , Kadushin James
Published: July 2005

Listeria monocytogenes
is a significant cause of food-borne disease and mortality; therefore, epidemiological investigations of this pathogen require subtyping methods that are rapid, discriminatory, and reproducible. Although conventional microarray subtyping analysis has been shown to be both high resolution and genetically informative, it is still relatively low throughput and technically challenging. Suspension microarray technology eliminates the technical issues associated with planar microarrays and allows high-throughput subtyping of
L. monocytogenes
strains. In this study, a suspension array assay using dendrimer signal amplification allowed rapid and accurate serovar identification of
L. monocytogenes
strains using genomic DNA as a target. The ability to subtype genomic DNA without PCR amplification allows probes to be designed for many different regions within the bacterial genome and should allow high-resolution subtyping not possible with multiplex PCR.
Comparison between the Hybrid Capture II Test and a PCR-Based Human Papillomavirus Detection Method for Diagnosis and Posttreatment Follow-Up of Cervical Intraepithelial Neoplasia
Authors: Söderlund-Strand Anna , Rymark Per , Andersson Pia , Dillner Joakim , Dillner Lena
Published: July 2005

Human papillomavirus (HPV) infection is the major cause of cervical cancer and its precursor, cervical intraepithelial neoplasia (CIN), and HPV testing has therefore been proposed for improved triaging and follow-up of women treated for CIN. We compared two common HPV DNA detection tests (Hybrid Capture II [HCII] and PCR-enzyme immunosorbent assay (EIA) using the primers GP5+/GP6+ followed by HPV typing with reverse dot blot hybridization) for sensitivity and specificity for detection of CIN and of CIN recurrence after treatment. Two hundred and thirty-nine women referred to the Department of Obstetrics and Gynaecology in Västerås, Sweden, were enrolled because of atypical Pap smears; 177 of these were later treated for dysplasia by conization or loop diathermy. Samples for HPV DNA testing were taken before and 4 to 6 months after treatment. There was substantial agreement between the HCII and PCR-EIA (kappa, 0.70 before treatment and 0.72 after treatment). The sensitivity for histopathologically confirmed CIN III was 100.0% for PCR-EIA and 95.6% for HCII. For patients with CIN II or worse (CIN II+), the sensitivities were 92.9% (PCR-EIA) and 91.8% (HCII). The specificities for CIN II+ in the pretreatment setting were 30.4% for PCR-EIA and 24.1% for HCII. After treatment, the sensitivities for CIN III in cytology were 100.0% by both methods, and for CIN II+, sensitivities were 80.0% by both methods. The specificities for CIN II+ in the posttreatment setting were 83.5% for PCR and 85.4% for HCII. In conclusion, the sensitivities of both PCR-EIA and HCII are high and almost equal, suggesting that both methods are suitable as tools for detection and posttreatment follow-up of CIN II-III.
Flow Cytometry-Based Assay for Titrating Dengue Virus
Authors: Lambeth C. R. , White L. J. , Johnston R. E. , de Silva A. M.
Published: July 2005

Plaque assays for titrating dengue virus (DENV) are time-consuming and not suitable for strains that do not plaque. Fluorescence-activated cell sorting (FACS) has been used to detect DENV-infected cells. Here we describe a FACS-based assay for titrating DENV. We determined that at 24 h postinfection, the number of infected cells detected by FACS represented the first round of infection and therefore could be used as a readout of the number of infectious particles in the inoculum. When the titers of different laboratory and clinical strains of DENV were compared using FACS, plaque, and endpoint dilution assays, for most strains the FACS titers were comparable to titers obtained by plaque or endpoint dilution assays. The FACS assay is an improvement over the plaque assay because the infection period is reduced from 5 to 7 days to 24 h and the assay can be used to titrate clinical isolates that frequently do not form clear plaques on cell monolayers. The novel FACS-based methods described here will facilitate laboratory studies of dengue.
Extended-Spectrum-β-Lactamase-Producing
Enterobacteriaceae
in Yaounde, Cameroon
Authors: Gangoué-Piéboji Joseph , Bedenic Branka , Koulla-Shiro Sinata , Randegger Corinne , Adiogo Dieudonné , Ngassam Pierre , Ndumbe Peter , Hächler Herbert
Published: July 2005

Organisms producing extended-spectrum β-lactamases (ESBLs) have been reported in many countries, but there is no information on the prevalence of ESBL-producing members of the family
Enterobacteriaceae
in Cameroon. A total of 259
Enterobacteriaceae
strains were isolated between 1995 and 1998 from patients at the Yaounde Central Hospital in Cameroon. Enterobacterial isolates resistant to extended-spectrum cephalosporin and monobactam were screened for ESBL production by the double-disk (DD) synergy test. Thirty-one (12%) of these
Enterobacteriaceae
strains were shown to be positive by the DD synergy test, suggesting the presence of ESBLs. Resistance to oxyimino-cephalosporins and monobactams of 12 (38.7%) of the 31 strains—i.e., 6
Klebsiella pneumoniae
, 4
Escherichia coli
, 1
Citrobacter freundii
, and 1
Enterobacter cloacae
strain—was transferred to
E. coli
HK-225 by conjugation. Resistance to gentamicin, gentamicin plus trimethoprim-sulfamethoxazole, or trimethoprim-sulfamethoxazole was cotransferred into 6, 2, and 1 of these transconjugants, respectively. All 12 transconjugants were resistant to amoxicillin, piperacillin, all of the cephalosporins, and aztreonam but remained susceptible to cefoxitin and imipenem. Crude extracts of β-lactamase-producing transconjugants were able to reduce the diameters of inhibition zones around disks containing penicillins, narrow- to expanded-spectrum cephalosporins or monobactams when tested against a fully susceptible
E. coli
strain but had no effect on such zones around cefoxitin, imipenem, and amoxicillin-clavulanate disks. The β-lactamases produced by the 12 tranconjugants turned out to be SHV-12 by DNA sequencing. Therefore, the ESBL SHV-12 is described for the first time in Cameroon.
Comparative Assessment of the Gelatin Particle Agglutination Test and an Enzyme-Linked Immunosorbent Assay for Diagnosis of Strongyloidiasis
Authors: Sithithaworn J. , Sithithaworn P. , Janrungsopa T. , Suvatanadecha K. , Ando K. , Haswell-Elkins M. R.
Published: July 2005

The performances of the gelatin particle agglutination test (GPAT) and enzyme-linked immunosorbent assay (ELISA) for the diagnosis of strongyloidiasis with reference to the results of the agar plate culture technique (APCT) were evaluated with samples from 459 individuals from communities in northeast Thailand where strongyloidiasis is endemic. The prevalence of strongyloidiasis in five sample groups determined by GPAT varied between 29.3 and 61.5% (mean, 38.8%). ELISA and APCT, employed concurrently, gave lower prevalence rates of 27.5% (range, 21.6 to 42.1%) and 22.7% (range, 12.7 to 53.8%), respectively. By using APCT as the standard method, the sensitivity of GPAT was generally higher than that of ELISA (81 versus 73%). The specificity of GPAT was slightly lower than that of ELISA (74 versus 86%). The resulting GPAT titers exhibited positive linear relationships with the ELISA values (optical density at 490 nm) (
P
< 0.05), which suggests that the GPAT titer also reflects the levels of specific antibody comparable to those reflected by the ELISA values. Based on the relative ease and simplicity of use of the technique as well as the acceptable rates of sensitivity and specificity of the test, GPAT is more practical for screening for strongyloidiasis than the conventional ELISA.
Countrywide Spread of Community- and Hospital-Acquired Extended-Spectrum β-Lactamase (CTX-M-15)-Producing
Enterobacteriaceae
in Lebanon
Authors: Moubareck Carole , Daoud Ziad , Hakimé Noha I. , Hamzé Monzer , Mangeney Nicole , Matta Hiam , Mokhbat Jacques E. , Rohban Raymond , Sarkis Dolla Karam , Doucet-Populaire Florence
Published: July 2005

A prospective study was carried out to assess the extent of carriage of extended-spectrum β-lactamase (ESBL)-producing
Enterobacteriaceae
at both hospital and community levels in Lebanon. A total of 1,442 fecal samples were collected from hospital-based patients and 58 from health care workers of six Lebanese tertiary care general hospitals located in different areas of Lebanon between January and March 2003. A total of 382 fecal samples were also collected from healthy subjects between April and June 2003. The samples analysis led to the identification of 118 strains as ESBL producers based on the synergistic effects between clavulanate and selected β-lactams (ceftazidime and cefotaxime). These strains were isolated from 72 subjects: 61 patients, 2 health care workers, and 9 healthy subjects. One representative strain per subject was selected, and a total of 72 nonduplicate ESBL producers, including a high majority of
Escherichia coli
(
n
= 56),
Klebsiella pneumoniae
(
n
= 9),
Enterobacter cloacae
(
n
= 6), and
Citrobacter freundii
(
n
= 1), were characterized. The molecular analysis revealed that the majority of the strains (83%) express CTX-M-15 ESBL (pI 8.6). SHV-5a ESBL (pI 8.2) was produced by 18% of the strains. DNA macrorestriction analysis of ESBL-producing
E. coli
presented 38 different genotypes, revealing the absence of clonal link among these strains. In addition to the fact that the present study highlights the emergence and the countrywide dissemination of CTX-M-15-producing
E. coli
in Lebanon, it represents the first report of an SHV-5a-producing
C. freundii
.
Uncultivated Phylotypes and Newly Named Species Associated with Primary and Persistent Endodontic Infections
Authors: Siqueira J. F. , Rôças I. N.
Published: July 2005

Endodontic infections have been traditionally studied by culture methods, but recent reports showing that over 50% of the oral microbiota is still uncultivable (B. J. Paster et al., J. Bacteriol. 183:3770-3783, 2001) raise the possibility that many endodontic pathogens remain unknown. This study intended to investigate the prevalence of several uncultivated oral phylotypes, as well as newly named species in primary or persistent endodontic infections associated with chronic periradicular diseases. Samples were taken from the root canals of 21 untreated teeth and 22 root-filled teeth, all of them with radiographic evidence of periradicular bone destruction. Genomic DNA was isolated directly from each sample, and 16S rRNA gene-based nested or heminested PCR assays were used to determine the presence of 13 species or phylotypes of bacteria. Species-specific primers had already been validated in the literature or were developed by aligning closely related 16S rRNA gene sequences. Species specificity for each primer pair was confirmed by running PCRs against a panel of several oral bacteria and by sequencing DNA from representative positive samples. All species or phylotypes were detected in at least one case of primary infections. The most prevalent species or phylotypes found in primary infections were
Dialister invisus
(81%),
Synergistes
oral clone BA121 (33%), and
Olsenella uli
(33%). Of the target bacteria, only these three species were detected in persistent infections. Detection of uncultivated phylotypes and newly named species in infected root canals suggests that there are previously unrecognized bacteria that may play a role in the pathogenesis of periradicular diseases.
Genetic Divergence of
Campylobacter fetus
Strains of Mammal and Reptile Origins
Authors: Tu Zheng-Chao , Eisner William , Kreiswirth Barry N. , Blaser Martin J.
Published: July 2005

Campylobacter fetus
is a gram-negative bacterial pathogen of both humans and animals. Two subspecies have been identified,
Campylobacter fetus
subsp.
fetus
and
Campylobacter fetus
subsp.
venerealis
, and there are two serotypes, A and B. To further investigate the genetic diversity among
C. fetus
strains of different origins, subspecies, and serotypes, we performed multiple genetic analyses by utilizing random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and DNA-DNA hybridization. All 10 primers used for the RAPD analyses can distinguish
C. fetus
strains of reptile and mammal origin, five can differentiate between
C. fetus
subsp.
fetus
and
C. fetus
subsp.
venerealis
strains, and four showed differences between type A and type B isolates from mammals. PFGE with SmaI and SalI digestion showed varied genome patterns among different
C. fetus
strains, but for mammalian
C. fetus
isolates, genome size was well conserved (mean, 1.52 ± 0.06 Mb for SmaI and 1.52 ± 0.05 Mb for SalI). DNA-DNA hybridization demonstrated substantial genomic-homology differences between strains of mammal and reptile origin. In total, these data suggest that
C. fetus
subsp
fetus
strains of reptile and mammal origin have genetic divergence more extensive than that between the two subspecies and that between the type A and type B strains. Combining these studies with sequence data, we conclude that there has been substantial genetic divergence between
Campylobacter fetus
of reptile and mammal origin. Diagnostic tools have been developed to differentiate among
C. fetus
isolates for taxonomic and epidemiologic uses.
Emergence of Virulent Methicillin-Resistant
Staphylococcus aureus
Strains Carrying Panton-Valentine Leucocidin Genes in The Netherlands
Authors: Wannet W. J. B. , Spalburg E. , Heck M. E. O. C. , Pluister G. N. , Tiemersma E. , Willems R. J. L. , Huijsdens X. W. , de Neeling A. J. , Etienne J.
Published: July 2005

Methicillin-resistant
Staphylococcus aureus
(MRSA) strains carrying the Panton-Valentine leucocidin (PVL) genes have been reported worldwide and are a serious threat to public health. The PVL genes encode a highly potent toxin which is involved in severe skin infections and necrotizing pneumonia, even in previously healthy individuals. We assessed the prevalence of PVL-positive MRSA in The Netherlands for two periods of time: (i) 1987 through 1995 and (ii) 2000 and 2002, and determined their characteristics by using multilocus sequence typing and staphylococcal chromosome cassette (SCC
mec
) typing. It was found that up to 15% of all MRSA isolates detected in The Netherlands harbored the PVL genes. Most PVL-positive MRSA isolates were obtained from severe soft tissue infections in relatively young individuals. The first PVL-positive MRSA described in The Netherlands, isolated in 1988, was a single-locus variant of the “Berlin” epidemic MRSA clone. The 20 PVL-positive MRSA isolates studied in 2000 and 2002 consisted of five different sequence types (STs) that belonged to four clonal complexes. One of the STs, ST80, is considered to be a widespread European clone and was the most predominant ST (60%) in this study, while ST37 had never been found to be associated with PVL-positive MRSA. Most isolates harbored SCC
mec
type IV, a supposed marker for community-acquired MRSA. The number and type of virulence-associated genes varied among the different STs.
A Panton-Valentine Leucocidin (PVL)-Positive Community-Acquired Methicillin-Resistant
Staphylococcus aureus
(MRSA) Strain, Another Such Strain Carrying a Multiple-Drug Resistance Plasmid, and Other More-Typical PVL-Negative MRSA Strains Found in Japan
Authors: Takizawa Yoko , Taneike Ikue , Nakagawa Saori , Oishi Tomohiro , Nitahara Yoshiyuki , Iwakura Nobuhiro , Ozaki Kyoko , Takano Misao , Nakayama Teruko , Yamamoto Tatsuo
Published: July 2005

Community-acquired methicillin-resistant
Staphylococcus aureus
(CA-MRSA) was collected from children with bullous impetigo in 2003 and 2004. One strain collected in 2003 was Panton-Valentine leucocidin (PVL) positive. In 2004, a multiple-drug-resistant PVL
+
CA-MRSA strain was isolated from an athlete with a cutaneous abscess. These strains were analyzed by multilocus sequence typing,
spa
typing,
agr
typing, coagulase typing, staphylococcal cassette chromosome
mec
(SCC
mec
) typing, PCR assay for 30 virulence genes, drug susceptibility testing, pulsed-field gel electrophoresis, and for plasmids. The two Japanese PVL
+
CA-MRSA strains belonged to the globally extant (“pandemic”) sequence type 30 (ST30) with SCC
mec
IV. A transmissible, multiple-drug resistance plasmid emerged in such ST30 strains. The PVL
−
CA-MRSA strains (“domestic” CA-MRSA) accumulated for bullous impetigo, exhibiting new genotypes. Hospital-acquired MRSA of ST91 (but not pandemic ST5) shared common features with the PVL
−
CA-MRSA strain.
Differential Distribution and Expression of Panton-Valentine Leucocidin among Community-Acquired Methicillin-Resistant
Staphylococcus aureus
Strains
Authors: Saïd-Salim Battouli , Mathema Barun , Braughton Kevin , Davis Stacy , Sinsimer Daniel , Eisner William , Likhoshvay Yekaterina , DeLeo Frank R. , Kreiswirth Barry N.
Published: July 2005

Community-acquired methicillin-resistant
Staphylococcus aureus
(CA-MRSA) is an emerging threat worldwide. CA-MRSA strains differ from hospital-acquired MRSA strains in their antibiotic susceptibilities and genetic backgrounds. Using several genotyping methods, we clearly define CA-MRSA at the genetic level and demonstrate that the prototypic CA-MRSA strain, MW2, has spread as a homogeneous clonal strain family that is distinct from other CA-MRSA strains. The Panton-Valentine leucocidin (PVL)-encoding genes,
lukF
and
lukS
, are prevalent among CA-MRSA strains and have previously been associated with CA-MRSA infections. To better elucidate the role of PVL in the pathogenesis of CA-MRSA, we first analyzed the distribution and expression of PVL among different CA-MRSA strains. Our data demonstrate that PVL genes are differentially distributed among CA-MRSA strains and, when they are present, are always transcribed, albeit with strain-to-strain variability of transcript levels. To directly test whether PVL is critical for the pathogenesis of CA-MRSA, we evaluated the lysis of human polymorphonuclear leukocytes (PMNs) during phagocytic interaction with PVL-positive and PVL-negative CA-MRSA strains. Unexpectedly, there was no correlation between PVL expression and PMN lysis, suggesting that additional virulence factors underlie leukotoxicity and, thus, the pathogenesis of CA-MRSA.
Spatial Organization and Composition of the Mucosal Flora in Patients with Inflammatory Bowel Disease
Authors: Swidsinski Alexander , Weber Jutta , Loening-Baucke Vera , Hale Laura P. , Lochs Herbert
Published: July 2005

The composition and spatial organization of the mucosal flora in biopsy specimens from patients with inflammatory bowel disease (IBD; either Crohn's disease or ulcerative colitis), self-limiting colitis, irritable-bowel syndrome (IBS), and healthy controls were investigated by using a broad range of fluorescent bacterial group-specific rRNA-targeted oligonucleotide probes. Each group included 20 subjects. Ten patients who had IBD and who were being treated with antibiotics were also studied. Use of nonaqueous Carnoy fixative to preserve the mucus layer was crucial for detection of bacteria adherent to the mucosal surface (mucosal bacteria). No biofilm was detectable in formalin-fixed biopsy specimens. Mucosal bacteria were found at concentrations greater than 10
9
/ml in 90 to 95% of IBD patients, 95% of patients with self-limiting colitis, 65% of IBS patients, and 35% of healthy controls. The mean density of the mucosal biofilm was 2 powers higher in IBD patients than in patients with IBS or controls, and bacteria were mostly adherent.
Bacteroides fragilis
was responsible for >60% of the biofilm mass in patients with IBD but for only 30% of the biofilm mass in patients with self-limiting colitis and <15% of the biofilm mass in patients with IBS. In contrast, bacteria which positively hybridized with the probe specific for
Eubacterium rectale
-
Clostridium coccoides
accounted for >40% of the biofilm in IBS patients but for <15% of the biofilm in IBD patients. In patients treated with (5-ASA) or antibiotics, the biofilm could be detected with 4,6-diamidino-2-phenylindole but did not hybridize with fluorescence in situ hybridization probes. A
Bacteroides fragilis
biofilm is the main feature of IBD. This was not previously recognized due to a lack of appropriate tissue fixation. Both 5-ASA and antibiotics suppress but do not eliminate the adherent biofilm.
Rapid Diagnosis of Bacterial Meningitis by Real-Time PCR and Fluorescence In Situ Hybridization
Authors: Poppert Sven , Essig Andreas , Stoehr Barbara , Steingruber Adelinde , Wirths Beate , Juretschko Stefan , Reischl Udo , Wellinghausen Nele
Published: July 2005

Real-time PCR and fluorescence in situ hybridization (FISH) were evaluated as rapid methods for the diagnosis of bacterial meningitis and compared to standard diagnostic procedures. For PCR, a LightCycler approach was chosen, implementing eubacterial and specific PCR assays for the most relevant bacteria. For FISH, a similar probe set containing eubacterial and specific probes was composed of published and newly designed probes. Both methods were evaluated by use of cerebrospinal fluid (CSF) samples from patients with suspected bacterial meningitis. For all microscopy- and culture-positive samples (
n
= 28), the eubacterial PCR was positive. In addition, all identifiable pathogens were detected with specific PCR assays, according to an algorithm based on the Gram stain. The FISH method detected the pathogen in 13 of 18 positive samples. While the FISH method remained negative for all microscopy- and culture-negative samples (
n
= 113), the eubacterial PCR was positive for five of these samples. Sequencing of the amplicon revealed the presence of
Neisseria meningitidis
,
Streptococcus agalactiae
, and
Haemophilus influenzae
in three of these five samples. In addition, samples with discordant results by culture and microscopy were successfully investigated by PCR (10 samples) and FISH (5 samples). In conclusion, PCR is a highly sensitive tool for rapid diagnosis of bacterial meningitis. FISH is less sensitive but is useful for the identification of CSF samples showing bacteria in the Gram stain. Based on our results, an approach for laboratory diagnosis of meningitis including PCR and FISH is discussed.
Molecular Diversity of
Bacillus anthracis
in Italy
Authors: Fasanella Antonio , Van Ert Matthew , Altamura Settimia A. , Garofolo Giuliano , Buonavoglia Canio , Leori Guido , Huynh Lynn , Zanecki Shaylan , Keim Paul
Published: July 2005

We used multiple-locus variable-number tandem repeat analysis (MLVA) to type 64
Bacillus anthracis
isolates from outbreaks that have occurred during the past 40 years in Italy. MLVA of the 64 isolates revealed 10 unique genotypes; 9 of these genotypes and the majority of isolates (63/64) belonged to the previously described genetic cluster A1.a. Within the A1.a isolates, two previously described genotypes (G1 and G3), which differ by a single mutation in the pX01 locus, account for the majority of isolates in the country (53/63). The low diversity of
B. anthracis
genotypes in Italy suggests a single, dominant historical introduction, followed by limited localized differentiation.
Potential Secondary Pathogenic Role for Bovine Herpesvirus 4
Authors: Donofrio Gaetano , Cavirani Sandro , van Santen Vicky , Flammini Cesidio Filippo
Published: July 2005

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with no clear disease association. Previous studies have demonstrated that macrophages can harbor persistent BoHV-4. We found that the addition of prostaglandin E2 (PGE2) to bovine macrophage cells persistently infected with BoHV-4 increases viral replication. Because opportunistic infection can increase PGE2 production, we propose a link between opportunistic infection, PGE2 production, and BoHV-4 replication.
Clinical Use of 16S rRNA Gene Sequencing To IdentifyMycoplasma felisandM. gateaeAssociated with Feline Ulcerative Keratitis
Authors: Gray Larry D. , Ketring Kerry L. , Tang Yi-Wei
Published: July 2005

Routine bacterial cultures of corneal scrapings from seven cats with either ulcerative feline keratitis, keratomalacia, or both yielded colonies which were identified by 16S rRNA gene sequencing asMycoplasma felis(six cases) andMycoplasma gateae(one case). Identification of the pathogens allowed the use of less empirical and more organism-specific therapy.
Prevalence of SCC
mec
Type IV in Nosocomial Bloodstream Isolates of Methicillin-Resistant
Staphylococcus aureus
Authors: Trindade Priscila de A. , Pacheco Renata L. , Costa Silvia F. , Rossi Flavia , Barone Antonio A. , Mamizuka Elsa M. , Levin Anna S.
Published: July 2005

Over a period of 7 months, 151 consecutive methicillin-resistant
Staphylococcus aureus
blood isolates were evaluated. None was community acquired. Twenty (13%) were susceptible to four or more antimicrobials, and 95% of these isolates were identified as SCC
mec
type IV. Molecular typing demonstrated four patterns, with one predominant pattern. Although usually community acquired, SCC
mec
type IV in our setting is clearly nosocomial.
Sensitivity and Specificity of a Rapid rRNA Gene Probe Assay for Simultaneous Identification of
Staphylococcus aureus
and Detection of
mecA
Authors: Kaplan Shannon , Marlowe Elizabeth M. , Hogan James J. , Doymaz Mehmet , Bruckner David A. , Simor Andrew E.
Published: July 2005

rRNA gene sequences were used for identification and target adequacy controls in a DNA probe assay to identify isolates as
Staphylococcus
and, more specifically, as
S. aureus
within 1 hour.
mecA
status was simultaneously determined using a specific DNA probe. The target adequacy control guarded against false-negative
mecA
results.
Rapid Detection of Human Metapneumovirus Strains in Nasopharyngeal Aspirates and Shell Vial Cultures by Monoclonal Antibodies
Authors: Percivalle Elena , Sarasini Antonella , Visai Livia , Revello M. Grazia , Gerna Giuseppe
Published: July 2005

Monoclonal antibodies to human metapneumovirus (hMPV) were developed for direct fluorescent antibody (DFA) staining of nasopharyngeal aspirates from 40 infants with respiratory infections, as well as for hMPV identification in shell vial cultures. With reference to reverse transcription-PCR, DFA staining showed sensitivity, specificity, and positive and negative predictive values of 73.9%, 94.1%, 94.4%, and 72.7%, respectively. Monoclonal antibodies are useful for direct hMPV detection.
Human Clinical Isolates ofCorynebacterium diphtheriaeandCorynebacterium ulceransCollected in Canada from 1999 to 2003 but Not Fitting Reporting Criteria for Cases of Diphtheria
Authors: DeWinter Leanne M. , Bernard Kathryn A. , Romney Marc G.
Published: July 2005

A 5-year collection ofCorynebacterium diphtheriaeandCorynebacterium ulceranshuman clinical isolates yielded nine isolates from blood cultures of patients with invasive infections, stressing the importance ofC. diphtheriaeas a serious blood-borne pathogen. Seven percent ofC. diphtheriaeand 100% ofC. ulceransisolates produced diphtheria toxin, demonstrating that toxigenic corynebacteria continue to circulate.
Improved Sensitivity of Sputum Smear Microscopy after Processing Specimens with C
18
-Carboxypropylbetaine To Detect Acid-Fast Bacilli: a Study of United States-Bound Immigrants from Vietnam
Authors: Laserson K. F. , Yen N. T. N. , Thornton C. G. , Mai V. T. C. , Jones W. , An D. Q. , Phuoc N. H. , Trinh N. A. , Nhung D. T. C. , Lien T. X. , Lan N. T. N. , Wells C. , Binkin N. , Cetron M. , Maloney S. A.
Published: July 2005

The goal of this study was to evaluate the effect of the specimen-processing method that uses the detergent C
18
-carboxypropylbetaine (CB-18) on the sensitivity of acid-fast bacillus (AFB) staining. Vietnamese immigrants with abnormal chest radiographs provided up to three sputum specimens, which were examined for acid-fast bacilli by use of direct auramine and Ziehl-Neelsen staining. The remaining sputum was split; half was cultured, and the other half was incubated with CB-18 for 24 h, centrifuged, and examined for AFB by both staining methods. CB-18 processing improved the sensitivity of AFB staining by 20 to 30% (only differences in auramine sensitivity were statistically significant) but reduced specificity by ≈20% (
P
< 0.05). These findings have direct utility for overseas migrant tuberculosis screening programs, for which maximizing test sensitivity is a major objective.
Interpretive Criteria for Use of AccuProbe for Identification ofMycobacterium aviumComplex Directly from 7H9 Broth Cultures
Authors: Cloud Joann L. , Carroll Karen C. , Cohen Samuel , Anderson Clint M. , Woods Gail L.
Published: July 2005

Rapid identification ofMycobacterium aviumcomplex (MAC) is possible by use of AccuProbe (Gen-Probe, San Diego, Calif.). To evaluate the reliability of the MAC AccuProbe for testing 7H9 cultures inoculated with broth from MGIT cultures positive for acid-fast bacilli or growth on a solid medium, we compared probe results to results obtained by sequencing a portion of the 16S rRNA gene. Isolates were sequenced if the MAC probe result was <100,000 relative light units (RLU) or if it was ≥100,000 RLU and the colony morphology was not classic or there were two colony types. For the 1,389 cultures tested in phase 1, conducted to evaluate cutoff values for the MAC probe in testing of 7H9 cultures inoculated with broth from MGIT cultures, the sensitivity and specificity of the MAC AccuProbe were 97.7% and 88.8%, respectively, according to the manufacturer's interpretive criteria (≥30,000 RLU is positive). If the cutoff for a positive result were 80,000 RLU, the specificity would be 100% and the sensitivity 92.3%. Of the 344 isolates in phase 2, which was conducted to confirm the 80,000-RLU cutoff for a positive result and therefore included only isolates with a MAC probe result of ≤100,000 RLU, 13 of 16 with results of ≥30,000 but <80,000 RLU were identified as mycobacteria other than MAC, including fiveMycobacterium tuberculosiscomplex isolates. These data support the use of 80,000 RLU as the cutoff for a positive result in testing of 7H9 broth cultures with the MAC AccuProbe.
Bacteriology of Acute and Chronic Ethmoid Sinusitis
Authors: Brook Itzhak
Published: July 2005

Aspirates from 26 acutely and 17 chronically infected ethmoid sinuses were studied. Thirty-seven aerobes and 10 anaerobes were recovered from isolates from patients with acute sinusitis.
Streptococcus pneumoniae
and
Haemophilus influenzae
were predominant. Twenty-seven aerobes and 41 anaerobes were found in isolates from patients with chronic sinusitis. The predominant isolates were anaerobic gram-negative bacilli and
Peptostreptococcus
spp.
Use of a Single Serum Sample for Diagnosis of Acute Toxoplasmosis in Pregnant Women and Other Adults
Authors: Press Cynthia , Montoya Jose G. , Remington Jack S.
Published: July 2005

Using a single serum sample for testing for immunoglobulin G (IgG)
Toxoplasma
antibodies, differences in sensitivity of the dye test (which measures primarily IgG antibodies) and an IgG enzyme immunoassay were found useful for very early diagnosis of acute
Toxoplasma gondii
infection.
Frequent Detection of Epstein-Barr Virus and Cytomegalovirus but Not JC Virus DNA in Cerebrospinal Fluid Samples from Human Immunodeficiency Virus-Infected Patients in Northern Thailand
Authors: Rojanawiwat Archawin , Miura Toshiyuki , Thaisri Hansa , Pathipvanich Panita , Umnajsirisuk Sittichai , Koibuchi Tomohiko , Vongsheree Suthon , Iwamoto Aikichi , Ariyoshi Koya , Sawanpanyalert Pathom
Published: July 2005

Applying nested-PCRs, we frequently detected DNA of Epstein-Barr virus and cytomegalovirus but not JC virus in cerebrospinal fluid samples from 140 human immunodeficiency virus-infected patients with central nervous system symptoms in northern Thailand. Despite the low incidence of primary central nervous system lymphoma or cytomegalovirus encephalitis among Thai AIDS patients, Epstein-Barr virus and cytomegalovirus infections in the central nervous system are common.
Evaluation of seaFAST, a Rapid Fluorescent In Situ Hybridization Test, for Detection of
Helicobacter pylori
and Resistance to Clarithromycin in Paraffin-Embedded Biopsy Sections
Authors: Morris Julie M. , Reasonover Alisa L. , Bruce Michael G. , Bruden Dana L. , McMahon Brian J. , Sacco Frank D. , Berg Douglas E. , Parkinson Alan J.
Published: July 2005

A commercially available rapid fluorescent in situ hybridization (FISH) test, (seaFAST
H. pylori
Combi-Kit; SeaPro Theranostics International, Lelystad, The Netherlands) was used to simultaneously detect the presence of
Helicobacter pylori
and determine clarithromycin susceptibility in paraffin-embedded biopsy sections. The FISH method was found to be 97% sensitive, 94% specific for the detection of
H. pylori
and comparable to agar dilution for the detection of resistance to clarithromycin.
DiskDiffusion Testing Using
Candida
sp. Colonies Taken Directlyfrom CHROMagar
Candida
Medium May Decrease Time Required To ObtainResults
Authors: Klevay Michael , Ebinger Alex , Diekema Daniel , Messer Shawn , Hollis Richard , Pfaller Michael
Published: July 2005

We
compared results of disk diffusion antifungal susceptibility testing
from
Candida
sp. strains passaged on CHROMagar and on potato
dextrose agar. The overall categorical agreements for fluconazole and
voriconazole disk testing were 95% and 98% with 0% and 0.5% very major
errors, respectively. Disk diffusion testing by the CLSI (formerly
NCCLS) M44-A method can be performed accurately by taking inocula
directly from
CHROMagar.
Evaluation of the COBAS TaqMan 48 Real-Time PCR System for Quantitation of Hepatitis B Virus DNA
Authors: Gordillo Rafael M. , Gutiérrez Juan , Casal Manuel
Published: July 2005

The purpose of this study is to evaluate the usefulness of the new real-time PCR COBAS TaqMan 48 analyzer, comparing it to the existing COBAS AMPLICOR HBV MONITOR based on conventional PCR technology. The study used 104 samples from different patients. No differences were found in the sensitivity of the tests. There was an excellent correlation between the sample with a viral load within the dynamic range of the two tests (
r
= 0.938). The COBAS TaqMan test has a wider linear range, and this fact enables quantifying of the viral load without diluting the sample.
First Infection with VanD-Type Glycopeptide-Resistant
Enterococcus faecium
in Europe
Authors: Lavigne Jean-Philippe , Marchandin Hélène , Bouziges Nicole , Sotto Albert
Published: July 2005

We report the first strain of glycopeptide-resistant
Enterococcus faecium
from Europe that contains a
vanD
allele isolated from blood cultures of an immunocompromised patient hospitalized in a French university hospital. Based on phenotypic results, PCR sequencing, pulsed-field gel electrophoresis, and Southern blotting, the isolate was assigned to
E. faecium
with a chromosomally located VanD allele most closely related to the VanD1 allele.
Detection of
Tropheryma whipplei
DNA in Clinical Specimens by LightCycler Real-Time PCR
Authors: Sloan Lynne M. , Rosenblatt Jon E. , Cockerill Franklin R.
Published: July 2005

A real-time PCR method using the LightCycler (Roche Applied Science, Indianapolis, IN) was compared to a conventional PCR assay for the detection of
Tropheryma whipplei
in 321 clinical specimens. The LightCycler method had sensitivity and specificity comparable to the conventional method but required considerably less labor and time (3 h versus 3 to 5 days).
Nosocomial Outbreak of Pediatric Gastroenteritis Caused by CTX-M-14-Type Extended-Spectrum β-Lactamase-Producing Strains of
Salmonella enterica
Serovar London
Authors: Yong Dongeun , Lim Young Sik , Yum Jong Hwa , Lee Hyukmin , Lee Kyungwon , Kim Eui-Chong , Lee Bok-Kwon , Chong Yunsop
Published: July 2005

CTX-M-14-type extended-spectrum β-lactamase was first detected in
Salmonella enterica
serovar London strains which were isolated from three hospitalized pediatric patients with gastroenteritis. The isolates had pulsed-field gel electrophoresis patterns identical to those of the previously isolated antimicrobial-susceptible strains from community-acquired gastroenteritis, suggesting the susceptible clone acquired the resistance.
Isolation of an Atypical Strain ofActinobacillus pleuropneumoniaeSerotype 1 with a Truncated Lipopolysaccharide Outer Core and No O-Antigen
Authors: Jacques Mario , Labrie Josée , St. Michael Frank , Cox Andrew D. , Paradis Marie-Anne , Dick C. Paul , Klopfenstein Christian , Broes André , Fittipaldi Nahuel , Gottschalk Marcelo
Published: July 2005

A field isolate ofActinobacillus pleuropneumoniae, the causative agent of porcine fibrinohemorrhagic necrotizing pleuropneumonia, was sent to the diagnostic laboratory for serotyping. The isolate presented a clear reaction, with both polyclonal antibodies against serotype 1 and monoclonal antibodies against the capsular polysaccharide of serotype 1. It also exhibited a PCR profile of Apx toxins expected for serotype 1. The isolate, however, failed to react with monoclonal antibodies against the O-antigen of serotype 1 lipopolysaccharide (LPS), suggesting a rough phenotype. The lipid A-core region of the isolate migrated faster than the corresponding region of the serotype 1 reference strain S4074 by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the presence of a truncated core. Sugar analysis and mass spectrometry analysis of the O-deacylated LPS from the field isolate were consistent with the absence of O-antigen and truncation of the outer core compared to the wild-type reference strain. Experimental infection of pigs confirmed the virulence of the isolate. This is the first report of an isolate ofA. pleuropneumoniaeserotype 1 with a truncated outer core and a rough LPS phenotype. Veterinary diagnostic laboratories should be vigilant, since infections caused by such an isolate will not be detected by serological tests based on LPS O-antigen.
Description of
Kingella potus
sp. nov., an Organism Isolated from a Wound Caused by an Animal Bite
Authors: Lawson Paul A. , Malnick Henry , Collins Matthew D. , Shah Jayesh J. , Chattaway Marie A. , Bendall Richard , Hartley John W.
Published: July 2005

We report the isolation and characterization of a hitherto unknown gram-negative, rod-shaped
Neisseria
-like organism from an infected wound resulting from a bite from a kinkajou. Based on both phenotypic and phylogenetic evidence, it is proposed that the unknown organism be classified as a new species,
Kingella potus
sp. nov.
Does Resistance to Pyrazinamide Accurately Indicate the Presence of
Mycobacterium bovis
?
Authors: de Jong Bouke C. , Onipede Anthony , Pym Alex S. , Gagneux Sebastien , Aga Roxanne S. , DeRiemer Kathryn , Small Peter M.
Published: July 2005

Mycobacterium bovis
is best identified by screening those isolates of the
Mycobacterium tuberculosis
complex that have any pyrazinamide (PZA) resistance, using a confirmatory test such as spoligotyping, biochemical testing, or genomic deletion analysis. The sensitivity for detection of
M. bovis
is lowered to 82% when only PZA-monoresistant isolates are screened.
Bacteremia Caused by
Janibacter melonis
Authors: Elsayed Sameer , Zhang Kunyan
Published: July 2005

We report a case of bacteremia caused by
Janibacter melonis
, a recently described aerobic actinomycete originally isolated from a spoiled oriental melon. Our patient's blood culture isolate was identified by partial 16S rRNA gene sequencing. This is the first report of the recovery of
Janibacter
species from humans.
First Case of Human Cryptococcosis Due to
Cryptococcus neoformans
var.
gattii
in Spain
Authors: Colom M. Francisca , Frasés Susana , Ferrer Consuelo , Jover Alejandro , Andreu Mariano , Reus Sergio , Sánchez Manuel , Torres-Rodríguez Josep M.
Published: July 2005

We report the first case of human cryptococcosis due to
Cryptococcus neoformans
var.
gattii
described in our country, which was presented as brain cryptococcoma in an immunocompetent patient. An extensive sampling of the patient's environment was carried out to find the source of infection.
Isolation of
Actinobaculum schaalii
and
Actinobaculum urinale
from a Patient with Chronic Renal Failure
Authors: Fendukly Faiz , Osterman Björn
Published: July 2005

We report on the isolation of two species of
Actinobaculum
from blood culture of a patient with chronic renal failure. The two isolates were distinct with regard to their morphological and biochemical characteristics. Subsequent 16S rRNA gene sequencing classified the two species as
Actinobaculum schaalii
and
A. urinale
.