bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of Polymorphic Microsatellite Markers for Typing
Penicillium marneffei
Isolates
Authors: Lasker Brent A. , Ran Yuping 
Published: April 2004

ABSTRACT

Penicillium marneffei
is an emerging opportunistic dimorphic fungal pathogen that is endemic in Southeast Asia. A typing method based on the analysis of size polymorphisms in microsatellite loci was investigated. Three loci available from the GenBank database were identified to harbor microsatellites. PCR primers flanking the microsatellite repeats were designed with one primer in the set fluorescently labeled. PCR products were then sized by automated capillary electrophoresis. As expected for a haploid fungus, a single band was observed for each microsatellite locus for all isolates. Polymorphic microsatellite marker (PMM) analysis detected a total of 22 different allelic types for 35 isolates of
P. marneffei
with a high discriminatory power (
D
= 0.956). Microsatellites I, II, and III detected 14, 10, and 7 alleles, respectively. The reproducibility of length polymorphisms was confirmed by using different DNA preparations from the same isolate or by repeated runs from the same DNA preparation. PMM profiles for eight isolates passaged in vitro for 7 to 8 weeks were identical to the original culture, demonstrating short-term stability and reproducibility. PCR products were not observed for other dimorphic fungi or human DNA. Comparison of allelic frequencies in isolates obtained from China and Thailand identified distinct allele combinations, suggesting the potential geographic isolation of populations. Due to the high discriminatory power, reproducibility, and potential for high throughput, PMM analysis may provide a good typing method for epidemiologic and surveillance investigations of
P. marneffei.
Epidemiology and Clinical Course of
Burkholderia cepacia
Complex Infections, Particularly Those Caused by Different
Burkholderia cenocepacia
Strains, among Patients Attending an Italian Cystic Fibrosis Center
Authors: Manno Graziana , Dalmastri Claudia , Tabacchioni Silvia , Vandamme Peter , Lorini Renata , Minicucci Laura , Romano Luca , Giannattasio Alessandro , Chiarini Luigi , Bevivino Annamaria 
Published: April 2004

ABSTRACT

In this study, the epidemiology of
Burkholderia cepacia
complex (Bcc) recovered from the sputum of 75 patients attending the Genoa Cystic Fibrosis (CF) Center at the Gaslini Children's Hospital (Genoa, Italy) was investigated, and the clinical course of the CF patients infected with the different species and genomovars of Bcc was evaluated. All isolates were analyzed for genomovar status by
recA
gene polymorphism and subsequently random amplified polymorphic DNA fingerprinting.
Burkholderia cenocepacia
is the predominant species recovered from the CF patients infected with Bcc at the Genoa CF Center. Of the other eight species comprising the Bcc, only a few isolates belonging to
B. cepacia
genomovar I,
Burkholderia stabilis
, and
Burkholderia pyrrocinia
were found. Of the four
recA
lineages of
B. cenocepacia
, most patients were infected by epidemic strains belonging to lineages IIIA and IIID, whereas only a few patients harbored IIIB strains. Patient-to-patient spread of Bcc among CF patients was mostly associated with
B. cenocepacia
, in particular with strains belonging to
recA
lineages IIIA and IIID. The mortality of CF patients infected with Bcc at the Genoa CF Center was significantly higher than mortality among CF patients not infected with Bcc. All of the deaths were associated with the presence of
B. cenocepacia
, except the case of a patient infected with
B. cepacia
genomovar I. Within
B. cenocepacia
, infection with epidemic strains belonging to lineages IIIA and IIID was associated with higher rates of mortality than was infection with lineage IIIB strains. No significant differences in lung function, body weight, and mortality rate were observed between patients infected with epidemic strains belonging to either
B. cenocepacia
IIIA or
B. cenocepacia
IIID.
SYBR Green-Based Real-Time Quantitative PCR Assay for Detection of West Nile Virus Circumvents False-Negative Results Due to Strain Variability
Authors: Papin James F. , Vahrson Wolfgang , Dittmer Dirk P. 
Published: April 2004

ABSTRACT
Real-time quantitative PCR is used routinely for the high-throughput diagnosis of viral pathogens, such as West Nile virus (WNV). Rapidly evolving RNA viruses present a challenge for diagnosis because they accumulate mutations that may render them undetectable. To explore the effect of sequence variations on assay performance, we generated every possible single point mutation within the target region of the widely used TaqMan assay for WNV and found that the TaqMan assay failed to detect 47% of possible single nucleotide variations in the probe-binding site and was unable to detect any targets with more than two mutations. In response, we developed and validated a less expensive assay with the intercalating dye SYBR green. The SYBR green-based assay was as sensitive as the TaqMan assay for WNV. Importantly, it detected 100% of possible WNV target region variants. The assay developed here adds an additional layer of protection to guard against false-negative results that result from natural variations or drug-directed selection and provides a rapid means to identify such variants for subsequent detailed analysis.
Incidence of Bloodstream Infections Due to
Candida
Species and In Vitro Susceptibilities of Isolates Collected from 1998 to 2000 in a Population-Based Active Surveillance Program
Authors: Hajjeh Rana A. , Sofair Andre N. , Harrison Lee H. , Lyon G. Marshall , Arthington-Skaggs Beth A. , Mirza Sara A. , Phelan Maureen , Morgan Juliette , Lee-Yang Wendy , Ciblak Meral A. , Benjamin Lynette E. , Thomson Sanza Laurie , Huie Sharon , Yeo Siew Fah , Brandt Mary E. , Warnock David W. 
Published: April 2004

ABSTRACT

To determine the incidence of
Candida
bloodstream infections (BSI) and antifungal drug resistance, population-based active laboratory surveillance was conducted from October 1998 through September 2000 in two areas of the United States (Baltimore, Md., and the state of Connecticut; combined population, 4.7 million). A total of 1,143 cases were detected, for an average adjusted annual incidence of 10 per 100,000 population or 1.5 per 10,000 hospital days. In 28% of patients,
Candida
BSI developed prior to or on the day of admission; only 36% of patients were in an intensive care unit at the time of diagnosis. No fewer than 78% of patients had a central catheter in place at the time of diagnosis, and 50% had undergone surgery within the previous 3 months.
Candida albicans
comprised 45% of the isolates, followed by
C. glabrata
(24%),
C. parapsilosis
(13%), and
C. tropicalis
(12%). Only 1.2% of
C. albicans
isolates were resistant to fluconazole (MIC, ≥64 μg/ml), compared to 7% of
C. glabrata
isolates and 6% of
C. tropicalis
isolates. Only 0.9% of
C. albicans
isolates were resistant to itraconazole (MIC, ≥1 μg/ml), compared to 19.5% of
C. glabrata
isolates and 6% of
C. tropicalis
isolates. Only 4.3% of
C. albicans
isolates were resistant to flucytosine (MIC, ≥32 μg/ml), compared to <1% of
C. parapsilosis
and
C. tropicalis
isolates and no
C. glabrata
isolates. As determined by E-test, the MICs of amphotericin B were ≥0.38 μg/ml for 10% of
Candida
isolates, ≥1 μg/ml for 1.7% of isolates, and ≥2 μg/ml for 0.4% of isolates. Our findings highlight changes in the epidemiology of
Candida
BSI in the 1990s and provide a basis upon which to conduct further studies of selected high-risk subpopulations.
Comparison of Multiplex PCR Assay with Culture for Detection of Genital Mycoplasmas
Authors: Stellrecht Kathleen A. , Woron Amy M. , Mishrik Nada G. , Venezia Richard A. 
Published: April 2004

ABSTRACT

Ureaplasma
, spp.
Mycoplasma genitalium
, and
Mycoplasma hominis
are associated with infection of the genitourinary tract, reproductive failure, and neonatal morbidity and mortality. We have developed a multiplex PCR for the detection of these
Mycoplasmatales
in a single amplification reaction. The analytical sensitivities of this assay were 10.8, 10.8, and 8.8 CFU for each organism, respectively. This multiplex PCR was compared to culture on 26 cervical swabs, 2 vaginal swabs, 4 female urine specimens, 49 semen samples, 2 male urine specimens, and 1 nonspecified sample. A total of 21 specimens were culture positive (25%); 17 of these were PCR positive. An additional 11 specimens were PCR positive but culture negative. Of the 21 culture-positive specimens, 17 (81%) grew
Ureaplasma
spp. and 4 (19%) grew
Mycoplasma
spp. Of the 28 PCR-positive specimens,
Ureaplasma
spp. was detected in 23 (82%),
M. hominis
was detected in 3 (11%), and both were detected in 2 (7%). In a confirmatory analysis, all samples were tested by amplification of a second target of the ureaplasma genome. True-positive cases were defined as a positive result by culture or by both amplification assays. The multiplex PCR detected organisms in 26 of the 30 true-positive specimens, as well as in 2 other specimens. Based on a 36% prevalence of infection, the sensitivity, specificity, and positive and negative predictive values of multiplex PCR analyses were 87, 96, 94, and 93%, respectively. Multiplex PCR offers a rapid, sensitive, and easy method to detect genital mycoplasmas.
Development of a Nucleic Acid Sequence-Based Amplification Assay That Uses
gag
-Based Molecular Beacons To Distinguish between Human Immunodeficiency Virus Type 1 Subtype C and C′ Infections in Ethiopia
Authors: Ayele Workenesh , Pollakis Georgios , Abebe Almaz , Fisseha Bitew , Tegbaru Belete , Tesfaye Girma , Mengistu Yohannes , Wolday Dawit , van Gemen Bob , Goudsmit Jaap , Dorigo-Zetsma Wendelien , de Baar Michel P. 
Published: April 2004

ABSTRACT

A
gag
-based molecular beacon assay utilizing real-time nucleic acid sequence-based amplification technology has been developed to differentiate between the two genetic subclusters of human immunodeficiency virus type 1 (HIV-1) subtype C (C and C′) circulating in Ethiopia. Of 41 samples, 36 could be classified as C or C′ by sequencing of the
gag
gene. All 36 isolates were correctly identified by the
gag
beacon test. Three isolates with genomes that were recombinant in
gag
were unambiguously typed as belonging to the C′ subcluster. Further analysis revealed that these contained the most sequence homology with a reference subcluster C′ sequence in the target region of the beacon and hence were correct for the analyzed region. For one sample, sequencing and
gag
molecular beacon results did not match, while another isolate could not be detected at all by the beacon assay. Overall, high levels of sensitivity and specificity were achieved for both beacons (90.5% sensitivity and 100% specificity for the C beacon and 100% sensitivity and 95.2% specificity for the C′ beacon). The availability of a diagnostic test which can quickly and reliably discriminate between C and C′ HIV-1 infections in Ethiopia is an important first step toward studying their respective biological characteristics. As the assay is specific to the Ethiopian HIV-1 subtype C epidemic, it will contribute to characterizing the circulating viruses in this population, thereby generating the information necessary for the development of a potential efficacious HIV-1 vaccine appropriate for the Ethiopian context.
Detection of Multiple Macrolide- and Lincosamide-Resistant Strains of
Streptococcus pyogenes
from Patients in the Boston Area
Authors: Hasenbein Meredith E. , Warner John E. , Lambert Kathleen G. , Cole Sarah E. , Onderdonk Andrew B. , McAdam Alexander J. 
Published: April 2004

ABSTRACT

Macrolide (including erythromycin and azithromycin) and lincosamide (including clindamycin) antibiotics are recommended for treatment of penicillin-allergic patients with
Streptococcus pyogenes
pharyngitis. Resistance to erythromycin in
S. pyogenes
can be as high as 48% in specific populations in the United States. Macrolide and lincosamide resistance in
S. pyogenes
is mediated by several different genes. Expression of the
erm
(A) or
erm
(B) genes causes resistance to erythromycin and inducible or constitutive resistance to clindamycin, respectively, whereas expression of the
mef
(A) gene leads to resistance to erythromycin but not clindamycin. We studied the resistance of
S. pyogenes
to erythromycin and clindamycin at an urban tertiary-care hospital. Of 196 sequential isolates from throat cultures, 15 (7.7%) were resistant to erythromycin. Three of these were also constitutively resistant to clindamycin and had the
erm
(B) gene. Five of the erythromycin-resistant isolates were resistant to clindamycin upon induction with erythromycin and had the
erm
(A) gene. The remaining seven erythromycin-resistant isolates were susceptible to clindamycin even upon induction with erythromycin and had the
mef
(A) gene. Pulsed-field gel electrophoresis analysis and
emm
typing demonstrated that the erythromycin-resistant
S. pyogenes
comprised multiple strains. These results demonstrate that multiple mechanisms of resistance to macrolide and lincosamide antibiotics are present in
S. pyogenes
strains in the United States.
Rapid and Sensitive Method Using Multiplex Real-Time PCR for Diagnosis of Infections by Influenza A and Influenza B Viruses, Respiratory Syncytial Virus, and Parainfluenza Viruses 1, 2, 3, and 4
Authors: Templeton Kate E. , Scheltinga Sitha A. , Beersma Matthias F. C. , Kroes Aloys C. M. , Claas Eric C. J. 
Published: April 2004

ABSTRACT
Laboratory diagnosis of viral respiratory infections is generally performed by virus isolation in cell culture and immunofluorescent assays. Reverse transcriptase PCR is now recognized as a sensitive and specific alternative for detection of respiratory RNA viruses. A rapid real-time multiplex PCR assay was developed for the detection of influenza A and influenza B viruses, human respiratory syncytial virus (RSV), parainfluenza virus 1 (PIV1), PIV2, PIV3, and PIV4 in a two-tube multiplex reaction which used molecular beacons to discriminate the pathogens. A total of 358 respiratory samples taken over a 1-year period were analyzed by the multiplex assay. The incidence of respiratory viruses detected in these samples was 67 of 358 (19%) and 87 of 358 (24%) by culture and real-time PCR, respectively. Culture detected 3 influenza A virus, 2 influenza B virus, 57 RSV, 2 PIV1, and 2 PIV3 infections. All of these culture-positive samples and an additional 5 influenza A virus, 6 RSV, 2 PIV1, 1 PIV2, 1 PIV3, and 3 PIV4 infections were detected by the multiplex real-time PCR. The application of real-time PCR to clinical samples increases the sensitivity for respiratory viral diagnosis. In addition, results can be obtained within 6 h, which increases clinical relevance. Therefore, use of this real-time PCR assay would improve patient management and infection control.
Immunological Characterization of the Spike Protein of the Severe Acute Respiratory Syndrome Coronavirus
Authors: Lu Liqun , Manopo Ivanus , Leung Bernard P. , Chng Hiok Hee , Ling Ai Ee , Chee Li Lian , Ooi Eng Eong , Chan Shzu-Wei , Kwang Jimmy 
Published: April 2004

ABSTRACT
Severe acute respiratory syndrome (SARS) is a novel infectious disease caused by the SARS-associated coronavirus (SARS-CoV). There are four major structural proteins in the SARS-CoV, including the nucleocapsid, spike, membrane, and small envelope proteins. In this study, two sets of truncated fragments of spike protein were generated, the first were approximately 210-bp nonoverlapping fragments and the second were overlapping segments of 750 to 900 bp. From these 23 fragments, we identified a fragment of 259 amino acids (amino acids 441 to 700) that is a major immunodominant epitope. This fragment was highly expressed, and the purified fragment C could detect all 33 SARS patient serum samples tested, collected from 7 to 60 days after the onset of fever, but had no reactivity with all 66 healthy human serum samples tested. Thus, fragment C of spike protein was identified as an immunodominant antigen and could be used for serological detection of SARS-CoV infection.
Molecular Diagnosis of Human Adenoviruses D and E by a Phylogeny-Based Classification Method Using a Partial Hexon Sequence
Authors: Shimada Yasushi , Ariga Toshihide , Tagawa Yoshitsugu , Aoki Koki , Ohno Shigeaki , Ishiko Hiroaki 
Published: April 2004

ABSTRACT
Human adenoviruses (HAdVs) are the major causes of a variety of acute illnesses. Virus isolation and neutralization tests are usually done to identify the causative virus, but these tests are labor-intensive and time-consuming, and standardized antisera are in limited supply. This study investigated a rapid and reliable method of virus identification based on PCR and phylogenetic analysis. The phylogenetic tree constructed by neighbor joining on the basis of the newly determined partial hexon sequences from 33 prototypes of HAdV-D and -E, along with 11 available prototypes of HAdV-A to -C and -F from GenBank, allowed HAdVs to be grouped into six distinct clusters. These clusters correspond closely to the six newly designated species, HAdV-A to -F. The partial hexon sequences of 57 isolates from patients with acute conjunctivitis obtained over 20 years plus those of 44 prototype strains were analyzed. Each isolate formed a monophyletic cluster along with its respective prototype strain, allowing serotype identification. Partial-hexon-based classification appears to be an effective tool for studying the molecular epidemiology of HAdVs.
Immunoblot Method To Detect
Streptococcus pneumoniae
and Identify Multiple Serotypes from Nasopharyngeal Secretions
Authors: Bronsdon Melinda A. , O'Brien Katherine L. , Facklam Richard R. , Whitney Cynthia G. , Schwartz Benjamin , Carlone George M. 
Published: April 2004

ABSTRACT

Conventional culture techniques are limited in the ability to detect multiple
Streptococcus pneumoniae
serotypes in nasopharyngeal (NP) secretions. We developed an immunoblot (IB) method with monoclonal antibodies (MAbs) to detect
S. pneumoniae
and to identify serotypes. NP specimens stored in skim milk-tryptone-glucose-glycerol medium were assessed by the IB method and the reference culture method (RM). In the RM, four optochin-sensitive alpha-hemolytic colonies resembling pneumococci were typed by the Quellung reaction. In the IB method, a nitrocellulose membrane blot of surface growth was reacted with a pneumococcal surface adhesion (PsaA) MAb and visualized. Of 47 NP specimens, 32 (68%) were found to be positive and 13 (28%) were found to be negative for pneumococci by both methods; each method alone yielded one positive result. The sensitivity and specificity of the IB method for the detection of pneumococci were 97 and 93%, respectively. To identify serotypes, blots were tested with serotype-specific MAbs (4, 6A, 6B, 9V, 14, 18C, 19F, and 23F). To detect the remaining serotypes, positive serotype-specific replicate blots were compared visually to an original anti-PsaA-positive blot; four unidentified colonies were subcultured and serotyped by the Quellung reaction. Fifty-eight
S. pneumoniae
-positive NP specimens containing 69 pneumococcal strains (23 serotypes) were tested; 68 (98.6%) of the strains were detected by the IB method, and 66 (95.6%) were detected by the RM. For 11 specimens found to contain two serotypes, both methods detected both serotypes in 7 (63.6%), the IB method alone detected the two serotypes in 3 (27.3%), and the RM alone detected both serotypes in 1 (9%). The IB method identified multiple clones and minor populations of pneumococci in NP secretions. This method is useful for detecting specific serotypes and carriage of multiple serotypes in epidemiologic surveillance and carriage studies.
Improved Enzyme-Linked Immunosorbent Assay Using C-Terminal Truncated Recombinant Antigens of
Babesia bovis
Rhoptry-Associated Protein-1 for Detection of Specific Antibodies
Authors: Boonchit Suthisak , Xuan Xuenan , Yokoyama Naoaki , Goff Will L. , Waghela Suryakant D. , Wagner Gale , Igarashi Ikuo 
Published: April 2004

ABSTRACT

An enzyme-linked immunosorbent assay (ELISA) based on a recombinant rhoptry-associated protein-1 (RAP-1) of
Babesia bovis
has been previously developed, but it was imperfect because some cross-reactions were still present in
Babesia bigemina
-infected bovine sera. To improve its accuracy for the specific detection of the antibodies to
B. bovis
, we constructed three C-terminal truncated recombinant antigens of the RAP-1—rCT1 (amino acids [aa] 301 to 408), rCT2 (aa 388 to 490), and rCT3 (aa 466 to 565)—by using a baculovirus expression system and evaluated their diagnostic potentials using ELISA. rCT1 and rCT2 were better diagnostic antigens in their sensitivities and diagnostic efficiencies than rCT3, although none of the recombinant antigens showed any cross-reactivity to
B. bigemina
-infected bovine sera. These results confirmed that the N-terminal 300-aa region caused cross-reactivity of the entire RAP-1 antigen, and the C-terminal truncated recombinant antigens were shown to be useful reagents for species-specific serodiagnosis.
Reassessment of Sequence-Based Targets for Identification ofBacillusSpecies
Authors: Blackwood K. S. , Turenne C. Y. , Harmsen D. , Kabani A. M. 
Published: April 2004

ABSTRACTTheBacillusgenus is a large heterogeneous group in need of an efficient method for species differentiation. To determine the current validity of a sequence-based method for identification and provide contemporary data, PCR and sequencing of a 500-bp product encompassing the V1 to V3 regions of the 16S rRNA gene were undertaken using 65 of the 83 type strains of this genus. This region proved discriminatory between most species (70.0 to 100% similarity), the exceptions being clinically relevantB. cereusandB. anthracisas well as nonpathogenicB. psychrotoleransandB. psychrodurans.Consequently, 27 type and clinical strains from theB. cereusgroup were used to test alternate targets (rpoB,vrrA, and the 16S-23S spacer region) for identification. TherpoBgene proved the best alternate target, with a conserved 4-nucleotide difference betweenB. cereusandB. anthracis. The high 16S rRNA gene sequence similarities between some strains demonstrated the need for a polyphasic approach to the systematics of this genus. This approach is one focus of the Ribosomal Differentiation of Medical Microorganisms mandate. Accordingly, the 16S rRNA gene sequences generated in this study have been submitted for inclusion into its publicly accessible, quality-controlled database athttp://www.ridom_rdna.de/.