bluuebunny/crossref_metadata_2025_split huggingface dataset:
Serotyping
Streptococcus pneumoniae
by Multiplex PCR
Authors: Brito D. A. , Ramirez M. , de Lencastre H. 
Published: June 2003

ABSTRACT
The capsule is a major virulence factor of pneumococci, and it was shown that some capsular variants are associated with antimicrobial resistance and certain types of disease. Moreover, pneumococcal capsular typing has received renewed interest since the availability of conjugate vaccines, which include serotypes frequently associated with pediatric disease. Our aim was to develop a simple, reliable, and economical method for detecting epidemiologically important serotypes present in the proposed 11-valent conjugate vaccine. We designed primers based on the sequences available for the capsular types 1, 3, 4, 6B, 14, 18C, 19F, 19A, and 23F and combined them into seven multiplex PCRs. The method involves streamlined DNA template preparation and agarose gel electrophoresis to analyze the amplification products. A total of 446 pneumococci selected from among isolates colonizing the nasopharynx of children attending day care centers in Lisbon, Portugal, were typed both by conventional immunological techniques and by multiplex PCR. Capsular types identified by the PCR method invariably produced results concordant with the conventional serotyping technique. Even when the method presented does not fully type an isolate, the PCR data can guide the experimenter when using immunological serotyping. Multiplex PCR for the analysis of pneumococci provides an accurate, expeditious, and cost-effective way of reducing the number of strains that have to be serotyped by conventional immunological techniques.
Survey of
emm
Gene Sequences from Pharyngeal
Streptococcus pyogenes
Isolates Collected in Spain and Their Relationship with Erythromycin Susceptibility
Authors: Albertí Sebastián , García-Rey César , Domínguez María A. , Aguilar Lorenzo , Cercenado Emilia , Gobernado Miguel , García-Perea Adela 
Published: June 2003

ABSTRACT

We conducted a nationwide survey of the variable 5′
emm
(M protein gene) sequences from 614 pharyngeal
Streptococcus pyogenes
isolates susceptible (299 isolates) and resistant (315 isolates) to erythromycin that were isolated in Spain from 1996 to 1999. Almost 98% of these isolates had
emm
sequences in agreement with previously recorded M antigen association. We only identified a new 5′
emm
sequence in 17 isolates. Nine different
emm
types accounted for 85% of the
S. pyogenes
isolates susceptible to erythromycin. By contrast, only 3
emm
types accounted for 70% of the erythromycin-resistant isolates. Further characterization of these isolates by ribotyping and pulsed-field gel electrophoresis indicated that high frequency of erythromycin resistance in Spain is due to few clones.
Controlled Clinical Comparison of BacT/ALERT Standard Aerobic Medium with BACTEC Standard Aerobic Medium for Culturing Blood
Authors: Mirrett Stanley , Reller L. Barth , Petti Cathy A. , Woods Christopher W. , Vazirani Bindu , Sivadas Rekha , Weinstein Melvin P. 
Published: June 2003

ABSTRACT

Standard aerobic media are widely used for culturing blood with the BacT/ALERT (BioMérieux, Inc., Durham, N.C.) (BM) and BACTEC 9240 (BD Diagnostic Systems, Sparks, Md.) (BD) automated continuously monitoring instrument systems. Although similarly composed of soybean-casein digest broths, the formulations of the standard aerobic media available for these instruments differ from each other in supplements and in sodium polyanetholesulfonate concentration. Therefore, we compared the standard aerobic media available for these systems at two university hospitals. Blood samples from adult patients with suspected bloodstream infection were inoculated at the bedside into nonvented BM and BD standard aerobic blood culture bottles and incubated in their respective instruments. The laboratories received 6,743 pairs of bottles that were each filled with 8 to 12 ml of blood. A total of 523 isolates representing true infections were recovered from 257 patients; of these isolates, 348 were recovered from both the BD and the BM bottles, 108 were recovered from the BM bottles only, and 67 were recovered from the BD bottles only (
P
< 0.005). More staphylococci (
P
< 0.05), especially coagulase-negative staphylococci (
P
< 0.05), and yeasts (
P
< 0.01) were recovered from BM bottles than from BD bottles. Of 291 unimicrobial episodes of bloodstream infection, 220 were detected with both bottles, 41 were detected with the BM bottles only, and 30 were detected with the BD bottles only (difference not significant). Among 335 cultures that were positive in both bottles within the first 72 h of incubation, the median times to detection were 14 h for BM bottles and 13 h for BD bottles. Rates for false-positive results were 0.5% for BM bottles and 0.1% for BD bottles. One BM bottle and seven BD bottles yielded false-negative results. We conclude that the BM medium provides improved recovery of microorganisms, especially staphylococci and yeasts, compared with that provided by the BD medium.
Several
Salmonella enterica
subsp.
enterica
Serotype 4,5,12:i:− Phage Types Isolated from Swine Samples Originate from Serotype Typhimurium DT U302
Authors: de la Torre E. , Zapata D. , Tello M. , Mejía W. , Frías N. , García Peña F. J. , Mateu E. M. , Torre E. 
Published: June 2003

ABSTRACT

Pulsed-field gel electrophoresis, plasmid profiling, and phage typing were used to characterize and determine possible genetic relationships between 48
Salmonella enterica
subsp.
enterica
isolates of pig origin collected in Catalonia, Spain, from 1998 to 2000. The strains were grouped into 23 multidrug-resistant
fljB
-lacking
S. enterica
serovar 4,5,12:i:− isolates, 24
S. enterica
serovar Typhimurium isolates, and 1
S. enterica
serovar 4,5,12:−:− isolate. After combining the
Xba
I and
Bln
I macrorestriction profiles (
XB
profile), we observed 29 distinct subtypes which were grouped into seven main patterns. All 23 of the 4,5,12:i:− serovar strains and 10 serovar Typhimurium isolates were found to have pattern AR, and similarities of >78% were detected among the subtypes. Three of the serovar Typhimurium DT U302 strains (strains T3, T4, and T8) were included in the same 4,5,12:i:− serovar cluster and shared a plasmid profile (profile I) and a pattern of multidrug resistance (resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide, tetracycline, gentamicin, and trimethoprim-sulfamethoxazole) commonly found in monophasic isolates. This led us to the conclusion that strains of the
S. enterica
4,5,12:i:− serovar might have originated from an
S. enterica
serovar Typhimurium DT U302 strain.
Transient and PersistentHelicobacter pyloriColonization in Native American Children
Authors: Pérez-Pérez Guillermo I. , Sack R. Bradley , Reid Raymond , Santosham Mathuram , Croll Janne , Blaser Martin J. 
Published: June 2003

ABSTRACTHelicobacter pyloriis chiefly acquired in childhood, but the exact timing of acquisition is not well understood. The main goal of this study was to assessH. pyloriacquisition in a pediatric population. We studied two cohorts of Native American children: a birth cohort of 50 children and 58 older children (mean age, 53 months). We measured serum immunoglobulin G (IgG), IgM, and IgA antibodies toH. pyloriwhole-cell antigen and IgG antibodies to CagA. Among 44 birth cohort children monitored for more than 12 months, 24 (54.5%) had seroconversions, 7 (15.9%) were transient, and 17 (38.6%) were persistent. Among the older children, 49 (84.5%) of the 58 children were monitored for 1 year; 34 (69.4%) hadH. pyloriantibodies at study entry. During the next year, 7 (20.6%) children seroreverted, and of 15 initially negative children, 5 (33.3%) seroconverted. In both groups, evaluation of CagA antibodies increased the sensitivity ofH. pyloridetection. Serum pepsinogen I (PGI) levels inH. pylori-negative children rose significantly until age 6 months and remained constant for the next 19 months. At the time ofH. pyloriseroconversion, PGI peaked to levels significantly higher than in the never-seroconverted (P= 0.02) and the pre-seroconverted (P= 0.03) children, but then declined to levels paralleling those ofH. pylori-negative children. Thus,H. pyloriacquisition, accompanied by a transient PGI increase, was frequent in this population, especially in the second and third years of life, but often was brief.
Development of Group- and Serotype-Specific One-Step SYBR Green I-Based Real-Time Reverse Transcription-PCR Assay for Dengue Virus
Authors: Shu Pei-Yun , Chang Shu-Fen , Kuo Yu-Chung , Yueh Yi-Yun , Chien Li-Jung , Sue Chien-Lin , Lin Ting-Hsiang , Huang Jyh-Hsiung 
Published: June 2003

ABSTRACT

A quantitative one-step SYBR Green I-based reverse transcription (RT)-PCR system was developed for the detection and differentiation of four different dengue virus serotypes in acute-phase serum samples. A set of group- and serotype-specific primer pairs was designed against conserved sequences in the core region and evaluated for clinical diagnosis. A linear relationship was obtained between the amount of input RNA and cycle threshold (Ct) value over a range of 10 to 10
7
PFU per ml of cell culture-derived dengue viruses. The detection limit of the group-specific primer pair was between 4.1 and 43.5 PFU/ml for four dengue serotypes. The detection limit of each of the serotype-specific primer pairs was calculated to be 10 PFU/ml for dengue virus serotype 1 (DEN-1), 4.6 PFU/ml for DEN-2, 4.1 PFU/ml for DEN-3, and 5 PFU/ml for DEN-4. Comparisons between the one-step SYBR Green-based RT-PCR assay and the conventional cell culture method in the clinical diagnosis of dengue virus infection from acute-phase serum samples of confirmed dengue patients were performed. The results showed that 83 and 67% of 193 acute-phase serum samples tested were positive by the one-step SYBR Green-based RT-PCR method and cell culture method, respectively. Further analysis showed that the one-step SYBR Green-based RT-PCR method could detect twice as many acute-phase serum samples with positive dengue-specific immunoglobulin M (IgM) and/or IgG antibodies than cell culture method. Our results demonstrate the potential clinical application of the one-step SYBR Green I-based RT-PCR assay for the detection and differentiation of dengue virus RNA.
Limitations of TaqMan PCR for Detecting Divergent Viral Pathogens Illustrated by Hepatitis A, B, C, and E Viruses and Human Immunodeficiency Virus
Authors: Gardner Shea N. , Kuczmarski Thomas A. , Vitalis Elizabeth A. , Slezak Tom R. 
Published: June 2003

ABSTRACT
Recent events illustrate the imperative to rapidly and accurately detect and identify pathogens during disease outbreaks, whether they are natural or engineered. Particularly for our primary goal of detecting bioterrorist releases, detection techniques must be both species-wide (capable of detecting all known strains of a given species) and species specific. Due to classification restrictions on the publication of data for species that may pose a bioterror threat, we illustrate the challenges of finding such assays using five nonthreat organisms that are nevertheless of public health concern: human immunodeficiency virus (HIV) and four species of hepatitis viruses. Fluorogenic probe-based PCR assays (TaqMan; Perkin-Elmer Corp., Applied Biosystems, Foster City, Calif.) may be sensitive, fast methods for the identification of species in which the genome is conserved among strains, such as hepatitis A virus. For species such as HIV, however, the strains are highly divergent. We use computational methods to show that nine TaqMan primer and probe sequences, or signatures, are needed to ensure that all strains will be detected, but this is an unfeasible number, considering the cost of TaqMan probes. Strains of hepatitis B, C, and E viruses show intermediate divergence, so that two to three TaqMan signatures are required to detect all strains of each virus. We conclude that for species such as hepatitis A virus with high levels of sequence conservation among strains, signatures can be found computationally for detection by the TaqMan assay, which is a sensitive, rapid, and cost-effective method. However, for species such as HIV with substantial genetic divergence among strains, the TaqMan assay becomes unfeasible and alternative detection methods may be required. We compare the TaqMan assay with some of the alternative nucleic acid-based detection techniques of microarray, chip, and bead technologies in terms of sensitivity, speed, and cost.
Detection of Marek's Disease Virus DNA in Chicken but Not in Human Plasma
Authors: Hennig Holger , Osterrieder Nikolaus , Müller-Steinhardt Michael , Teichert Hanns-Martin , Kirchner Holger , Wandinger Klaus-Peter 
Published: June 2003

ABSTRACT
Marek's disease virus (MDV) causes a common lymphomatous and neuropathic disease in domestic chickens and, less commonly, turkeys and quail. It is a member of the α-herpesviruses and until now was considered to be strongly cell associated. In 1991, MDV was suggested to be the causative infectious agent of multiple sclerosis (MS) in humans. In a previous study, we investigated the leukocytes of 107 well-defined MS patients for the presence of MDV DNA but were unable to confirm a role for MDV in the pathogenesis of MS. A recent report (S. Laurent, E. Esnault, G. Dambrine, A. Goudeau, D. Choudat, and D. Rasschaert, J. Gen. Virol. 82:233-240, 2001) described the detection of MDV DNA in 20% of 202 human serum samples, regardless of whether the individuals were exposed to poultry. The detection of MDV DNA in chicken serum samples was reported as well. The aim of the present study was to investigate whether we can confirm the presence of MDV DNA in chickens and humans if we use plasma as the source for nucleic acid isolation. Leukocytes and plasma specimens from 16 chickens experimentally infected with MDV serotype 1 and plasma specimens from 300 volunteer blood donors were tested for MDV DNA by two different TaqMan PCR assays. MDV DNA was repeatedly found in the leukocytes as well as in the plasma specimens of all 16 animals. All human samples analyzed, however, tested negative by both assays. Accordingly, Marek's disease in chickens can be diagnosed by detection of MDV DNA in plasma as well as in leukocytes. Once again, we found no evidence for the spread of MDV to humans.
Development of a Heteroduplex Mobility Assay To Identify Field Isolates of Porcine Reproductive and Respiratory Syndrome Virus with Nucleotide Sequences Closely Related to Those of Modified Live-Attenuated Vaccines
Authors: Key K. F. , Guenette D. K. , Yoon K.-J. , Halbur P. G. , Toth T. E. , Meng X. J. 
Published: June 2003

ABSTRACTPorcine reproductive and respiratory syndrome has been devastating the swine industry since the late 1980s. The disease has been controlled, to some extent, through the use of modified live-attenuated (MLV) vaccines once available. However, such a practice periodically resulted in isolation or detection of vaccine-like viruses from pigs as determined by a partial genomic sequencing. In this study, we developed a heteroduplex mobility assay (HMA) for quickly identifying porcine reproductive and respiratory syndrome virus (PRRSV) isolates with significant nucleotide sequence identities (≥98%) with the modified live-attenuated vaccines. The major envelope gene (ORF5) of 51 PRRSV field isolates recovered before and after the introduction of the vaccines was amplified, denatured, and reannealed with the HMA reference vaccine strains Ingelvac PRRS MLV and Ingelvac PRRS ATP, respectively. Nine of the 51 field isolates and the VR2332 parent virus of Ingelvac PRRS MLV, which were all highly related to Ingelvac PRRS MLV with ≤2% nucleotide sequence divergence as determined by sequence analysis, were all identified by the HMA to form homoduplexes with the reference Ingelvac PRRS MLV. No homoduplex-forming field isolate was identified when Ingelvac PRRS ATP was used as the HMA reference except for its parent virus JA142. Other field isolates with more than 2% nucleotide sequence divergence with the respective reference vaccine strain resulted in the formation of heteroduplexes with reduced mobility in polyacrylamide gel electrophoresis. The HMA results also correlated well with the results of phylogenetic analyses. The data indicated that the HMA developed in the study may be a rapid and efficient method for large-scale screening of potential vaccine-like PRRSV field isolates for further genetic characterization.
Comparison of Commercial DNA Extraction Kits for Extraction of Bacterial Genomic DNA from Whole-Blood Samples
Authors: Smith K. , Diggle M. A. , Clarke S. C. 
Published: June 2003

ABSTRACT
The demand for molecular diagnostic tests in medical microbiology has highlighted the need for efficient methods of DNA extraction. In addition, it is preferable for these methods to be automated. An example of such a requirement is for the confirmation of meningococcal disease where rapid, sensitive, and specific procedures are required for public health management purposes. Previous studies have shown that whole blood is the preferred method for the isolation of bacterial DNA in meningococcal disease, and in this study, we compare five commercially available kits for the extraction of bacterial genomic DNA from whole-blood samples. These include kits in a 96-well binding plate, 96-well filter plate, and metallic bead formats. The method for all five kits is described, and the sensitivity, specificity, ease of automation, and overall efficiency are determined.
Infection of a Ventriculoatrial Shunt with Phenotypically Variable
Staphylococcus epidermidis
Masquerading as Polymicrobial Bacteremia Due to Various Coagulase-Negative Staphylococci and
Kocuria varians
Authors: Ben-Ami Ronen , Navon-Venezia Shiri , Schwartz David , Carmeli Yehuda 
Published: June 2003

ABSTRACT

The diagnosis of bloodstream infection with coagulase-negative staphylococci is frequently based on the isolation of the same organism from more than one blood culture. Phenotypic variation is a common characteristic of pathogenic strains of
Staphylococcus epidermidis
which may affect species identification by the microbiology laboratory. We describe a patient with a new onset of nephritis and gram-positive bacteremia. Gram-positive cocci grew in multiple blood cultures and were identified by the Vitek 2 system as
Kocuria varians
,
Staphylococcus hyicus
, and
S. epidermidis
. Bacterial isolates grew on blood agar and Congo red agar plates as two distinct morphotypes and exhibited phenotypic variation. Neither morphotype could be identified by the API-Staph assay. Cellular fatty acid analysis identified one of the morphotypes as
S. epidermidis
but could not identify the other morphotype. All isolates were found to be identical by pulsed-field gel electrophoresis, and both colonial morphotypes were identified as
S. epidermidis
by 16S ribosomal gene sequencing. Phenotypic variation of
S. epidermidis
may affect identification to the species level by phenotype-based identification systems. Caution should be exercised when differentiating between true infection and contamination based on strain identification.
Shiga Toxin 1c-Producing
Escherichia coli
Strains: Phenotypic and Genetic Characterization and Association with Human Disease
Authors: Friedrich Alexander W. , Borell Julia , Bielaszewska Martina , Fruth Angelika , Tschäpe Helmut , Karch Helge 
Published: June 2003

ABSTRACT

The distribution of the
stx
1c
allele among Shiga toxin (Stx)-producing
Escherichia coli
(STEC) and the virulence characteristics of
stx
1c
-harboring STEC are unknown. In this study, we identified
stx
1c
in 76 (54.3%) of 140
eae
-negative, but in none of 155
eae
-positive, human STEC isolates (
P
< 0.000001). The 76
stx
1c
-harboring
E. coli
isolates belonged to 22 serotypes, and each produced Stx1c as demonstrated by latex agglutination. Characterization of putative virulence factors demonstrated the presence of the locus of proteolysis activity (LPA) and the high-pathogenicity island in 65.8 and 21.1%, respectively, of the 76 Stx1c-producing
E. coli
isolates. Moreover, all but three of these strains contained
saa
, the gene encoding an STEC autoagglutinating adhesin. The virulence profiles of Stx1c-producing
E. coli
isolates were mostly serotype independent and heterogeneous. This enabled us to subtype the isolates within the same serotype. The individuals infected with Stx1c-producing
E. coli
strains were between 3 months and 72 years old (median age, 23.5 years) and usually had uncomplicated diarrhea or were asymptomatic. We conclude that Stx1c-producing
E
.
coli
strains represent a significant subset of
eae
-negative human STEC isolates, which belong to various serotypes and frequently possess LPA and
saa
as their putative virulence factors. The phenotypic and molecular characteristics determined in this study allow the subtyping of Stx1c-producing STEC in epidemiological and clinical studies.
Evaluation of Repetitive Element Sequence-Based PCR as a Molecular Typing Method for
Clostridium difficile
Authors: Spigaglia Patrizia , Mastrantonio Paola 
Published: June 2003

ABSTRACT

Repetitive element sequence-based PCR (rep-PCR) is a typing method that enables the generation of DNA fingerprinting that discriminates bacterial strains. In this study, we evaluated the applicability of rep-PCR in typing
Clostridium difficile
clinical isolates. The results obtained by rep-PCR were compared with those obtained by pulsed-field gel electrophoresis (PFGE) and PCR ribotyping. A high correspondence between pattern differentiations produced by rep-PCR and PFGE was observed, whereas PCR ribotyping showed a lower level of discriminatory power.
Genetic Relatedness of Levofloxacin-Nonsusceptible
Streptococcus pneumoniae
Isolates from North America
Authors: Johnson Crystal N. , Benjamin, William H. , Moser Stephen A. , Hollingshead Susan K. , Zheng Xiaotian , Crain Marilyn J. , Nahm Moon H. , Waites Ken B. 
Published: June 2003

ABSTRACT

We characterized 32 levofloxacin-nonsusceptible
Streptococcus pneumoniae
(LNSP) isolates obtained from a broad geographic region of North America over a 5-year period by using capsular serotypes, antimicrobial susceptibility profiles, BOX-PCR, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Sixteen international clones identified by the Pneumococcal Molecular Epidemiology Network also were included for comparison. Fifteen serotypes were represented, with serogroups 6, 9, 14, 19, and 23 accounting for 63% of isolates. Among isolates whose quinolone resistance-determining regions were sequenced, all contained
gyrA
and
parC
point mutations. Sixty-three percent were penicillin susceptible, and 84% were erythromycin susceptible. BOX-PCR analysis identified 39 different band patterns among 32 LNSP and 16 international clones and grouped 16 isolates, including 2 international clones, into seven unrelated groups of 2 to 4 isolates each. PFGE analysis identified 35 different band patterns among 32 LNSP and 16 international clones and grouped 21 isolates, including 3 international clones, into eight unrelated groups of 2 to 6 isolates each. MLST performed on 10 isolates identified five allelic profiles and separated 9 isolates into four groups of 2 to 3 isolates each. Overall, each typing method indicated that the LNSP were heterogeneous and that resistance to fluoroquinolones was not closely associated with a particular serotype or with coresistance to other antimicrobial classes and suggests that LNSP have likely arisen through independent mutational events as a result of selective pressure. However, seven LNSP were found to be related to three international clones by PFGE.