bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a Multilocus Sequence Typing Scheme for Characterization of Clinical Isolates of
Acinetobacter baumannii
Authors: Bartual Sergio G. , Seifert Harald , Hippler Corinna , Luzon M. Angeles Domínguez , Wisplinghoff Hilmar , Rodríguez-Valera Francisco
Published: September 2005

In this study a multilocus sequence typing (MLST) scheme for
Acinetobacter baumannii
was developed and evaluated by using 40 clinical
A. baumannii
isolates recovered from outbreaks in Spanish and German hospitals during the years 1990 to 2001, as well as isolates from other European hospitals and two DSMZ reference strains of
A. baumannii
. For comparison, two isolates of
Acinetobacter
species 13 (sensu Tjernberg and Ursing), two clinical isolates, and three DSMZ strains of
A. calcoaceticus
(both belonging to the
A. calcoaceticus
-
A. baumannii
complex) were also investigated. Primers were designed for conserved regions of housekeeping genes, and 305- to 513-bp internal fragments of seven such genes—
gltA
,
gyrB
,
gdhB
,
recA
,
cpn60
,
gpi
, and
rpoD
—were sequenced for all strains. The number of alleles at individual loci ranged from 6 to 12, and a total of 20 allelic profiles or sequence types were distinguished among the investigated
A. baumannii
strains. The MLST data were in high concordance with the epidemiologic typing results generated by pulsed-field gel electrophoresis and amplified fragment length polymorphism fingerprinting. The MLST scheme provides a high level of resolution and an excellent tool for studying the population structure and long-term epidemiology of
A. baumannii
.
Detection of Antibodies against Adenovirus Protein IX, Fiber, and Hexon in Human Sera by Immunoblot Assay
Authors: Bauer Ulrike , Flunker Gisela , Bruss Kornelia , Kallwellis Knut , Liebermann Herbert , Luettich Tanja , Motz Manfred , Seidel Werner
Published: September 2005

The 51 serotypes of human adenoviruses (HAdVs) of the genus
Mastadenovirus
are classified into the six species HAdV-A to HAdV-F. For the detection of genus- and species-specific antibodies in human sera an immunoblot assay was developed. The recombinant long fiber of HAdV-41[F] (Ad41Fi) and the native hexon of HAdV-5[C] were used as genus-specific antigens. The recombinant capsid protein IX (pIX) of HAdV-2 (Ad2pIX[C]) and HAdV-41 (Ad41pIX[F]), the C-terminal pIX part of HAdV-3 (Ad3pIXC[B]), and the fiber knob of HAdV-8 (Ad8FiKn[D]) were evaluated as representative species-specific antigens. Hence, the pIX amino acid sequences of numerous serotypes of all HAdV species were compared, and the cross-reactivities of pIX antigens with rabbit hyperimmune sera among HAdV-A to -F were analyzed. In an epidemiological study, 667 human patient sera, not selected for viral infection, were screened for adenovirus seroprevalence. The genus-specific antibody prevalences directed against the Ad41Fi and HAdV-5 hexon were 82.8 and 98.8%, respectively. The species-specific antibody prevalence of 44.7% against Ad2pIX[C], 36.6% against Ad41pIX[F], 26.4% against Ad8FiKn[D], and 18% against Ad3pIXC[B] showed an age-dependent distribution and correlated well with the frequency of isolated serotypes of the respective species in earlier studies (except HAdV-D). In conclusion, the immunoblot assay using pIX, fiber, and hexon antigens represents a valuable and new serological tool for refined adenovirus diagnosis as shown in an epidemiological study.
Multilocus Sequence Typing of Methicillin-Resistant
Staphylococcus aureus
from an Area of Low Endemicity by Real-Time PCR
Authors: Berglund Carolina , Mölling Paula , Sjöberg Lennart , Söderquist Bo
Published: September 2005

A protocol for multilocus sequence typing (MLST) of methicillin-resistant
Staphylococcus aureus
(MRSA) was adapted to real-time LightCycler System PCR for efficient and rapid amplification of seven housekeeping genes in the same PCR run and real-time detection of the products. The method was evaluated on a representative and well-characterized collection of clinical MRSA isolates (
n
= 57) obtained from an area of low endemicity. Twenty sequence types (STs) and nine clonal complexes were identified. Combining STs and the staphylococcal cassette chromosome
mec
(SCC
mec
) type identified 27 different genotypes, and type IV SCC
mec
was present in 11 different STs. The presence of the Panton Valentine leukocidin (PVL) genes was found in isolates of four different STs. Eleven different STs were found among the community-acquired as well as among the hospital-acquired MRSA. The genetic heterogeneity was also denoted by pulsed-field gel electrophoresis analysis that showed 24 different pulsotypes among the 57 MRSA isolates. The presence of more than one different type of SCC
mec
in the same ST indicates that the MRSA clones have arisen at several occasions in the same genetic background by independent acquisition of SCC
mec
into methicillin-sensitive strains. This circumstance shows the importance of combining MLST data with SCC
mec
-typing results when investigating the origins of MRSA.
Detection and Characterization of Human Group C Rotaviruses in Bangladesh
Authors: Rahman Mustafizur , Banik Sukalyani , Faruque Abu S. G. , Taniguchi Koki , Sack David A. , Van Ranst Marc , Azim Tasnim
Published: September 2005

Group C rotaviruses were detected by reverse transcription-PCR in 14 (2.3%) of 611 group A rotavirus-negative stool specimens from the patients admitted to the International Centre for Diarrhoeal Disease Research, Bangladesh hospital, Dhaka, Bangladesh, during July to December 2003. The low rate of detection suggested that infection with group C rotaviruses was an uncommon cause of hospitalization due to gastroenteritis. In addition, coinfections with pathogenic enteric bacteria were frequently observed in group C rotavirus-infected patients. Nucleotide sequence comparison of the VP4, VP6, and VP7 genes revealed that the Bangladeshi group C rotaviruses were most similar to Nigerian group C rotavirus strains. Phylogenetic analysis suggested that all human group C rotaviruses, including the strains isolated in our study, clustered in a monophyletic branch, which was distantly related to the branch comprised of animal group C rotaviruses.
Breakpoints for Predicting
Pseudomonas aeruginosa
Susceptibility to Inhaled Tobramycin in Cystic Fibrosis Patients: Use of High-Range Etest Strips
Authors: Morosini María I. , García-Castillo María , Loza Elena , Pérez-Vázquez María , Baquero Fernando , Cantón Rafael
Published: September 2005

Inhaled administration of tobramycin assures high concentrations in cystic fibrotic lungs, improving the therapeutic ratio over that of parenteral tobramycin levels, particularly against
Pseudomonas aeruginosa
. Conventional Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards) breakpoints only consider parenteral levels and do not take into account these high antimicrobial concentrations. The Spanish Antibiogram Committee (The MENSURA Group) has tentatively defined specific breakpoint values for inhaled tobramycin when testing
P. aeruginosa
isolates from cystic fibrosis (CF) patients (susceptible, ≤64 μg/ml; resistant, ≥128 μg/ml). The antimicrobial susceptibilities of 206 prospectively collected CF
P. aeruginosa
isolates were determined by the reference agar dilution method. For tobramycin, the performance of high range tobramycin Etest strips (AB Biodisk, Solna, Sweden) and conventional tobramycin disks were assessed with the same collection. Applying MENSURA proposed breakpoints, 95.1% of the strains were categorized as susceptible to tobramycin, either using agar dilution or Etest high-range strips (99% categorical agreement between both methods). With CLSI breakpoints, susceptibility rates decreased to 79.1 and 81.1% for agar dilution and Etest strips, respectively (83.5% categorical agreement). Minor, major, and very major errors for Etest strips (CLSI criteria) were 13.6, 1.2, and 14.8%, respectively. Upon applying the new proposed criteria for inhaled tobramycin, only one major and one very major error were observed with Etest strips. Whenever inhaled tobramycin is considered for therapy, we suggest that
P. aeruginosa
strains from CF patients categorized as intermediate or resistant to tobramycin according to the CLSI criteria should be retested with high-range Etest strips and recategorized using MENSURA interpretive criteria. CLSI breakpoints should still be followed when intravenous tobramycin is used in CF patients, particularly during the course of exacerbations.
Microbiologic Response to Treatment of Bacterial Vaginosis with Topical Clindamycin or Metronidazole
Authors: Austin M. N. , Beigi R. H. , Meyn L. A. , Hillier S. L.
Published: September 2005

To compare the frequencies, concentrations, and antimicrobial susceptibilities of vaginal microbes isolated from women with bacterial vaginosis (BV) before and after therapy, 119 nonpregnant women aged 18 to 45 with clinical and Gram stain evidence of BV were randomized to receive intravaginal clindamycin or metronidazole. Vaginal swabs were collected at baseline and 7 to 12 days, 35 to 45 days, and 70 to 90 days following therapy for quantitative vaginal culture. For the 99 women completing all four visits, statistical analyses were performed comparing differences in vaginal microflora between the two treatment arms and between visits in the same treatment group. Antimicrobial susceptibility testing using the agar dilution method was performed for anaerobic gram-negative rods. Although both therapies resulted in decreased colonization by
Gardnerella vaginalis
and
Mycoplasma hominis
, only metronidazole treatment resulted in a significant decrease in the frequency and concentration of
Prevotella bivia
and black-pigmented
Prevotella
species. Of the 865 anaerobic gram-negative rods evaluated for susceptibility, only 3 (0.3%) were resistant to metronidazole, whereas clindamycin resistance increased significantly for
P. bivia
and black-pigmented anaerobic gram-negative rods persisting following clindamycin therapy. Clindamycin-resistant subpopulations of
P. bivia
and black-pigmented
Prevotella
species emerged 7 to 12 days after therapy even among women colonized initially by clindamycin-susceptible strains. These resistant subpopulations persisted at high frequencies (42 to 50%) 70 to 90 days following therapy. The two topical agents for treatment of BV have differing microbiologic effects on the vaginal microflora. The emergence of clindamycin-resistant anaerobic gram-negative rods following therapy is of concern.
Use of rMPB70 Protein and ESAT-6 Peptide as Antigens for Comparison of the Enzyme-Linked Immunosorbent, Immunochromatographic, and Latex Bead Agglutination Assays for Serodiagnosis of Bovine Tuberculosis
Authors: Koo Hye Cheong , Park Yong Ho , Ahn Jongsam , Waters W. Ray , Palmer Mitch V. , Hamilton Mary Jo , Barrington George , Mosaad Abdelaziz A. , Park Kun Taek , Jung Woo Kyung , Hwang In Yeong , Cho Sang-Nae , Shin Sang Jae , Davis William C.
Published: September 2005

Current assays used to detectMycobacterium bovisinfection lack accuracy, especially for recently infected animals, or are impractical for rapid field diagnostic applications. To overcome these limitations with serological assays, a synthetic peptide derived from early secretory antigenic target 6 (ESAT6-p) and a recombinant major secreted immunogenic protein (rMPB70) ofM. boviswere used in an enzyme-linked immunosorbent assay (EIA), an immunochromatographic assay (ICGA), and a latex bead agglutination assay (LBAA). Sera from noninfected,M. bovis-infected, orM. aviumsubsp.paratuberculosis-infected (by natural and experimental routes) animals were evaluated. Receiver operating characteristic analysis comparing optical density values from the EIA with results of bacterial culture or skin test, the reference test, established suitable cutoff values for assessing sensitivity and specificity. The EIA and LBAA, respectively, had sensitivities of 98.6 and 94.8%, specificities of 98.5 and 92.6%, and kappa values of 0.97 and 0.88 with ESAT6-p. The EIA, ICGA, and LBAA, respectively, had sensitivities of 96.8, 83.0, and 86.7%, specificities of 90.1, 99.4, and 97.8%, and kappa values of 0.87, 0.85, and 0.83 with rMPB70. Examination of serial samples of sera collected from experimentallyM. bovis-infected cattle and deer revealed that ESAT6-p-specific responses developed early after infection whereas responses to rMPB70 developed later in the course of disease. The advantage of the LBAA and ICGA as initial tests for multiple species is a rapid reaction obtained in 2 to 3 h by LBAA or 20 min by ICGA without species-specific secondary antibodies under field conditions, thus allowing immediate segregation of suspect animals for further testing before culling.
Rapid Identification of
Candida
Species and Other Clinically Important Yeast Species by Flow Cytometry
Authors: Page Brent T. , Kurtzman Cletus P.
Published: September 2005

Two rapid diagnostic assays, utilizing two different Luminex flow cytometry methods, were developed for identification of clinically important ascomycetous yeast species. Direct hybridization and allele-specific primer extension methods were both successful in establishing a DNA-based assay that can rapidly and accurately identify
Candida albicans
,
Candida krusei
,
Candida parapsilosis
,
Candida glabrata
, and
Candida tropicalis
as well as other clinical species. The direct hybridization assay was designed to identify a total of 19 ascomycetous yeast species, and the allele-specific primer extension assay was designed to identify a total of 34 species. Probes were validated against 438 strains representing 303 species. From culture to identification, the allele-specific primer extension method takes 8 h and the direct hybridization method takes less than 5 h to complete. These assays represent comprehensive, rapid tests that are well suited for the clinical laboratory.
Evaluation of tRNA Gene PCR for Identification of
Mollicutes
Authors: Stakenborg Tim , Vicca Jo , Verhelst Rita , Butaye Patrick , Maes Dominiek , Naessens Anne , Claeys Geert , De Ganck Catharine , Haesebrouck Freddy , Vaneechoutte Mario
Published: September 2005

We evaluated the applicability of tRNA gene PCR in combination with fluorescent capillary electrophoresis with an ABI310 genetic analyzer (Applied Biosystems, Calif.) for the identification of different mollicute species. A total of 103 strains and DNA extracts of 30 different species belonging to the genera
Acholeplasma
,
Mycoplasma
, and
Ureaplasma
were studied. Reproducible peak profiles were generated for all samples, except for one
M. genitalium
isolate, the three
M. gallisepticum
isolates, and 8 of the 24
Ureaplasma
cultures, where no amplification could be obtained. Clustering revealed numerous discrepancies compared to the identifications that had been previously obtained by means of biochemical and serological tests. Final identification was obtained by 16S rRNA gene amplification followed by sequence analysis and/or restriction digestion. This confirmed the identification obtained by tRNA gene PCR in all cases. Seven samples yielded an unexpected tRNA gene PCR profile. Sequence analysis of the 16S rRNA genes showed that six of these samples were mixed and that one had a unique sequence that did not match any of the published sequences, pointing to the existence of a not-yet-described species. In conclusion, we found tRNA gene PCR to be a rapid and discriminatory method to correctly identify a large collection of different species of the class of
Mollicutes
and to recognize not-yet-described groups.
Microsatellite Marker Analysis as a Typing System for
Candida glabrata
Authors: Foulet F. , Nicolas N. , Eloy O. , Botterel F. , Gantier J.-C. , Costa J.-M. , Bretagne S.
Published: September 2005

Candida glabrata
is one of the most important causes of nosocomial fungal infection. We investigated, using a multiplex PCR, three polymorphic microsatellite markers,
RPM2
,
MTI
, and
ERG3
, in order to obtain a rapid genotyping method for
C. glabrata
. One set of primers was designed for each locus, and one primer of each set was dye labeled to read PCR signals using an automatic sequencer. Eight reference strains including other
Candida
species and 138 independent
C. glabrata
clinical isolates were tested. The clinical isolates were collected from different anatomical sites of adult patients either hospitalized in different wards of two different hospitals or not hospitalized. Since
C. glabrata
is haploid, one single PCR product for each PCR set was obtained and assigned to an allele. The numbers of different alleles were 5, 7, and 15 for the
RPM2
,
MTI
, and
ERG3
loci, respectively. The number of allelic associations was 21, leading to a discriminatory power of 0.84. The markers were stable after 25 subcultures, and the amplifications were specific for
C. glabrata
. A factorial correspondence analysis did not indicate any correlation between the 21 multilocus genotypes and the clinical data (source, sex, ward, anatomical sites). Microsatellite marker analysis is a rapid and reliable technique to investigate clinical issues concerning
C. glabrata
. However, its discriminatory power should be improved by testing other polymorphic microsatellite loci.
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

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

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

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

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

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

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

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

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

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

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

No 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

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

A 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

This 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.
Clonal Analysis of
Staphylococcus epidermidis
Isolates Carrying or Lacking Biofilm-Mediating Genes by Multilocus Sequence Typing
Authors: Kozitskaya Svetlana , Olson Michael E. , Fey Paul D. , Witte Wolfgang , Ohlsen Knut , Ziebuhr Wilma
Published: September 2005

Staphylococcus epidermidis
is part of the normal microflora of the human skin but is also a leading cause of device-associated infections in critically ill patients. Commensal and clinical
S. epidermidis
isolates differ in their ability to form biofilms on medical devices; the synthesis of biofilms is mediated by the
icaADBC
operon. Currently, the epidemiological relatedness between
ica-
positive and -negative isolates is not known; neither is it known whether the
ica
genes can spread to biofilm-negative strains through horizontal gene transfer. In this study, multilocus sequence typing (MLST) was employed for the clonal analysis of 118
S. epidermidis ica
-positive and -negative strains. MLST revealed that the majority of
ica
-positive and -negative strains were closely related and formed a single clonal complex. Within this complex one sequence type (ST27) was identified which contained exclusively
ica-
positive isolates and represented the majority of clinical strains tested. ST27 and related
ica
-positive clones carried different SCC
mec
cassettes (conferring methicillin resistance) and the insertion sequence IS
256
. The findings suggest that the
S. epidermidis
infections analyzed in this report are mainly caused by a single clone (ST27) which occurs preferentially in hospitals and differs from clones in the community. It is hypothesized that the successful establishment of ST27 within nosocomial environments has been facilitated by the presence of genes encoding biofilm and resistance traits.
Production and Validation of the Use of Gamma Phage for Identification of
Bacillus anthracis
Authors: Abshire T. G. , Brown J. E. , Ezzell J. W.
Published: September 2005

Gamma phage specifically lyses vegetative cells of
Bacillus anthracis
and serves as part of the basis for identification of isolates from agar cultures. We report our study to standardize gamma phage production and preparation and to validate the assay for routine use. Unstable phage preparations were largely reduced through propagation of phage on blood agar cultures of the avirulent
B. anthracis
strain CDC684 and were adequately stable for extended storage beyond 1 to 2 years at 4°C, provided that the preparation initially gave rise to clearly discernible plaques (macroplaques, 5 to 10 mm in diameter) on dilution at 1:8 or greater during potency testing with the Sterne strain or its equivalent. The primary intent of the assay was to test nonhemolytic, ground-glass-appearing bacterial
B. anthracis
-like colonies arising from culture of clinical or nonclinical samples on 5% sheep blood agar. Specifically, the assay was designed to show clear or primarily clear circular zones of lysis on bacterial lawns at the site of gamma phage inoculation after incubation at 35 °C ± 2°C for 20 h. When tested with 51
B. anthracis
strains and 49 similar non-
B. anthracis Bacillus
species, the analytical specificity was >95%, a value that is intentionally low because our study design included two rare nonsusceptible
B. anthracis
strains as well as a rare susceptible non-
B. anthracis
strain,
B. cereus
ATCC 4342. Repeatability, day-to-day precision, and analyst-to-analyst precision were superior. The assay was rugged to variations among phage lots, phage concentration, amounts of bacterial inoculum, and incubation times as short as 6 to 8 h. System suitability evaluation showed improved robustness when bacterial lawns were tested with high- and low-density inoculum using the first and second quadrants of a serial four-quadrant streak on 5% sheep blood agar plates.
Reproducibility of Low Galactomannan Enzyme Immunoassay Index Values Tested in Multiple Laboratories
Authors: Upton Arlo , Gugel Anja , Leisenring Wendy , Limaye Ajit , Alexander Barbara , Hayden Randall , Marr Kieren A.
Published: September 2005

The Platelia galactomannan enzyme immunoassay is a commercially available nonculture method for diagnosing invasive aspergillosis. Recently, steps have been taken to improve sensitivity; specifically, a low (0.5 to 0.7) galactomannan index (GMI) value to determine positivity has been validated by multiple groups. We evaluated the intra-assay and interassay reproducibility at low index levels using three different kit lots on three different days in three different microbiology laboratories. Clinical and spiked sera were blinded and sent with galactomannan enzyme immunoassay (EIA) kits to the participating laboratories. We also prospectively collected data on all galactomannan EIAs performed between 1 September 2003 and 21 July 2004 at the University of Washington Medical Center microbiology laboratory to assess reproducibility of clinical samples analyzed in “real time.” From the multilaboratory study, a total of 836 results were available for evaluation. Reproducibility was excellent between laboratories and on different days. Significant variability was seen between runs/lots, which may in part be associated with different threshold control values in different kits. Among the 1,410 clinical samples that were prospectively analyzed, 168 (90%) were confirmed to be positive on repeat testing (GMI, ≥0.5). Among the 19 (10.2%) initially positive samples not confirmed on repeat testing, the majority had a GMI at the threshold of the assay (between 0.5 and 0.7). Our findings suggest that the Platelia galactomannan immunoassay has good reproducibility. However, changes in GMI levels when different kit lots are used, and single samples with low-positive (GMI of 0.5 to 0.7) indices, should be interpreted with caution.
Relationships between Total Plasma Load of Torquetenovirus (TTV) and TTV Genogroups Carried
Authors: Maggi Fabrizio , Andreoli Elisabetta , Lanini Letizia , Fornai Claudia , Vatteroni Marialinda , Pistello Mauro , Presciuttini Silvano , Bendinelli Mauro
Published: September 2005

In 239 torquetenovirus-positive people, multiple-genogroup infections were common and associated with higher viral loads than would be expected from simple additive effects. The latter observation was restricted to the infections which included both genogroups 1 and 3, pointing to the possible existence of some kind of infection facilitation between these genogroups.