bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of a Multiplex Molecular Beacon Platform for Rapid Detection of Methicillin and Vancomycin Resistance in
Staphylococcus aureus
Authors: Sinsimer Daniel , Leekha Surbhi , Park Steven , Marras Salvatore A. E. , Koreen Larry , Willey Barbara , Naidich Steve , Musser Kimberlee A. , Kreiswirth Barry N. 
Published: September 2005

ABSTRACT

Drug resistance, particularly vancomycin and methicillin resistance, in
Staphylococcus aureus
continues to emerge as a significant public health threat in both the hospital and community settings. In addition to the limited treatment options,
S. aureus
strains acquire and express numerous virulence factors that continue to increase its ability to cause a wide spectrum of human disease. As a result, empirical treatment decisions are confounded and there is a heightened need for a diagnostic test (or assay) to rapidly identify antibiotic resistance and specific virulence determinants and indicate the appropriate treatment. To that end we developed a platform using multiplex molecular beacon probes with real-time PCR for the rapid detection of drug resistance-determining genes and virulence factors in
S. aureus
. In this study, we demonstrate the specificity and sensitivity of our platform for detection of the genes conferring methicillin (
mecA
) and vancomycin (
vanA
) resistance as well as a gene encoding the virulence factor Panton-Valentine leucocidin (
lukF
) in
S. aureus
isolates.
Anticoagulant Carryover May Influence Clot Formation in Direct Tube Coagulase Tests from Blood Cultures
Authors: Varettas Kerry , Mukerjee Chinmoy , Taylor Peter C. 
Published: September 2005

ABSTRACT

The tube coagulase test (TCT) performed directly from positive blood culture bottles has been used to reduce the turnaround time for identifying
Staphylococcus aureus
. Most reports have shown the test to be specific but often lacking sufficient sensitivity to be useful. In a prospective study of blood culture bottles (BCB) signaling positive, with a Gram-stained smear showing gram-positive cocci resembling staphylococci, the sensitivity of the direct TCT was improved by diluting the BCB broth 1:10 in saline before inoculating 0.1 ml into 1.0 ml of 10% pooled human plasma. It was hypothesized that the improved sensitivity might be explained by reduced carryover of the anticoagulant sodium polyanetholesulfonate (SPS) used in blood culture media. By titrating the inoculum size and the concentration of SPS in an in vitro checkerboard assay, it was shown that concentrations of SPS >0.0008% prevented plasma coagulation. The 1:10 dilution of blood culture broth reduced the amount of residual SPS carried over to the TCT to a level (0.0005%) that did not impair plasma coagulation. The direct TCT inoculated with a 1:10 saline dilution of blood culture broth achieved 100% specificity and sensitivity within 4 h of inoculation without reducing the quality or quantity of coagulum.
Genotyping of B and Non-B Subtypes of Human Immunodeficiency Virus Type 1
Authors: Tong C. Y. W. , Mullen J. , Kulasegaram R. , De Ruiter A. , O'Shea S. , Chrystie I. L. 
Published: September 2005

ABSTRACT

Current HIV-1 genotyping assays were developed using subtype B viruses prevalent in Western countries. It is not clear whether these assays are appropriate for use among African patients, who are likely to be infected with non-B subtypes. We evaluated the Bayer TRUGENE HIV-1 genotyping (TG) assay using prospectively collected samples from HIV-1-infected individuals who acquired infection in either sub-Saharan Africa or the West (Europe, North America, and Australia). Plasma samples from 208 individuals with an HIV-1 viral load of >1,000 copies/ml were tested using version 1 primers supplied with the TG assay. If these failed, an alternative primer set version 1.5 was used. Of the 208 individuals, the likely origin of infection was Africa (
n
= 104), Western (
n
= 87) and “Others” (i.e., all other geographic locations or origin not certain;
n
= 17). Among the three groups, the version 1 primers were successful in 85 (82%), 77 (89%), and 13 (76%) individuals, respectively (
P
= 0.1). Of the remaining 32 samples, 30 were successfully amplified by using the version 1.5 primers. HIV-1 subtypes deduced from the reverse transcriptase sequences correlated with the likely origin of infection: Africa (28A, 3B, 33C, 13D, 6G, 4J, 2K, 5CRF01_AE, and 10CRF02_AG), Western (86B and 1K), and Others (1A and 16B). The success of the version 1 primers correlated with viral load (
P
< 0.014) and not with HIV-1 subtypes. A protocol based on version 1 primers, followed by 1.5 primers, was successful in sequencing 99% of the samples in this cohort.
Prevalence of Toxic Shock Syndrome Toxin 1-Producing
Staphylococcus aureus
and the Presence of Antibodies to This Superantigen in Menstruating Women
Authors: Parsonnet Jeffrey , Hansmann Melanie A. , Delaney Mary L. , Modern Paul A. , DuBois Andrea M. , Wieland-Alter Wendy , Wissemann Kimberly W. , Wild John E. , Jones Michaelle B. , Seymour Jon L. , Onderdonk Andrew B. 
Published: September 2005

ABSTRACT

Menstrual toxic shock syndrome (mTSS) is thought to be associated with colonization with toxic shock syndrome toxin 1 (TSST-1)-producing
Staphylococcus aureus
in women with insufficient antibody titers. mTSS has been associated with menstruation and tampon use, and although it is rare, the effects can be life threatening. It remains of interest because of the widespread use of tampons, reported to be about 70% of women in the United States, Canada, and much of Western Europe. This comprehensive study was designed to determine
S. aureus
colonization and TSST-1 serum antibody titers in 3,012 menstruating women in North America between the ages of 13 and 40, particularly among age and racial groups that could not be assessed reliably in previous small studies. One out of every four subjects was found to be colonized with
S. aureus
in at least one of three body sites (nose, vagina, or anus), with approximately 9% colonized vaginally. Eighty-five percent of subjects had antibody titers (≥1:32) to TSST-1, and the vast majority (81%) of teenaged subjects (13 to 18 years) had already developed antibody titers. Among carriers of toxigenic
S. aureus
, a significantly lower percentage of black women than of white or Hispanic women were found to have antibody titers (≥1:32) to TSST-1 (89% versus 98% and 100%). These findings demonstrate that the majority of teenagers have antibody titers (≥1:32) to TSST-1 and are presumed to be protected from mTSS. These findings also suggest that black women may be more susceptible to mTSS than previously thought.
Discordance between Viral Loads Determined by Roche COBAS AMPLICOR Human Immunodeficiency Virus Type 1 Monitor (Version 1.5) Standard and Ultrasensitive Assays Caused by Freezing Patient Plasma in Centrifuged Becton-Dickinson Vacutainer Brand Plasma Preparation Tubes
Authors: Salimnia Hossein , Moore Ellen C. , Crane Lawrence R. , MacArthur Rodger D. , Fairfax Marilynn R. 
Published: September 2005

ABSTRACT

The Roche COBAS AMPLICOR human immunodeficiency virus type 1 (HIV-1) Monitor (version 1.5) standard and ultrasensitive viral load assays often gave discordant results, with viral loads from the standard assay exceeding those from the ultrasensitive assay by more than 0.5 log
10
for approximately 20% of specimens received. We began studies to determine the extent, magnitude, and reproducibility of the discordance between the assays and to discover and eliminate the cause of this discordance. Until then, we revised our standard operating procedure to include both standard and ultrasensitive testing on all specimens submitted for viral load determinations. Discordant results usually recurred on retesting. They were most prevalent for specimens with ultrasensitive viral loads of <1,000 and rare for specimens with viral loads of >10,000. Often, standard assay results exceeded those of the ultrasensitive assay by 50- to 100-fold. At higher viral loads, the difference between the standard and ultrasensitive assays persisted, but the percent difference was smaller and rarely caused discordance. The proportion of discordant results was significantly higher in specimens from pediatric patients than in specimens from adults. The ultrasensitive viral load determinations generally agreed with the results of the B-DNA (Bayer) viral load assays. If the plasma was transferred from the centrifuged plasma preparation tubes before freezing, standard and ultrasensitive results were concordant with each other and with values determined on plasma from lavender-topped EDTA tubes.
Comparison of Two Probes for Testing Susceptibilities of Pathogenic Yeasts to Voriconazole, Itraconazole, and Caspofungin by Flow Cytometry
Authors: Pina-Vaz Cidália , Costa-de-Oliveira Sofia , Rodrigues Acácio G. , Espinel-Ingroff Ana 
Published: September 2005

ABSTRACT

A cytometric approach to determine the susceptibilities of
Candida
spp. and
Cryptococcus neoformans
to voriconazole, itraconazole, and caspofungin is described. A total of 63 clinical isolates with different susceptibility patterns were exposed for 1, 2, 4, and 6 h to serial concentrations of each antifungal agent, followed by staining with two fluorescent probes: propidium iodide (PI) and FUN-1. FUN-1 was able to identify the susceptibility patterns of the assayed strains to the three agents after 1 h. PI penetrated a maximum of 50% of the cells treated with PI, at the highest concentration of caspofungin, 16 μg/ml, after 6 h of incubation (this percentage varied with the strain and was drug concentration and time of incubation dependent) and did not stain cells treated with high concentrations of either azole after 6 h. The use of FUN-1 appears to be an excellent fast and reliable alternative to the classical dilution method for determining the susceptibility of
Candida
spp. and
C. neoformans
to these three antifungal agents.
Diagnosis of Neuroparacoccidioidomycosis by Detection of Circulating Antigen and Antibody in Cerebrospinal Fluid
Authors: Silva Silvia Helena Marques da , Colombo Arnaldo Lopes , Blotta Maria Heloisa Souza Lima , Queiroz-Telles Flávio , Lopes José Daniel , Camargo Zoilo Pires de 
Published: September 2005

ABSTRACT

Neuroparacoccidioidomycosis (neuroPCM) is the central nervous system infection by the fungus
Paracoccidioides brasiliensis
. Its diagnosis is a difficult task that depends on neuroimaging techniques such as computed tomography and magnetic resonance imaging. However, the detection of circulating
P. brasiliensis
antigens in body fluids by inhibition enzyme-linked immunosorbent assay (inh-ELISA) has provided encouraging results. In this study, 14 cerebrospinal fluid (CSF) and 11 serum samples of patients with neuroPCM were analyzed by inh-ELISA for detection of circulating glycoprotein antigens of 43 kDa (gp43) and 70 kDa (gp70). Circulating gp43 and gp70 antigens were detected in all CSF samples from patients with neuroPCM at mean concentrations of 19.3 and 6.8 μg/ml, respectively. In addition, both gp43 and gp70 antigens were detected in 10 of 11 serum samples analyzed at mean concentrations of 4.6 and 4.0 μg/ml, respectively. By immunodiffusion test, CSF samples were determined to be negative in 13 of 14 samples. The detection of anti-gp43 and anti-gp70 antibodies by conventional ELISA showed positive results for all CSF samples, with titers ranging from 1:50 to 1:51,200. Therefore, the high sensitivity of the inh-ELISA technique in detecting gp43 and gp70 antigens in the CSF of neuroPCM patients strongly indicates that this assay can be considered as a powerful diagnostic tool. In addition, the finding of anti-gp43 and anti-gp70 antibodies in CSF samples by conventional ELISA also seems to be a promising diagnostic method for this special modality of PCM.
Properties of Novel International Drug-Resistant Pneumococcal Clones Identified in Day-Care Centers of Lisbon, Portugal
Authors: Sousa Natacha G. , Sá-Leão Raquel , Crisóstomo M. Inês , Simas Carla , Nunes Sónia , Frazão Nelson , Carriço João A. , Mato Rosario , Santos-Sanches Ilda , de Lencastre Hermínia 
Published: September 2005

ABSTRACT

In this study, 61 drug-resistant
Streptococcus pneumoniae
strains were characterized by multilocus sequence typing (MLST). These strains were representatives of 26 major clones (defined using pulsed-field gel electrophoresis) accounting for 93% of the 1,285 drug-resistant
Streptococcus pneumoniae
isolates recovered from the nasopharynges of healthy children attending day-care centers in Lisbon during 2001 to 2003. Using MLST, 13 of the 26 clones were found to be identical or closely related to 11 Pneumococcal Molecular Epidemiology Network (PMEN) clones, 4 clones were found to be unique as there were no identical or highly related allelic profiles deposited in the MLST database, and the remaining 9 clones had sequence types that matched or differed at a single or double locus from allelic profiles available in the MLST database. These nine clones were of serotypes 33F, 10A, 19A, 19F, 6A, 20, 24F, and 3, one was nontypeable, and, by MLST, they were found to be identical or highly related to isolates from disease origin that were dispersed internationally. Since the majority of these clones had serotypes that are not included in the 7-valent conjugate pneumococcal vaccine, monitoring of these clones is important for surveying their possible spread in the future. We propose the inclusion of these novel international clones in the PMEN.
Comparison of a 3-Set Genotyping System with Multilocus Sequence Typing for
Streptococcus agalactiae
(Group B Streptococcus)
Authors: Sun Ying , Kong Fanrong , Zhao Zuotao , Gilbert Gwendolyn L. 
Published: September 2005

ABSTRACT

Group B streptococcus (GBS;
Streptococcus agalactiae
) is the most common cause of neonatal and obstetric sepsis and is an increasingly important cause of septicemia in elderly individuals and immunocompromised patients. Epidemiological studies of GBS infections require comprehensive typing systems that provide information about variable characteristics, such as antigenic type, virulence, or antibiotic resistance, as well as the “backbone” structure or the genetic lineage of isolates. We have previously described a 3-set genotyping system that identifies the molecular serotype (MS) or molecular serosubtype (msst), the protein gene profile, and the presence of several mobile genetic elements (F. Kong, D. Martin, G. James, and G. L. Gilbert, J. Med. Microbiol. 52:337-344, 2003). In this study, 83 clinical GBS isolates which had been previously studied by multilocus sequence typing (MLST) (N. Jones, J. F. Bohnsack, S. Takahashi, K. A. Oliver, M. S. Chan, F. Kunst, P. Glaser, C. Rusniok, D. W. Crook, R. M. Harding, N. Bisharat, and B. G. Spratt, J. Clin. Microbiol. 41:2530-2536, 2003) were examined by using the 3-set genotyping system. Genotypes were assigned to five isolates that were nontypeable by conventional serotyping. There were 27 “3-set” genotypes, 24 multilocus sequence types (STs), and 35 unique combinations (or strains), of which the 4 most common, msst III-2 (ST-17), msst III-1 (ST-19), Ia-1 (ST-23), and V-1 (ST-1), accounted for more than 60% of isolates. The 83 isolates were grouped into seven clusters, with a good correlation between the multilocus STs and the genotypes. The combination of 3-set genotyping and MLST adds discriminatory power to strain typing of GBS, which will be useful for future studies of the epidemiology and pathogenesis of GBS disease.
Multispacer Typing of
Rickettsia prowazekii
Enabling Epidemiological Studies of Epidemic Typhus
Authors: Zhu Yong , Fournier Pierre-Edouard , Ogata Hiroyuki , Raoult Didier 
Published: September 2005

ABSTRACT

Currently, there is no tool for typing
Rickettsia prowazekii
, the causative agent of epidemic typhus, currently considered a potential bioterrorism agent, at the strain level. To test if the multispacer typing (MST) method could differentiate strains of
R. prowazekii
, we amplified and sequenced the 25 most variable intergenic spacers between the
R. prowazekii
and
R. conorii
genomes in five strains and 10 body louse amplicons of
R. prowazekii
from various geographic origins. Two intergenic spacers, i.e.,
rpmE
/tRNA
fMet
and
serS
/
virB4
, were variable among tested
R. prowazekii
isolates and allowed identification of three and two genotypes, respectively. When the genotypes obtained from the two spacers were combined, we identified four different genotypes. MST demonstrated that several
R. prowazekii
strains circulated in human body lice during an outbreak of epidemic typhus in Burundi. This may help to discriminate between natural and intentional outbreaks. Our study supports the usefulness of MST as a versatile method for rickettsial strain genotyping.
Diagnosis of and Screening for Cytomegalovirus Infection in Pregnant Women
Authors: Munro S. C. , Hall B. , Whybin L. R. , Leader L. , Robertson P. , Maine G. T. , Rawlinson W. D. 
Published: September 2005

ABSTRACTNo single diagnostic test for cytomegalovirus (CMV) infection is currently available for pregnant women at all stages of gestation. Improved accuracy in estimating the timing of primary infections can be used to identify women at higher risk of giving birth to congenitally infected infants. A diagnostic algorithm utilizing immunoglobulin G (IgG), IgM, and IgG avidity was used to prospectively screen serum from 600 pregnant women enrolled from two groups: ≤20 weeks gestation (n= 396) or >20 weeks gestation (n= 204). PCR testing of urine and/or blood was performed on all seropositive women (n= 341). The majority (56.8%) of women were CMV IgG seropositive, with 5.5% being also CMV IgM positive. In the IgM-positive women, 1.2% had a low-avidity IgG, indicating a primary CMV infection and a high risk of intrauterine transmission. Two infants with asymptomatic CMV infection were born of mothers who had seroconverted in the second trimester of pregnancy. Baseline, age-stratified CMV serostatus was established from 1,018 blood donors. Baseline seropositivity from a blood donor population increased with age from 34.9% seroprevalence at less than 20 years of age to 72% seroprevalence at 50 years of age. Women at high risk of intrauterine transmission of CMV were identified at all stages of gestation. Women infected with CMV during late gestation may be more likely to transmit the virus, so failure to detect seroconversions in late gestation may result in failure to detect infected neonates.
Multilocus Sequence Typing of
Staphylococcus aureus
Isolated from High-Somatic-Cell-Count Cows and the Environment of an Organic Dairy Farm in the United Kingdom
Authors: Smith E. M. , Green L. E. , Medley G. F. , Bird H. E. , Dowson C. G. 
Published: September 2005

ABSTRACT

An outbreak of
Staphylococcus aureus
intramammary infections on an organic dairy farm was monitored for 10 months. Environmental and milk samples were collected from a total of 26 cows and a group of 21 purchased heifers about to be introduced into the milking herd. There was variation in the rate of isolation of
S. aureus
(9.5 to 43.8%) from individual mammary quarters, although no
S. aureus
isolates were detected in the milk samples collected from the heifers. One hundred ninety-one
S. aureus
isolates were detected from cow milk samples (
n
= 182), milking machine clusters (
n
= 4), farm personnel (
n
= 4), and the environment (
n
= 1). Multilocus sequence typing (MLST) had a typeability of 100% when it was applied to the 191 isolates. Among the 191 isolates there was limited strain diversity, with seven sequence types (STs) dominated by two strains with closely related STs that differed at a single locus. Within individual mammary quarters there were naturally occurring dual infections, although this was identified in only 0.4% of milk samples. Different strains were associated with variable persistence within quarters. MLST is clearly a very suitable tool for the differentiation and analysis of
S. aureus
populations detected on dairy cattle farms.
Multilocus Sequence Typing of Intercontinental BovineStaphylococcus aureusIsolates
Authors: Smith E. M. , Green L. E. , Medley G. F. , Bird H. E. , Fox L. K. , Schukken Y. H. , Kruze J. V. , Bradley A. J. , Zadoks R. N. , Dowson C. G. 
Published: September 2005

ABSTRACTA total of 258 bovine-associatedStaphylococcus aureusisolates from the United States, Chile, and the United Kingdom, plus the reference isolateS. aureusNewbould 305 (NCIMB 702892), were analyzed by multilocus sequence typing (MLST). A collection of previously characterized United Kingdom isolates were also included in the analysis. The results demonstrated that MLST is suitable for the differentiation of bovineS. aureusisolates from various sites (milk, teat skin, milking machine unit liners, hands, and bedding) and countries. The theory of the host specificity ofS. aureusis supported by the detection of a previously undescribed clonal complex that comprised 87.4% of the isolates studied, with representatives from all geographic locations investigated. This suggests that a single clonal group has achieved a widespread distribution and is responsible for the majority of infections. Some sequence types (STs; ST25, ST115, ST124, and ST126) demonstrated site specificity, as they were significantly (P< 0.05) associated with milk or teat skin.
Development of a Nested PCR Method Targeting a Unique Multicopy Element, ISMap02, for Detection ofMycobacterium aviumsubsp.paratuberculosisin Fecal Samples
Authors: Stabel J. R. , Bannantine J. P. 
Published: September 2005

ABSTRACTThis study describes the development of a nested PCR assay that uses a unique element (ISMap02) forMycobacterium aviumsubsp.paratuberculosisthat is present at six copies within the genome. In addition, the sensitivity of the assay with this element was compared to the sensitivity of detection of the IS900element in both conventional and real-time PCR assays. The specificity of the ISMap02element was evaluated by PCR of the DNA extracted from isolates ofM. aviumsubsp.paratuberculosisandM. aviumsubsp.avium, as well as DNA fromM. fortuitum,M. scofulaceum,M. phlei,M. smegmatis, andM. gordonae. OnlyM. aviumsubsp.paratuberculosisDNA was detectable after amplification with the ISMap02primers. The sensitivity of detection for the ISMap02element in either a conventional or a real-time PCR format was less than 100 fg DNA or 102CFU/ml in serial titration curves with pure bacteria. These results were comparable to those obtained for the IS900element. Experimental spiking of a negative fecal sample followed byM. aviumsubsp.paratuberculosisDNA extraction resulted in detection thresholds of 102CFU/g for the IS900element and 103CFU/g for the ISMap02element by using a real-time PCR format, but this sensitivity dropped 10-fold for both elements in a conventional PCR format. Analyses of fecal samples obtained from naturally infected animals demonstrated a sensitivity for the detection ofM. aviumsubsp.paratuberculosisDNA by use of the ISMap02element similar to that achieved by use of the IS900element when it was used in a conventional PCR format. The real-time PCR format improved the levels of detection of both elements, but not to a significant degree. In conclusion, the ISMap02element provides a very sensitive and specific alternative as a diagnostic reagent for use in PCR assays for the detection of paratuberculosis.