bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multilocus Sequence Typing for Characterization of Methicillin-Resistant and Methicillin-Susceptible Clones ofStaphylococcus aureus
Authors: Enright Mark C. , Day Nicholas P. J. , Davies Catrin E. , Peacock Sharon J. , Spratt Brian G. 
Published: December 2019

ABSTRACTA multilocus sequence typing (MLST) scheme has been developed forStaphylococcus aureus. The sequences of internal fragments of seven housekeeping genes were obtained for 155S. aureusisolates from patients with community-acquired and hospital-acquired invasive disease in the Oxford, United Kingdom, area. Fifty-three different allelic profiles were identified, and 17 of these were represented by at least two isolates. The MLST scheme was highly discriminatory and was validated by showing that pairs of isolates with the same allelic profile produced very similarSmaI restriction fragment patterns by pulsed-field gel electrophoresis. All 22 isolates with the most prevalent allelic profile were methicillin-resistantS. aureus(MRSA) isolates and had allelic profiles identical to that of a reference strain of the epidemic MRSA clone 16 (EMRSA-16). Four MRSA isolates that were identical in allelic profile to the other major epidemic MRSA clone prevalent in British hospitals (clone EMRSA-15) were also identified. The majority of isolates (81%) were methicillin-susceptibleS. aureus(MSSA) isolates, and seven MSSA clones included five or more isolates. Three of the MSSA clones included at least five isolates from patients with community-acquired invasive disease and may represent virulent clones with an increased ability to cause disease in otherwise healthy individuals. The most prevalent MSSA clone (17 isolates) was very closely related to EMRSA-16, and the success of the latter clone at causing disease in hospitals may be due to its emergence from a virulent MSSA clone that was already a major cause of invasive disease in both the community and hospital settings. MLST provides an unambiguous method for assigning MRSA and MSSA isolates to known clones or assigning them as novel clones via the Internet.
Comparison of Different PCR Approaches for Typing of
Francisella tularensis
Strains
Authors: de la Puente-Redondo V. A. , del Blanco N. García , Gutiérrez-Martín C. B. , García-Peña F. J. , Ferri E. F. Rodríguez 
Published: December 2019

ABSTRACT

In this study, we evaluated three PCR methods for epidemiological typing of
Francisella tularensis
: repetitive extragenic palindromic element PCR (REP-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), and random amplified polymorphic DNA (RAPD) assay with both M13 and T3-T7 primers. The analysis was performed with 40 strains of
F. tularensis
isolated from hares, humans, ticks, and a vole. On the basis of the combination of REP, ERIC, and RAPD fingerprints,
F. tularensis
strains were divided into 17 genetic groups (designated A to Q), and one
Francisella novicida
strain was classified in group R. The
F. novicida
strain is of special concern, since previous genetic methods have been unable to clearly distinguish between
F. tularensis
and
F. novicida
. The
F. tularensis
isolates recovered from hares were included in groups A to J, M, and P; those recovered from humans were included in groups A, D, G, J, L, O, and N; those isolated from ticks were included in groups B and Q; and that recovered from a vole was in group K. The diversities calculated for the 40
F. tularensis
isolates, according to Simpson's index, were 0.14 for REP-PCR, 0.52 for ERIC-PCR, 0.39 for RAPD assay with the M13 primer (RAPD/M13-PCR), and 0.65 for RAPD/T3-T7-PCR, and the diversity increased up to 0.90 when ERIC-PCR, RAPD/M13-PCR, and RAPD/T3-T7-PCR were combined. Our results suggest that although limited genetic heterogeneity among
F. tularensis
strains was observed, this small variation is enough to validate the PCR methods used in this study and their combinations, because they can provide safe, useful, and rapid tools for the typing of
F. tularensis
.
Failure of an Automated Blood Culture System To Detect Nonfermentative Gram-Negative Bacteria
Authors: Klaerner H.-G. , Eschenbach U. , Kamereck K. , Lehn N. , Wagner H. , Miethke T. 
Published: December 2019

ABSTRACT

During a 1-year study we observed that both aerobic and anaerobic blood culture bottles from patients were negative by the BacT/Alert system during a 7-day incubation period. However, upon subcultivation of negative bottles, growth of
Pseudomonas aeruginosa
was detectable. In an attempt to explain this observation, aerobic BacT/Alert Fan bottles were seeded with a defined inoculum (0.5 McFarland standard; 1 ml) of
Escherichia coli
,
Klebsiella pneumoniae
,
Serratia marcescens
,
P. aeruginosa
,
Stenotrophomonas maltophilia
, or
Acinetobacter baumannii
. Half of the inoculated bottles were loaded into the BacT/Alert system immediately, and the remainder were preincubated for 4, 8, 16, and 24 h at 36°C. With preincubation all bottles seeded with the
Enterobacteriaceae
signaled positive during the next 1.5 h. Organisms in bottles seeded with the nonfermentative species
P. aeruginosa
and
A. baumannii
remained undetected by the BacT/Alert system for 7 days.
S. maltophilia
was detected if the preincubation time was equal or less than 8 h. Without preincubation all bottles seeded with the
Enterobacteriaceae
or nonfermentative species signaled positive. Since nonfermentative species seem to enter a state of bacteriostasis within the preincubation period, we reasoned that an unknown factor is consumed. Accordingly, a smaller inoculum should allow the detection of nonfermentative species, even after preincubation, and serial dilutions of
P. aeruginosa
were detected in preincubated bottles. In this case preincubated bottles signaled positive faster than bottles without preincubation. We conclude that all bottles from clinical settings should be subcultured prior to loading to avoid false negatives. An alternative may be preincubation at room temperature.
Simultaneous Detection of Multiplex-Amplified Human Immunodeficiency Virus Type 1 RNA, Hepatitis C Virus RNA, and Hepatitis B Virus DNA Using a Flow Cytometer Microsphere-Based Hybridization Assay
Authors: Defoort J.-P. , Martin M. , Casano B. , Prato S. , Camilla C. , Fert V. 
Published: December 2019

ABSTRACT

The feasibility of performing a multiplex assay for the detection of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) RNAs and hepatitis B virus (HBV) DNA is demonstrated. This assay is based (i) on the coamplification of a 142-bp fragment from the
gag
region of the HIV-1 genome and a 142-bp HIV-1 quantitation standard fragment, a 244-bp fragment from the 5′ noncoding region of the HCV genome, and a 104-bp fragment from the pre-C and C gene regions of the HBV genome, using three sets of specific primers; (ii) on the capacity of these four biotinylated PCR products to hybridize to their specific oligonucleotide probe-coated microspheres; and (iii) on the ability of the flow cytometer to discriminate between distinct fluorescent-microsphere categories. Absence of cross-hybridization between the unrelated oligonucleotide probes and PCR products generated by the multiplex reverse transcription-PCR (RT-PCR) and the highly sensitive detection method allowed us to assess unambiguously the HIV-1 viral load and the infectious status of 35 serologically well-established clinical samples and 20 seronegative blood donor plasma samples tested. The results indicate that multiplex RT-PCR and flow cytometer microsphere-based hybridization assays, when combined, provide a rapid, sensitive, and specific method for the quantitation and detection of the major viral agents of infectious diseases in a single plasma sample.
Multicenter Evaluation of the AMPLICOR and Automated COBAS AMPLICOR CT/NG Tests for Detection of
Chlamydia trachomatis
Authors: Van Der Pol Barbara , Quinn Thomas C. , Gaydos Charlotte A. , Crotchfelt Kimberly , Schachter Julius , Moncada Jeanne , Jungkind D. , Martin David H. , Turner Buffy , Peyton Cynthia , Jones Robert B. 
Published: December 2019

ABSTRACT

The fully automated COBAS AMPLICOR CT/NG and semiautomated AMPLICOR CT/NG tests were evaluated in a multicenter trial for the ability to detect
Chlamydia trachomatis
infections. Test performance compared to that of culture was evaluated for 2,236 matched endocervical swab and urine specimens obtained from women and for 1,940 matched urethral swab and urine specimens obtained from men. Culture-negative, PCR-positive specimens that tested positive in a direct fluorescent-antibody test or in a confirmatory PCR test for an alternative target sequence were resolved as true positives. The overall prevalences of chlamydia were 2.4% in women and 7.2% in men. The COBAS AMPLICOR and AMPLICOR formats yielded concordant results for 98.1% of the specimens. With the infected patient as the reference standard, the resolved sensitivities of COBAS AMPLICOR were 89.7% for endocervical swab specimens, 89.2% for female urine specimens, 88.6% for male urethral swab specimens, and 90.3% for male urine specimens. When results were analyzed as if only a single test had been performed on a single specimen type, the resolved sensitivity was always higher. The resolved specificities of PCR were 99.4% for endocervical swab specimens, 99.0% for female urine specimens, 98.7% for male urethral swab specimens, and 98.4% for male urine specimens. The internal control revealed that 2.4% of the specimens were inhibitory when initially tested. Nevertheless, valid results were obtained for 98.6% of the specimens because 59.1% of the inhibitory specimens were not inhibitory when a second aliquot was tested. The COBAS AMPLICOR and AMPLICOR CT/NG tests for
C. trachomatis
exhibited equally high sensitivity and specificity with both urogenital swab and urine specimens and thus are well suited for screening for
C. trachomatis
infection.
Quantitative and Cost Comparison of Ultrasensitive Human Immunodeficiency Virus Type 1 RNA Viral Load Assays: Bayer bDNA Quantiplex Versions 3.0 and 2.0 and Roche PCR Amplicor Monitor Version 1.5
Authors: Elbeik Tarek , Charlebois Edwin , Nassos Patricia , Kahn James , Hecht Frederick M. , Yajko David , Ng Valerie , Hadley Keith 
Published: December 2019

ABSTRACT
Quantification of human immunodeficiency virus type 1 (HIV-1) RNA as a measure of viral load has greatly improved the monitoring of therapies for infected individuals. With the significant reductions in viral load now observed in individuals treated with highly active anti-retroviral therapy (HAART), viral load assays have been adapted to achieve greater sensitivity. Two commercially available ultrasensitive assays, the Bayer Quantiplex HIV-1 bDNA version 3.0 (bDNA 3.0) assay and the Roche Amplicor HIV-1 Monitor Ultrasensitive version 1.5 (Amplicor 1.5) assay, are now being used to monitor HIV-1-infected individuals. Both of these ultrasensitive assays have a reported lower limit of 50 HIV-1 RNA copies/ml and were developed from corresponding older generation assays with lower limits of 400 to 500 copies/ml. However, the comparability of viral load data generated by these ultrasensitive assays and the relative costs of labor, disposables, and biohazardous wastes were not determined in most cases. In this study, we used matched clinical plasma samples to compare the quantification of the newer bDNA 3.0 assay with that of the older bDNA 2.0 assay and to compare the quantification and costs of the bDNA 3.0 assay and the Amplicor 1.5 assay. We found that quantification by the bDNA 3.0 assay was approximately twofold higher than that by the bDNA 2.0 assay and was highly correlated to that by the Amplicor 1.5 assay. Moreover, cost analysis based on labor, disposables, and biohazardous wastes showed significant savings with the bDNA 3.0 assay as compared to the costs of the Amplicor 1.5 assay.
Species Identification and Subtyping of
Ureaplasma parvum
and
Ureaplasma urealyticum
Using PCR-Based Assays
Authors: Kong Fanrong , Ma Zhenfang , James Gregory , Gordon Susanna , Gilbert Gwendolyn L. 
Published: December 2019

ABSTRACT

There is good evidence that the organism currently known as
Ureaplasma urealyticum
should be divided into two species—
U. parvum
(previously
U. urealyticum
biovar 1) and
U. urealyticum
(previously
U. urealyticum
biovar 2). In this study, we designed a series of primers, targeting the 16S rRNA gene and 16S rRNA-23S rRNA intergenic spacer regions, the urease gene subunits, and the 5′ ends of the multiple-banded antigen (MBA) genes, to identify and subtype these
Ureaplasma
species. All of the species-specific primer pairs could distinguish the two species, but only subtype-specific primer pairs targeting the MBA genes could distinguish subtypes within each species.
U. parvum
was separated into three subtypes, represented by serovars 1, 3/14, and 6.
U. urealyticum
was also separated into three subtypes by PCR and/or direct sequencing. Subtype 1 consisted of serovars 2, 5, 8, and 9; subtype 2 contained serovars 4, 10, 12, and 13; and subtype 3 contained serovars 7 and 11. A selection of primer pairs was used to identify and subtype 78 clinical ureaplasma isolates from vaginal swabs of pregnant women and to identify and subtype ureaplasmas directly in 185 vaginal swabs in which they had been previously detected.
U. parvum
was identified in 228 (87%) of 263 isolates or specimens, and
U. urealyticum
was identified in 50 (19%) (both were present in 6%). Serovars 3/14 (48%) and 1 (43%) were most common among
U. parvum
isolates, and subtypes 2 (62%) and 1 (34%) were most common among
U. urealyticum
isolates. This new PCR-based typing system will facilitate future studies of the relationship between individual
Ureaplasma
species or subtypes and human disease.
Detection and Identification of Human Parainfluenza Viruses 1, 2, 3, and 4 in Clinical Samples of Pediatric Patients by Multiplex Reverse Transcription-PCR
Authors: Aguilar Jose C. , Pérez-Breña María P. , García María L. , Cruz Nieves , Erdman Dean D. , Echevarría Juan Emilio 
Published: December 2019

ABSTRACT

We describe a multiplex reverse transcription-PCR (m-RT-PCR) assay that is able to detect and differentiate all known human parainfluenza viruses (HPIVs). Serial dilution experiments with reference strains that compared cell culture isolation and m-RT-PCR showed sensitivities ranging from 0.0004 50% tissue culture infective dose (TCID
50
) for HPIV type 4B (HPIV-4B) to 32 TCID
50
s for HPIV-3. As few as 10 plasmids containing HPIV PCR products could be detected in all cases. When 201 nasopharyngeal aspirate specimens from pediatric patients hospitalized for lower respiratory illness were tested, m-RT-PCR assay detected 64 HPIVs (24 HPIV-3, 23 HPIV-1, 10 HPIV-4, and 7 HPIV-2), while only 42 of them (21 HPIV-1, 14 HPIV-3, 6 HPIV-2, and 1 HPIV-4 isolates) grew in cell culture. Our m-RT-PCR assay was more sensitive than either cell culture isolation or indirect immunofluorescence with monoclonal antibodies for the detection of HPIV infections. Also, HPIV-4 was more frequently detected than HPIV-2 in this study, suggesting that it may have been underestimated as a lower respiratory tract pathogen because of the insensitivity of cell culture.
Detection of a Previously Unamplified Spacer within the DR Locus of
Mycobacterium tuberculosis
: Epidemiological Implications
Authors: Filliol Ingrid , Sola Christophe , Rastogi Nalin 
Published: December 2019

ABSTRACT

Spoligotyping, a method based on the variability of distribution of the 43 inter-direct repeat (DR) spacers of
Mycobacterium tuberculosis
and
Mycobacterium bovis
BCG, is useful to study the molecular epidemiology of bovine and human tuberculosis. Recently, a major family of
M. tuberculosis
clinical isolates named the Haarlem family, which did not contain spacers 31 and 33 to 36, was reported in a multicenter study. Independently, a data bank containing all the published spoligotypes showed that the two most prevalent spoligotypes in the world differed only by the presence or absence of spacer 31. A careful analysis of the DR locus sequence led us to hypothesize that spacer 31 may not have been amplified in some isolates with the primer sets DRa and DRb currently used for spoligotyping. Consequently, a modified spoligotyping method based on different combinations of the 36-bp DR and IS
6110
primers was devised that was able to discriminate between the left and the right parts of the DR locus and demonstrated the presence of the previously unamplified spacer 31 for some of the clinical isolates. By analogy, we suggest that a single-spacer difference in some epidemiologically linked cases of tuberculosis may simply arise due to the insertion of an extra copy of IS
6110
within the DR locus, leading to its asymmetrical disruption and subsequent lack of the DRa or DRb targets. The influence of the IS
6110
preferential insertion sites within the DR locus on spoligotyping results should be further investigated.
Multiple Clones within Multidrug-Resistant
Salmonella enterica
Serotype Typhimurium Phage Type DT104
Authors: Markogiannakis Antonis , Tassios Panayotis T. , Lambiri Maria , Ward Linda R. , Kourea-Kremastinou Jenny , Legakis Nicholas J. ,   The Greek Nontyphoidal Salmonella Study Group, Vatopoulos Alkiviadis C. 
Published: December 2019

ABSTRACT

Six distinct clones were present among Greek multidrug-resistant
Salmonella enterica
serotype Typhimurium phage type DT104, since isolates belonging to resistance phenotypes including the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline) core could be distinguished with respect to their pulsed-field gel electrophoresis patterns,
int
1 integron structures, and presence or absence of antibiotic resistance genes
ant
(3")-Ia,
pse
-1, and
tem
-1.
Genotyping of
Mycoplasma pneumoniae
Clinical Isolates Reveals Eight P1 Subtypes within Two Genomic Groups
Authors: Dorigo-Zetsma J. Wendelien , Dankert Jacob , Zaat Sebastian A. J. 
Published: December 2019

ABSTRACT

Three methods for genotyping of
Mycoplasma pneumoniae
clinical isolates were applied to 2 reference strains and 21 clinical isolates. By a modified restriction fragment length polymorphism (RFLP) analysis of PCR products of the
M. pneumoniae
cytadhesin P1 gene, 5 subtypes were discriminated among 13 P1 type 1 strains and 3 subtypes were discriminated among 8 P1 type 2 strains. Sequence analysis of the 16S-23S rRNA gene spacer region and part of the 23S rRNA gene revealed one nucleotide difference in the intergenic spacer region in 3 of the 21 isolates. In the 23S rRNA gene sequence of the 8 P1 type 2 strains an extra adenosine was present, but it was absent from the 13 P1 type 1 strains. On the basis of
M. pneumoniae
genome sequence data, primers were designed to amplify large interrepeat fragments by long PCR, and these fragments were subsequently analyzed by RFLP analysis. Only two types, long PCR types 1 and 2, could be discriminated among the
M. pneumoniae
isolates. All P1 type 1 strains were assigned to long PCR type 1, and all P1 type 2 strains were assigned to long PCR type 2. These data obtained by three independent typing methods thus confirm the existence of two distinct
M. pneumoniae
genomic groups but expand the possibility of strain typing on the basis of variations within their P1 genes.
Identification of the Major Spanish Clones of Penicillin-Resistant Pneumococci via the Internet Using Multilocus Sequence Typing
Authors: Zhou Jiaji , Enright Mark C. , Spratt Brian G. 
Published: December 2019

ABSTRACT

Multilocus sequence typing was used to characterize isolates of the major Spanish clones of penicillin-resistant and multiple-antibiotic-resistant
Streptococcus pneumoniae
. Isolates of the multidrug-resistant Spanish serotype 23F clone and serotype variants of this clone either had identical allelic profiles or their allelic profiles differed from this typical allelic profile at only one of the seven housekeeping loci. Similarly, isolates of the Spanish serotype 6B and 14 clones and the penicillin-resistant serotype 9V clone (and serotype variants of this clone) each had the same allelic profiles or profiles that differed at a single locus. Multilocus sequence typing therefore allows resistant pneumococci to be assigned to the Spanish clones if they have the typical allelic profile of the clone or if their profiles differ from that profile at a single locus. A few resistant isolates that had allelic profiles typical of that of a Spanish clone or whose profiles differed from that of the typical profile at only a single locus possessed penicillin-binding protein
pbp1a
,
pbp2b
, or
pbp2x
genes that differed from those that are characteristic of the clone. In most cases these isolates could be assigned as variant members of the clone. Since almost all serotype 9V isolates have very similar genotypes, independently emerging penicillin-resistant clones of this serotype will inevitably appear to be similar by molecular typing procedures. Analysis of the
pbp
genes, in addition to multilocus sequence typing (or any other molecular typing procedure), is therefore required to assign isolates unambiguously to the penicillin-resistant Spanish serotype 9V clone.
Early Aqueous Humor Analysis in Patients with Human Ocular Toxoplasmosis
Authors: Garweg Justus G. , Jacquier Patrick , Boehnke Matthias 
Published: December 2019

ABSTRACTTo evaluate the diagnostic sensitivity of a panel of laboratory tests for ocular toxoplasmosis performed at the time of presentation, paired samples of aqueous humor and serum were collected from 49 consecutive episodes of ocular toxoplasmosis with a clinical course of less than 3 weeks. Total immunoglobulin G (IgG) andToxoplasma gondii-specific IgG, IgM, and IgA were quantified by enzyme-linked immunosorbent assay. The avidity ofT. gondii-specific IgG was determined, and DNA extracted from aqueous humor was amplified for detection of a glycoprotein B gene sequence ofT. gondii. The diagnosis was confirmed for 73% (36 of 49) of the patients; this rate rose to 79.5% if data from a later analysis of aqueous humor derived from five of the negative patients were included. The analysis of serum (detection ofT. gondii-specific IgM and analysis of consecutive serum samples) alone did not contribute to the diagnosis. Calculation of local antibody production lacked diagnostic sensitivity when it was determined less than 3 weeks after the manifestation of clinical symptoms (28 of 49 patients [57%]), but this rose to 70% after an analysis of a second aqueous humor sample. The antibody avidity index attained diagnostic significance in only 8 of 43 instances (19%), andT. gondiiDNA was amplified from no more than 6 of 39 (16%) aqueous humor samples. However,T. gondii-specific IgA was found within the aqueous humors of 11 of 43 patients (26%); measurement of theT. gondii-specific IgA level thus contributed substantially to the diagnostic sensitivity of the laboratory tests.