bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Epidemiology of
Aspergillus fumigatus
Isolates Recovered from Water, Air, and Patients Shows Two Clusters of Genetically Distinct Strains
Authors: Warris Adilia , Klaassen Corné H. W. , Meis Jacques F. G. M. , de Ruiter Maaike T. , de Valk Hanneke A. , Abrahamsen Tore G. , Gaustad Peter , Verweij Paul E. 
Published: September 2003

ABSTRACT

There has been an increase in data suggesting that besides air, hospital water is a potential source of transmission of filamentous fungi, and in particular
Aspergillus fumigatus
. Molecular characterization of environmental and clinical
A. fumigatus
isolates, collected prospectively during an 18-month period, was performed to establish if waterborne fungi play a role in the pathogenesis of invasive aspergillosis. Isolates recovered from water (
n
= 54) and air (
n
= 21) at various locations inside and outside the hospital and from 15 patients (
n
= 21) with proven, probable, or possible invasive aspergillosis were genotyped by amplified fragment length polymorphism analysis. Based on genomic fingerprints, the environmental
A. fumigatus
isolates could be grouped into two major clusters primarily containing isolates recovered from either air or water. The genotypic relatedness between clinical and environmental isolates suggests that patients with invasive aspergillosis can be infected by strains originating from water or from air. In addition, 12 clusters with genetically indistinguishable or highly related strains were differentiated, each containing two to three isolates. In two clusters, clinical isolates recovered from patients matched those recovered from water sources, while in another cluster the clinical isolate was indistinguishable from one cultured from air. This observation might open new perspectives in the development of infection control measures to prevent invasive aspergillosis in high-risk patients. The genetic variability found between airborne and waterborne
A. fumigatus
strains might prove to be a powerful tool in understanding the transmission of invasive aspergillosis and in outbreak control.
Analysis of Loci Required for Determination of Serotype Antigenicity in
Streptococcus mutans
and Its Clinical Utilization
Authors: Shibata Yukie , Ozaki Kazuhisa , Seki Mitsuko , Kawato Takayuki , Tanaka Hideki , Nakano Yoshio , Yamashita Yoshihisa 
Published: September 2003

ABSTRACT

We recently identified the genes responsible for the serotype c-specific glucose side chain formation of rhamnose-glucose polysaccharide (RGP) in
Streptococcus mutans
. These genes were located downstream from the
rgpA
through
rgpF
locus that is involved in the synthesis of RGP. In the present study, the corresponding chromosomal regions were isolated from serotype e and f strains and characterized. The
rgpA
through
rgpF
homologs were well conserved among the three serotypes. By contrast, the regions downstream from the
rgpF
homolog differed considerably among the three serotypes. Replacement of these regions in the different serotype strains converted their serotypic phenotypes, suggesting that these regions participated in serotype-specific glucose side chain formation in each serotype strain. Based on the differences among the DNA sequences of these regions, a PCR method was developed to determine serotypes.
S. mutans
was isolated from 198 of 432 preschool children (3 to 4 years old). The serotypes of all but one
S. mutans
isolate were identified by serotyping PCR. Serotype c predominated (84.8%), serotype e was the next most common (13.3%), and serotype f occured rarely (1.9%) in Japanese preschool children. Caries experience in the group with a mixed infection by multiple serotypes of
S. mutans
was significantly higher than that in the group with a monoinfection by a single serotype.
Molecular Analysis of
Burkholderia cepacia
Complex Isolates from a Portuguese Cystic Fibrosis Center: a 7-Year Study
Authors: Cunha Mónica V. , Leitão Jorge H. , Mahenthiralingam Eshwar , Vandamme Peter , Lito Luís , Barreto Celeste , Salgado Maria José , Sá-Correia Isabel 
Published: September 2003

ABSTRACT

This work reports results of a systematic molecular analysis involving 113
Burkholderia cepacia
complex isolates obtained from 23 cystic fibrosis (CF) patients under surveillance over a 7-year period at the major Portuguese CF center, the Santa Maria Hospital in Lisbon. The majority of the isolates were serial isolates from persistently infected patients (more than one-half of the population examined). In agreement with previous studies,
B. cenocepacia
(formerly genomovar III) was the most prevalent species; it was isolated from 52% of the patients infected with
B. cepacia
complex isolates. Contrasting with previous studies, a very significant percentage of the Portuguese CF subpopulation examined was infected with
B. cepacia
genomovar I (36%) and
B. stabilis
(18%).
B. multivorans
was recovered from two of the infected patients. All four of the species or genomovars were associated with poor clinical outcome, including the cepacia syndrome, and gave rise to chronic and transient infections, with the clinical condition depending on the patient and other still-unidentified factors. The
B. cepacia
epidemic strain marker region was found exclusively in genomovar III strains, while
cblA
was detected in genomovars I and III, only. There was no clear relation between the presence of these markers and transmissibility. Altogether, our results indicate that the use of these markers or the genomovar status in identifying patients at higher risk for infection is uncertain.
Evaluation of Real-Time PCR for Detection of and Discrimination between
Bordetella pertussis
,
Bordetella parapertussis
, and
Bordetella holmesii
for Clinical Diagnosis
Authors: Templeton Kate E. , Scheltinga Sitha A. , van der Zee Anneke , Diederen Bram M. W. , Kruijssen Anna M. , Goossens Herman , Kuijper Ed , Claas Eric C. J. 
Published: September 2003

ABSTRACT

PCR is increasingly being used as a diagnostic test for the detection of
Bordetella pertussis
and
Bordetella parapertussis
DNA, as it has improved sensitivity and specificity in comparison to conventional techniques. The assay described here uses the two insertion sequences IS
481
and IS
1001
for
B. pertussis
and
B. parapertussis
, respectively, with detection by molecular beacons. The real-time PCR for IS
481
detects both
B. pertussis
and
Bordetella holmesii
, and the real-time PCR for IS
1001
detects both
B. parapertussis
and
B. holmesii
. By performing both assays discrimination between
B. pertussis
and
B. parapertussis
can be obtained. The sensitivity was 1 to 10 CFU/ml for
B. pertussis
, 10 CFU/ml for
B. parapertussis
, and 10 CFU/ml for
B. holmesii
in both assays. The clinical sensitivity of the
B. pertussis
assay was not affected by duplexing with an internal control PCR. Real-time PCR, conventional PCR, and culture were performed on 57 clinical samples. Eight of the 57 (14%) were found positive by culture, 19 of 57 (33%) were found positive by conventional PCR, and 22 of 57 (39%) were found positive by real-time PCR. One sample was inhibitory. When the
B. pertussis
assay was compared with a clinical standard for
B. pertussis
infection, sensitivity was 38, 83, and 100% and specificity was 100, 97, and 97% for culture, conventional PCR, and real-time PCR, respectively. The real-time PCR designed for
B. pertussis
and
B. parapertussis
provides sensitive and specific diagnosis of
B. pertussis
and
B. parapertussis
infections and is therefore suitable for implementation in the diagnostic laboratory.
Molecular Analysis of Cases of Italian Sheep Scrapie and Comparison with Cases of Bovine Spongiform Encephalopathy (BSE) and Experimental BSE in Sheep
Authors: Nonno Romolo , Esposito Elena , Vaccari Gabriele , Conte Michela , Marcon Stefano , Di Bari Michele , Ligios Ciriaco , Di Guardo Giovanni , Agrimi Umberto 
Published: September 2003

ABSTRACT

Concerns have been raised about the possibility that the bovine spongiform encephalopathy (BSE) agent could have been transmitted to sheep populations via contaminated feedstuffs. The objective of our study was to investigate the suitability of molecular strain typing methods as a surveillance tool for studying scrapie strain variations and for differentiating PrP
Sc
from sheep scrapie, BSE, and sheep BSE. We studied 38 Italian sheep scrapie cases from 13 outbreaks, along with a British scrapie case, an experimental ovine BSE, and 3 BSE cases, by analyzing the glycoform patterns and the apparent molecular masses of the nonglycosylated forms of semipurified, proteinase-treated PrP
Sc
. Both criteria were able to clearly differentiate sheep scrapie from BSE and ovine experimental BSE. PrP
Sc
from BSE and sheep BSE showed a higher glycoform ratio and a lower molecular mass of the nonglycosylated form compared to scrapie PrP
Sc
. Scrapie cases displayed homogeneous PrP
Sc
features regardless of breed, flock, and geographic origin. The glycoform patterns observed varied with the antibody used, but either a monoclonal antibody (MAb) (F99/97.6.1) or a polyclonal antibody (P7-7) was able to distinguish scrapie from BSE PrP
Sc
. While more extensive surveys are needed to further corroborate these findings, our results suggest that large-scale molecular screening of sheep populations for BSE surveillance may be eventually possible.
Ribosomal DNA Sequencing for Identification of Aerobic Gram-Positive Rods in the Clinical Laboratory (an 18-Month Evaluation)
Authors: Bosshard P. P. , Abels S. , Zbinden R. , Böttger E. C. , Altwegg M. 
Published: September 2003

ABSTRACT
We have evaluated over a period of 18 months the use of 16S ribosomal DNA (rDNA) sequence analysis as a means of identifying aerobic gram-positive rods in the clinical laboratory. Two collections of strains were studied: (i) 37 clinical strains of gram-positive rods well identified by phenotypic tests, and (ii) 136 clinical isolates difficult to identify by standard microbiological investigations, i.e., identification at the species level was impossible. Results of molecular analyses were compared with those of conventional phenotypic identification procedures. Good overall agreement between phenotypic and molecular identification procedures was found for the collection of 37 clinical strains well identified by conventional means. For the 136 clinical strains which were difficult to identify by standard microbiological investigations, phenotypic characterization identified 71 of 136 (52.2%) isolates at the genus level; 65 of 136 (47.8%) isolates could not be discriminated at any taxonomic level. In comparison, 16S rDNA sequencing identified 89 of 136 (65.4%) isolates at the species level, 43 of 136 (31.6%) isolates at the genus level, and 4 of 136 (2.9%) isolates at the family level. We conclude that (i) rDNA sequencing is an effective means for the identification of aerobic gram-positive rods which are difficult to identify by conventional techniques, and (ii) molecular identification procedures are not required for isolates well identified by phenotypic investigations.
Comparison of Serologic and Genetic
porB
-Based Typing of
Neisseria gonorrhoeae
: Consequences for Future Characterization
Authors: Unemo Magnus , Olcén Per , Albert Jan , Fredlund Hans 
Published: September 2003

ABSTRACT

Due to temporal changes in the epidemiology of gonorrhea, a precise characterization of
Neisseria gonorrhoeae
is essential. In the present study genetic heterogeneity in the
porB
genes of
N. gonorrhoeae
was examined, and serovar determination was compared to
porB
gene sequencing. Among 108
N. gonorrhoeae
isolates, phylogenetic analysis of the entire
porB
alleles (924 to 993 bp) identified 87 unique sequences. By analyzing only the four to six most heterogeneous
porB
gene regions (174 to 363 bp), 86 out of these 87 genetic variants were identified. Consequently, analysis of shorter highly variable regions of the
porB
gene generates high-level discriminatory ability as well as fast, objective, reproducible, and portable data for epidemiological characterization of
N. gonorrhoeae
. Regarding putative antigenic epitopes of PorB for Genetic Systems monoclonal antibodies (MAbs), some of the previous findings were confirmed, but new findings were also observed. For several of the MAbs, however, the precise amino acid residues of PorB critical for single-MAb reactivity were difficult to identify. In addition, repeated serovar determination of 108
N. gonorrhoeae
isolates revealed discrepancies for 34 isolates, mostly due to nonreproducible reactivity with single MAbs. Thus, the prospects of a genetic typing system with congruent translation of the serovar determination seem to be limited. In conclusion, analysis of short highly variable regions of the
porB
gene could form the basis for a fast molecular epidemiological tool for the examination of emergence and transmission of
N. gonorrhoeae
strains within the community.
Molecular Typing of, and Distribution of Genetic Markers among,
Burkholderia cepacia
Complex Isolates from Brazil
Authors: Detsika Maria G. , Corkill John E. , Magalhães Marcelo , Glendinning Kerry J. , Hart C. Anthony , Winstanley Craig 
Published: September 2003

ABSTRACT

PCR tests were used to assign genomovar status to 39 non-cystic fibrosis (non-CF) and 11 CF
Burkholderia cepacia
complex isolates from patients in hospitals in Recife, Brazil. Non-CF isolates were assigned to genomovar IIIA (71.8%), genomovar I (15.4%),
B. vietnamiensis
(7.7%), and
B. multivorans
(5.1%). CF isolates were assigned to genomovar IIIA (18.2%),
B. vietnamiensis
(18.2%), and genomovar I (9.1%). Six CF isolates sharing
recA
PCR-restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) patterns could not be assigned to a genomovar. 16S rDNA sequence obtained from these isolates indicated a closest relationship to
B. anthina
, but the
recA
sequence was equally divergent from several genomovars. PCR screening indicated the presence of
cblA
in only two isolates, whereas the
B. cepacia
epidemic strain marker was found in 22 of 28 genomovar IIIA isolates. A type III secretion gene was detected in all but genomovar I isolates. RAPD and PCR-RFLP assays, targeting both
recA
and
fliC
, indicated a large amount of genetic variability among the isolates, with many novel patterns being observed. Nine genomovar IIIA isolates from different non-CF patients and clinical sources had identical genotypes, indicating the presence of a common clone.
Detection of Immune-Complex-Dissociated Nonstructural-1 Antigen in Patients with Acute Dengue Virus Infections
Authors: Koraka Penelopie , Burghoorn-Maas Chantal P. , Falconar Andrew , Setiati Tatty E. , Djamiatun Kiss , Groen Jan , Osterhaus Albert D. M. E. 
Published: September 2003

ABSTRACT

Accurate and timely diagnosis of dengue virus (DEN) infections is essential for the differential diagnosis of patients with febrile illness and hemorrhagic fever. In the present study, the diagnostic value of a newly developed immune-complex dissociated nonstructural-1 (NS-1) antigen dot blot immunoassay (DBI) was compared to a commercially available DEN antigen detection kit (denKEY Blue kit; Globio Co., Beverly, Mass.) and a reverse transcription-PCR (RT-PCR) kit. Serial serum or plasma samples (
n
= 181) obtained from 55 acute DEN-infected patients were used. In samples obtained from 32 of these 55 DEN-infected patients, viral RNA could be detected by RT-PCR. DEN antigen was detected in only 10 of these 55 patient samples by using the denKEY kit. When these samples were treated with acid to release the immune-complex-associated NS-1 antigen for detection by DBI, 43 of these 55 patients were found to be positive for DEN NS-1 antigen. In nondissociated samples, 22 of these patients were found to be positive by the DBI. In the presence of DEN-specific immunoglobulin M antibodies, both viral RNA and DEN (NS-1) antigen could be detected. The number of positive samples identified by RT-PCR and DBI from these patients with primary DEN infections varied between 28 and 78%. In secondary DEN infections, the number of samples that tested positive by the DBI after immune-complex dissociation (DIS-DBI) was 25% higher than the number of samples that tested positive by RT-PCR and was 35% higher than that determined by nondissociated antigen (NDIS-DBI) detection. We conclude that the denKEY kit has limited diagnostic value for acute DEN infections compared to the RT-PCR and the NDIS-DBI and DIS-DBI methods. We clearly demonstrate that in secondary DEN infections the dissociation of NS-1 immune complexes is essential for early diagnosis of DEN infections.
Comparison of Quantitative Reverse Transcription-PCR to Viral Culture for Assessment of Respiratory Syncytial Virus Shedding
Authors: Falsey Ann R. , Formica Maria A. , Treanor John J. , Walsh Edward E. 
Published: September 2003

ABSTRACTRespiratory syncytial virus (RSV) has recently been recognized as a serious pathogen in elderly and immunocompromised adults. Diagnosis of acute infection in adults is often difficult due to the insensitivity of viral culture, and reverse transcription-PCR (RT-PCR) is a more sensitive alternative. The relationship of quantitative RT-PCR to viable virus has never been studied for RSV. Therefore, we compared a quantitative real-time RT-PCR with viral culture to assess viral load in adult volunteers challenged with the RSV A2 strain. Twelve of 13 volunteers were infected, and there was a high correlation (r= 0.84) between quantitative RT-PCR and viral titer by cell culture. However, RT-PCR was more sensitive, with 73 of 169 (43%) samples positive compared to 58 (34%) samples positive by culture. The correlation between the two tests was highest early in the course of viral shedding (r= 0.91, days 0 to 6), whereas during days 7 to 13, there was more variability (r= 0.70). All subjects were culture negative by day 11, whereas one subject remained RT-PCR positive on day 12. All subjects were RT-PCR negative at day 28 postinfection. Quantitative RT-PCR has an excellent correlation with viral titers, as measured by culture, and should be a useful tool for future studies addressing viral load and disease pathogenesis.
Intraspecies Genotype Variability of the Microsporidian ParasiteEncephalitozoon hellem
Authors: Haro María , del Águila Carmen , Fenoy Soledad , Henriques-Gil Nuno 
Published: September 2003

ABSTRACTSeven isolates ofEncephalitozoon hellemfrom human immunodeficiency virus-positive patients were genotyped through a series of markers: the internal transcribed spacer (ITS) of ribosomal DNA, the polar tube protein (PTP) gene, and two intergenic spacers (IGS-TH and IGS-HZ) whose polymorphism is newly reported. The genome markers were all analyzed at three levels: PCR amplification followed by polyacrylamide gel electrophoresis, single-strand conformation analysis (SSCA), and DNA sequencing. The polymorphisms detected involve insertions/deletions and point mutations. SSCA can distinguish any pair of sequences, even those differing by a single base pair. The different isolates studied fit into the previously described ITS genotype 1A, except one which seems to be a 2A derivative variant (2D). When PTP and the new markers IGS-TH and IGS-HZ were analyzed, most of the isolates displayed different genotypes, demonstrating thatE. hellemhas a strong intraspecies variability. A set of markers such as those used here may be very useful in genotyping of clinical samples and in the assessment of epidemiological relationships amongE. hellemstrains.
Development and Evaluation of a Seminested PCR for Detection and Differentiation ofBabesia gibsoni(Asian Genotype) andB. canisDNA in Canine Blood Samples
Authors: Birkenheuer Adam J. , Levy Michael G. , Breitschwerdt Edward B. 
Published: September 2003

ABSTRACTCanine babesiosis has recently been recognized as an emerging infectious disease of dogs in North America. We sought to develop a seminested PCR to detect and differentiateBabesia gibsoni(Asian genotype),B. canissubsp.vogeli,B. canissubsp.canis, andB. canissubsp.rossiDNA in canine blood samples. An outer primer pair was designed to amplify an ∼340-bp fragment of the 18S rRNA genes fromB. gibsoni(Asian genotype),B. canissubsp.vogeli,B. canissubsp.rossi, andB. canissubsp.canisbut not mammalian DNA. Forward primers were designed that would specifically amplify a smaller fragment from each organism in a seminested PCR. The practical limit of detection was 50 organisms/ml of mock-infected EDTA anticoagulated whole blood. The primer pair also amplified an ∼370-bp fragment of theB. gibsoni(USA/California genotype) 18S rRNA gene from the blood of an experimentally infected dog with a high percentage of parasitemia. Amplicons were not detected when DNA extracted from the blood of a dog that was naturally infected withTheileria annaeat a low percentage of parasitemia was amplified. Due to limited sensitivity, this test is not recommended for the routine diagnosis ofB. gibsoni(USA/California genotype) orT. annae. The PCR test did not amplifyToxoplasma gondii,Neospora caninum,Leishmania infantum,Cryptosporidium parvum, or canine DNA under any of the conditions tested. The seminested PCR test was able to detect and discriminateB. gibsoni(Asian genotype),B. canissubsp.vogeli,B. canissubsp.canis, andB. canissubsp.rossiDNA in blood samples from infected dogs.
Antimicrobial Susceptibility and Serotype Distribution of
Streptococcus pneumoniae
and Molecular Characterization of Multidrug-Resistant Serotype 19F, 6B, and 23F Pneumococci in Northern Thailand
Authors: Watanabe Hiroshi , Asoh Norichika , Hoshino Kazuhiko , Watanabe Kiwao , Oishi Kazunori , Kositsakulchai Weerayut , Sanchai Tippaya , Kunsuikmengrai Khemrassamee , Kahintapong Sumpun , Khantawa Banyong , Tharavichitkul Prasit , Sirisanthana Thira , Nagatake Tsuyoshi 
Published: September 2003

ABSTRACT

Penicillin-resistant
Streptococcus pneumoniae
is widely spread worldwide. Our study was undertaken to examine the susceptibility and serotypes of
S. pneumoniae
in northern Thailand. Ninety-three
S. pneumoniae
strains were isolated from 93 patients at Chiang Mai University Hospital, Chiang Mai, Thailand, from September 1999 to June 2000. The strains were isolated from sputum (
n
= 51), blood (
n
= 15), nasopharynges (
n
= 14), and other sources (e.g., pus, ears, ascites, and cerebrospinal fluid) (
n
= 13). Of the 93 isolates, 29 (31.2%) were susceptible, 24 (25.8%) showed intermediate resistance (MIC, 0.12 to 1.0 μg/ml), and 40 (43.0%) were fully resistant (MIC, ≥2.0 μg/ml) to penicillin G. Seven (46.7%) from blood, 5 (35.7%) from nasopharynges, 15 (29.4%) from sputum, and 2 (15.4%) from other sources were susceptible isolates. Serotyping with the use of antiserum revealed differences in the predominant types that were susceptible (6A, 11A, and 19A), intermediately resistant (6B and 23F), and fully resistant (6B, 19F, and 23F). Molecular typing by pulsed-field gel electrophoresis of multidrug-resistant pneumococci showed four patterns (A, B, C, and D) for 16 isolates of serotype 19F, with pattern B being predominant (12 isolates). This finding was different from that with the Taiwan multidrug-resistant serotype 19F clone. Eleven isolates of serotype 6B all showed pattern E, and nine isolates of serotype 23F showed two patterns (F and G), with pattern F being predominant (seven isolates). This finding was similar to that with the Spanish multidrug-resistant serotype 23F clone. Our results indicated that the resistance of pneumococci to antibiotics in northern Thailand is progressing rapidly and that effort should be intensified to prevent any spread of pandemic multidrug-resistant serotypes 19F, 6B, and 23F.