bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of Time-Saving Flow Cytometry for Rapid Determination of Resistance of Human Cytomegalovirus to Ganciclovir
Authors: Lee Gyu-Cheol , Lee Dong-Gun , Choi Su-Mi , Yoo Jin-Hong , Park Sun-Hee , Choi Jung-Hyun , Min Woo-Sung , Cho Ok-Hee , Lee Chan-Hee , Shin Wan-Shik 
Published: October 2005

ABSTRACT

There are two ways to assess the susceptibility of human cytomegalovirus (HCMV) to ganciclovir (GCV): one is a genotypic test that detects resistance-related mutations and the other is a phenotypic test that actually assesses susceptibility. The advantages of genotyping the UL97 gene are its rapidity and accuracy. However, to detect novel mutations or mutations affecting the UL54 DNA polymerase, a phenotypic test such as the plaque reduction assay (PRA) is also required. To avoid the shortcomings of PRA such as its time-consuming nature and labor-intensiveness, we developed a time-saving fluorescence-activated cell sorting (TS-FACS) technique. We obtained a GCV 50% inhibitory concentration (IC
50
) from five clinical isolates and an HCMV laboratory strain (AD169) and compared the results with those from the PRA. The laboratory strain and three clinical isolates were sensitive to GCV. Although there was a minor discrepancy in the case of one of the three isolates, the GCV IC
50
values obtained by TS-FACS analysis correlated well with the results of the PRA. The remaining two isolates were resistant to GCV; one was GCV resistant due to the mutation M460V, and the GCV IC
50
results obtained by TS-FACS analysis and by PRA were also comparable. The advantages of TS-FACS analysis are the shorter time required, the possibility of automation, and its comparability to PRA, considered the gold standard. Thus, TS-FACS analysis may be useful as an alternative to PRA in the clinic.
Phylogenetic Characterization of Canine Distemper Viruses Detected in Naturally Infected Dogs in North America
Authors: Pardo Ingrid D. R. , Johnson Gayle C. , Kleiboeker Steven B. 
Published: October 2005

ABSTRACT
In 2004, six puppies and one adult dog from a total of four premises were subjected to necropsy evaluation. For five of the seven dogs, disease caused by canine distemper virus (CDV) infection was suspected based on clinical signs. In all of the dogs, a diagnosis of CDV infection was established by the presence of compatible gross and histologic lesions, immunohistochemical labeling for CDV antigen, and detection of CDV RNA by reverse transcription-PCR. To further characterize the CDV strains detected in the four cases, complete gene sequences were determined for the hemagglutinin (H) and fusion (F) protein genes, while partial gene sequencing was performed for the phosphoprotein gene. A total of 4,508 bases were sequenced for the CDV strains detected from each of the four cases. Two cases were found to have identical sequences except for 2 bases in the intergenic region of the F and H genes. Phylogenetic analysis strongly suggested an evolutionary relationship between sequences detected in these two cases and those of phocine distemper virus 2 and two other strains of CDV not previously detected in the continental United States. Clear phylogenetic relationships were not established for viruses detected in the two additional cases; however, one strain showed similarity to CDV strains detected in a panda from China. Importantly, the three CDV strains detected were demonstrated to be genetically distinct from known vaccine strains and strains previously reported in the continental United States.
Novel Multiplex PCR Assay for Characterization and Concomitant Subtyping of Staphylococcal Cassette ChromosomemecTypes I to V in Methicillin-ResistantStaphylococcus aureus
Authors: Zhang Kunyan , McClure Jo-Ann , Elsayed Sameer , Louie Thomas , Conly John M. 
Published: October 2005

ABSTRACTStaphylococcal cassette chromosomemec(SCCmec) typing is essential for understanding the molecular epidemiology of methicillin-resistantStaphylococcus aureus(MRSA). SCCmecelements are currently classified into types I to V based on the nature of themecandccrgene complexes, and are further classified into subtypes according to their junkyard region DNA segments. Previously described traditional SCCmecPCR typing schemes require multiple primer sets and PCR experiments, while a previously published multiplex PCR assay is limited in its ability to detect recently discovered types and subtypes such as SCCmectype V and subtypes IVa, b, c, and d. We designed new sets of SCCmectype- and subtype-unique and specific primers and developed a novel multiplex PCR assay allowing for concomitant detection of the methicillin resistance (mecAgene) (also serving as an internal control) to facilitate detection and classification of all currently described SCCmectypes and subtypes I, II, III, IVa, b, c, d, and V. Our assay demonstrated 100% sensitivity and specificity in accurately characterizing 54 MRSA strains belonging to the various known SCCmectypes and subtypes, when compared with previously described typing methods. Further application of our assay in 453 randomly selected local clinical isolates confirmed its feasibility and practicality. This novel assay offers a rapid, simple, and feasible method for SCCmectyping of MRSA, and may serve as a useful tool for clinicians and epidemiologists in their efforts to prevent and control infections caused by this organism.
Diagnosis of Cerebral Toxoplasmosis in AIDS Patients in Brazil: Importance of Molecular and Immunological Methods Using Peripheral Blood Samples
Authors: Colombo Fabio A. , Vidal José E. , Oliveira Augusto C. Penalva de , Hernandez Adrián V. , Bonasser-Filho Francisco , Nogueira Roberta S. , Focaccia Roberto , Pereira-Chioccola Vera Lucia 
Published: October 2005

ABSTRACTCerebral toxoplasmosis is the most common cerebral focal lesion in AIDS and still accounts for high morbidity and mortality in Brazil. Its occurrence is more frequent in patients with low CD4+T-cell counts. It is directly related to the prevalence of anti-Toxoplasma gondiiantibodies in the population. Therefore, it is important to evaluate sensitive, less invasive, and rapid diagnostic tests. We evaluated the value of PCR using peripheral blood samples on the diagnosis of cerebral toxoplasmosis and whether its association with immunological assays can contribute to a timely diagnosis. We prospectively analyzed blood samples from 192 AIDS patients divided into two groups. The first group was composed of samples from 64 patients with cerebral toxoplasmosis diagnosed by clinical and radiological features. The second group was composed of samples from 128 patients with other opportunistic diseases. Blood collection from patients with cerebral toxoplasmosis was done before or on the third day of anti-toxoplasma therapy. PCR forT. gondii, indirect immunofluorescence, enzyme-linked immunosorbent assay, and an avidity test for toxoplasmosis were performed on all samples. The PCR sensitivity and specificity for diagnosis of cerebral toxoplasmosis in blood were 80% and 98%, respectively. Patients with cerebral toxoplasmosis (89%) presented higher titers of anti-T. gondiiIgG antibodies than patients with other diseases (57%) (P< 0.001). These findings suggest the clinical value of the use of both PCR and high titers of anti-T. gondiiIgG antibodies for the diagnosis of cerebral toxoplasmosis. This strategy may prevent more invasive approaches.
Clinical and Molecular Analysis of Extended-Spectrum β-Lactamase-Producing Enterobacteria in the Community Setting
Authors: Arpin Corinne , Dubois Véronique , Maugein Jeanne , Jullin Jacqueline , Dutilh Brigitte , Brochet Jean-Philippe , Larribet Gilberte , Fischer Isabelle , Quentin Claudine 
Published: October 2005

ABSTRACT

During a previous survey, five extended-spectrum β-lactamase (ESBL)-producing enterobacteria (ESBLE) (two
Enterobacter aerogenes
isolates expressing TEM-24b, two
Escherichia coli
isolates expressing TEM-21 or TEM-24b, and one
Klebsiella pneumoniae
isolate expressing SHV-4/TEM-15) responsible for urinary tract infections (UTIs) were found among 1,584 strains collected from community patients. The aim of the present study was to elucidate the route of emergence of these typically nosocomial organisms in the community. Thus, the files of the five patients were analyzed over at least a decade, and potentially related ESBLE from hospitals or clinics were examined. Their enzymes were characterized at a molecular level, and the strains were typed by amplified-primed PCR, enterobacterial repetitive intergenic consensus PCR, and restriction plasmid profile. All patients (C1 to C5) had risk factors for ESBLE acquisition, including past history of hospitalization (2.5 to 23 months before). Four (C1 and C3 to C5) had previously received antibiotics (concomitantly to 35 months earlier), two (C1 and C3) had indwelling urinary catheters and recurrent UTIs, and three (C2, C3, and C5) formerly experienced ESBLE-induced UTIs (2 to 11 months before). The same ESBLE and/or an identical or similar ESBL-encoding plasmid was identified in the hospital ward (C1 to C4) or in a clinic (C5) where the patients had previously resided. Patients C1 and C4, infected with different ESBLE carrying a closely related plasmid, were hospitalized in the same unit. Persistence of ESBLE over at least a 5-year period was demonstrated for patient C3. Thus, community-acquired UTIs in these patients likely resulted from nosocomially acquired ESBLE or an ESBL-encoding plasmid followed by a prolonged digestive carriage.
Real-Time Reverse Transcription PCR for Detection and Quantitative Analysis of Equine Influenza Virus
Authors: Quinlivan Michelle , Dempsey Eugene , Ryan Fergus , Arkins Sean , Cullinane Ann 
Published: October 2005

ABSTRACTEquine influenza is a cause of epizootic respiratory disease of the equine. The detection of equine influenza virus using real-time Light Cycler reverse transcription (RT)-PCR technology was evaluated over two influenza seasons with the analysis of 171 samples submitted for viral respiratory disease. Increased sensitivity was found in overall viral detection with this system compared to Directigen Flu A and virus isolation, which were 40% and 23%, respectively, that of the RT-PCR. The assay was also evaluated as a viable replacement for the more traditional methods of quantifying equine influenza virus, 50% egg infectious dose and 50% tissue culture infectious dose. There was a significant positive correlation (P< 0.05) between the quantitative RT-PCR and both of these assays.
Validation of a Rapid and Reliable Test for Diagnosis of Chagas' Disease by Detection of
Trypanosoma cruzi
-Specific Antibodies in Blood of Donors and Patients in Central America
Authors: Ponce Carlos , Ponce Elisa , Vinelli Elizabeth , Montoya Alberto , de Aguilar Vilma , Gonzalez Antonio , Zingales Bianca , Rangel-Aldao Rafael , Levin Mariano J. , Esfandiari Javan , Umezawa Eufrosina S. , Luquetti Alejandro O. , da Silveira José Franco 
Published: October 2005

ABSTRACT
In this study we compared the performance of the Chagas Stat-Pak rapid immunochromatographic test with a standard enzyme-linked immunosorbent assay (ELISA) in the serodiagnosis of Chagas' disease in Central America. Out of 3,400 blood donor samples, 156 (4.6%) were positive in both assays. Three sera out of 2,084 samples from reference laboratories were negative with the rapid test but positive with the ELISA (99.8% agreement). Agreement of 100% between the two tests was observed with 339 additional sera from patients with cardiopathies and 175 sera from potential blood donors in emergency surgical cases occurring on weekends or at night. In conclusion, Chagas Stat-Pak showed 99.6% and 99.9% sensitivity and specificity, respectively, when assayed with 5,998 serum samples. It is a sensitive and specific alternative to the ELISA, as required in medical emergencies and blood screenings in Central America.
Antibiotic Susceptibilities of
Pseudomonas aeruginosa
Isolates Derived from Patients with Cystic Fibrosis under Aerobic, Anaerobic, and Biofilm Conditions
Authors: Hill Dominic , Rose Barbara , Pajkos Aniko , Robinson Michael , Bye Peter , Bell Scott , Elkins Mark , Thompson Barbara , MacLeod Colin , Aaron Shawn D. , Harbour Colin 
Published: October 2005

ABSTRACT

Recent studies have determined that
Pseudomonas aeruginosa
can live in a biofilm mode within hypoxic mucus in the airways of patients with cystic fibrosis (CF).
P. aeruginosa
grown under anaerobic and biofilm conditions may better approximate in vivo growth conditions in the CF airways, and combination antibiotic susceptibility testing of anaerobically and biofilm-grown isolates may be more relevant than traditional susceptibility testing under planktonic aerobic conditions. We tested 16 multidrug-resistant isolates of
P. aeruginosa
derived from CF patients using multiple combination bactericidal testing to compare the efficacies of double and triple antibiotic combinations against the isolates grown under traditional aerobic planktonic conditions, in planktonic anaerobic conditions, and in biofilm mode. Both anaerobically grown and biofilm-grown bacteria were significantly less susceptible (
P
< 0.01) to single and combination antibiotics than corresponding aerobic planktonically grown isolates. Furthermore, the antibiotic combinations that were bactericidal under anaerobic conditions were often different from those that were bactericidal against the same organisms grown as biofilms. The most effective combinations under all conditions were colistin (tested at concentrations suitable for nebulization) either alone or in combination with tobramycin (10 μg ml
−1
), followed by meropenem combined with tobramycin or ciprofloxacin. The findings of this study illustrate that antibiotic sensitivities are dependent on culture conditions and highlight the complexities of choosing appropriate combination therapy for multidrug-resistant
P. aeruginosa
in the CF lung.
Transmission of
Burkholderia cepacia
Complex: Evidence for New Epidemic Clones Infecting Cystic Fibrosis Patients in Italy
Authors: Campana S. , Taccetti G. , Ravenni N. , Favari F. , Cariani L. , Sciacca A. , Savoia D. , Collura A. , Fiscarelli E. , De Intinis G. , Busetti M. , Cipolloni A. , d'Aprile A. , Provenzano E. , Collebrusco I. , Frontini P. , Stassi G. , Trancassini M. , Tovagliari D. , Lavitola A. , Doherty C. J. , Coenye T. , Govan J. R. W. , Vandamme P. 
Published: October 2005

ABSTRACT

To analyze national prevalence, genomovar distribution, and epidemiology of the
Burkholderia cepacia
complex in Italy, 225 putative
B. cepacia
complex isolates were obtained from 225 cystic fibrosis (CF) patients attending 18 CF centers. The genomovar status of these isolates was determined by a polyphasic approach, which included whole-cell protein electrophoresis and
recA
restriction fragment length polymorphism (RFLP) analysis. Two approaches were used to genotype
B. cepacia
complex isolates: BOX-PCR fingerprinting and pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments. A total of 208 (92%) of 225 isolates belonged to the
B. cepacia
complex, with
Burkholderia cenocepacia
as the most prevalent species (61.1%). Clones delineated by PFGE were predominantly linked to a single center; in contrast, BOX-PCR clones were composed of isolates collected either from the same center or from different CF centers and comprised multiple PFGE clusters. Three BOX-PCR clones appeared of special interest. One clone was composed of 17
B. cenocepacia
isolates belonging to
recA
RFLP type H. These isolates were collected from six centers and represented three PFGE clusters. The presence of insertion sequence IS
1363
in all isolates and the comparison with PHDC reference isolates identified this clone as PHDC, an epidemic clone prominent in North American CF patients. The second clone included 22 isolates from eight centers and belonged to
recA
RFLP type AT. The genomovar status of strains with the latter RFLP type is not known. Most of these isolates belonged to four different PFGE clusters. Finally, a third clone comprised nine
B. pyrrocinia
isolates belonging to
recA
RFLP type Se13. They represented three PFGE clusters and were collected in three CF centers.
Comparison of Genetic Backgrounds of Methicillin-Resistant and -Susceptible
Staphylococcus aureus
Isolates from Portuguese Hospitals and the Community
Authors: de Sousa M. Aires , Conceição T. , Simas C. , de Lencastre H. 
Published: October 2005

ABSTRACT

In order to understand the origins of the dominant methicillin-resistant
Staphylococcus aureus
(MRSA) clones in Portuguese hospitals, we compared the genetic backgrounds of nosocomial MRSA with methicillin-susceptible
S. aureus
(MSSA) isolates from the same hospitals (
n
= 155) and from the community (
n
= 157) where they were located. Pulsed-field gel electrophoresis,
spa
typing, multilocus sequence typing, and
agr
type analysis revealed that the genetic backgrounds correspondent to the dominant MRSA clones in Portuguese hospitals during the last 15 years (Iberian ST247, Brazilian ST239, and EMRSA-15 ST22) were scarcely or not found among the present MSSA collection. The four major MSSA clones encountered (A-ST30, B-ST34, C-ST5, and H-ST45) correspond, or are very similar, to the background of other international MRSA pandemic clones, i.e., EMRSA-16, New York/Japan, Pediatric, and Berlin clones. However, with the exception of the Pediatric clone, none of these MRSA clones has been detected in Portugal. Our findings suggest the three major MRSA clones identified in Portuguese hospitals have not originated from the introduction of SCC
mec
into dominant MSSA backgrounds present in the Portuguese nosocomial or community environment but were probably imported from abroad. In contrast, the MRSA Pediatric clone might have originated in our country by the acquisition of SCC
mec
type IV into MSSA clone C. Furthermore, we provide evidence that the introduction of SCC
mec
into sensitive clones is most likely a relatively infrequent event that seems to depend not exclusively on the presence of a successful MSSA lineage.
Rapid Genotyping of Hepatitis C Virus by Primer-Specific Extension Analysis
Authors: Antonishyn Nick A. , Ast Vivian M. , McDonald Ryan R. , Chaudhary Rabindra K. , Lin Lisa , Andonov Anton P. , Horsman Greg B. 
Published: October 2005

ABSTRACT

Quick and accurate genotyping of hepatitis C virus (HCV) is becoming increasingly important for clinical management of chronic infection and as an epidemiological marker. Furthermore, the incidence of HCV infection with mixed genotypes has clinical significance that is not addressed by most genotyping methods. We have developed a fluorescence-based genotyping assay called primer-specific extension analysis (PSEA) for the most prevalent HCV genotypes and have demonstrated the capacity of PSEA-HCV for detecting mixed-genotype HCV infections. PSEA-HCV detects genotype-specific sequence differences in the 5′ untranslated region of HCV in products amplified by the COBAS AMPLICOR HCV Test, v2.0. Simulated mixed HCV infection of plasma with RNase-resistant RNA controls demonstrates that PSEA-HCV can detect as many as five genotypes in one specimen. Furthermore, in dual-genotype simulations, PSEA-HCV can unequivocally detect both genotypes, with one genotype representing only 3.1% of the mixture (313/10,000 IU in starting plasma). Compared to INNO-LiPA HCV II, both assays determined the same genotype for 191/199 (96%) patient specimens (175 subtype and 16 genotype-only identifications). Following the initial evaluation, PSEA-HCV was used routinely to genotype HCV from patient specimens submitted to our laboratory (
n
= 312). Seventeen (5.4%) mixed infections were identified. The distribution of single-infection HCV genotypes in our population was 60.9% type 1 (
n
= 190), 12.8% type 2 (
n
= 40), 20.2% type 3 (
n
= 63), 0.3% type 4 (
n
= 1), and 0.3% other (
n
= 1). In conclusion, PSEA-HCV provides an inexpensive, high-throughput screening tool for rapid genotyping of HCV while reliably identifying mixed HCV infections.
Characterization of New Recombinant Noroviruses
Authors: Ambert-Balay K. , Bon F. , Le Guyader F. , Pothier P. , Kohli E. 
Published: October 2005

ABSTRACT
Noroviruses are important etiologic agents of acute gastroenteritis and show great genetic diversity. To characterize more fully previously detected strains that could not be assigned unequivocally to one particular genotype based on the RNA polymerase, we have sequenced a region in the capsid gene and, in some cases, in the junction between open reading frame 1 (ORF1) and ORF2. The results allowed us to identify several recombinant noroviruses: GGIIb viruses were detected for the first time in France in August 2000 and then spread through France and to Europe during the following winter. Here we present the characterization of three other probable GII recombinants which showed different phylogenetic positions depending on their ORF1 and ORF2 sequences. Analysis of the region located between ORF1 and ORF2 by a nucleotide identity window search showed a sudden shift in similarities. Moreover, recombination breakpoints were identified upstream and downstream of the beginning of ORF2 by using a statistical test, thus confirming the involvement of this region in recombination. Unlike GGIIb, the three recombinants described here do not seem to have diffused widely in the community: one was found in a waterborne outbreak, and the other two were found in sporadic cases. Recombination is important for the evolution of RNA viruses and has already been described for noroviruses. Our results suggest that recombination is not a rare phenomenon among noroviruses, but not all these presumed recombinants that formed during RNA replication are able to spread widely.
Molecular Characterization of Hand Flora and Environmental Isolates in a Community Setting
Authors: Pancholi Preeti , Healy Mimi , Bittner Tracy , Webb Renee , Wu Fan , Aiello Allison , Larson Elaine , Latta Phyllis Della 
Published: October 2005

ABSTRACT

We analyzed 69 bacterial isolates, comprising seven species of gram-negative bacterial rods and three species of coagulase-negative staphylococci, recovered from both the hands of caretakers and their environment in households sampled in upper Manhattan. Repetitive sequence-based PCR and dendrogram analysis were used to determine strain similarity. Greater than 25% of individual species of
Acinetobacter
,
Enterobacter
, and coagulase-negative staphylococci recovered from the hands and immediate environment within each household shared the same genotype. This study is the first to demonstrate the frequency of bacteria shared within community households. These strains may serve as potential reservoirs for either community- or hospital-acquired infections.