bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of Antifungal Activities and 16S Ribosomal DNA Sequences of Clinical and Environmental Isolates of
Stenotrophomonas maltophilia
Authors: Minkwitz Arite , Berg Gabriele 
Published: July 2002

ABSTRACT

In recent years, the gram-negative bacterium
Stenotrophomonas maltophilia
has become increasingly important in biotechnology and as a nosocomial pathogen, giving rise to a need for new information about its taxonomy and epidemiology. To determine intraspecies diversity and whether strains can be distinguished based on the sources of their isolation, 50
S. maltophilia
isolates from clinical and environmental sources, including strains of biotechnological interest, were investigated. The isolates were characterized by in vitro antagonism against pathogenic fungi and the production of antifungal metabolites and enzymes. Phenotypically the strains showed variability that did not correlate significantly with their sources of isolation. Clinical strains displayed remarkable activity against the human pathogenic fungus
Candida albicans
. Antifungal activity against plant pathogens was more common and generally more severe from the environmental isolates, although not exclusive to them. All isolates, clinical and environmental, produced a range of antifungal metabolites including antibiotics, siderophores, and the enzymes proteases and chitinases. From 16S ribosomal DNA sequencing analysis, the isolates could be separated into three clusters, two of which consisted of isolates originating from the environment, especially rhizosphere isolates, and one of which consisted of clinical and aquatic strains. In contrast to the results of other recent investigations, these strains could be grouped based on their sources of isolation, with the exception of three rhizosphere isolates. Because there was evidence of nucleotide signature positions within the sequences that are suitable for distinguishing among the clusters, the clusters could be defined as different genomovars of
S. maltophilia
. Key sequences on the 16S ribosomal DNA could be used to develop a diagnostic method that differentiates these genomovars.
Genetic Analysis for Virulence Factors in
Escherichia coli
O104:H21 That Was Implicated in an Outbreak of Hemorrhagic Colitis
Authors: Feng Peter , Weagant Stephen D. , Monday Steven R. 
Published: July 2002

ABSTRACT

Isolates of enterohemorrhagic
Escherichia coli
(EHEC) of serotype O104:H21 implicated in a 1994 outbreak of hemorrhagic colitis in Montana were analyzed for the presence of trait EHEC virulence markers. By using a multiplex PCR that specifically amplifies several genes, the O104:H21 strains were found to carry only the Shiga toxin 2 gene (
stx2
) and to express Stx2. They did not have the
eaeA
gene for γ-intimin, which is typically found in O157:H7, or the α- or β-intimin derivatives, which are common in other EHEC and enteropathogenic
E. coli
serotypes. Results of the multiplex PCR also indicated that the
ehxA
gene for enterohemolysin was absent from O104:H21. This, however, was not consistent with the results of a phenotypic assay that showed them to be hemolytic or a PCR analysis with another set of
ehxA
-specific primers, which indicated the presence of
ehxA
. To resolve this discrepancy, the
ehxA
region in O104:H21 and O157:H7 strains, to which the multiplex PCR primers anneal, was cloned and sequenced. Comparison of the sequences showed that the upstream primer binding site in the
ehxA
gene of O104:H21 was not identical to that of O157:H7. Specifically, there were several base mutations, including an A-to-G substitution at the 3′ end of the primer binding site. These base mutations are presumably not unique to O104:H21, since other enterohemolytic serotypes were also not detected with the
ehxA
primers used in the multiplex PCR. Comparison of the
ehxA
sequences of O104:H21 strains with those of other Stx-producing
E. coli
strains showed that they more closely resembled those of O8:H19 strains, which have cluster II
ehxA
genes, than those of O157:H7 strains, which have cluster I
ehxA
sequences. By modifying the upstream
ehxA
primer, the multiplex PCR was able to detect
ehxA
genes in both O157:H7 and O104:H21 strains.
Extraction and Genotyping of
Cryptosporidium parvum
DNA from Fecal Smears on Glass Slides Stained Conventionally for Direct Microscope Examination
Authors: Amar C. , Pedraza-Dı́az S. , McLauchlin J. 
Published: July 2002

ABSTRACT

A method was developed for extracting cryptosporidial DNA from stained fecal smears on glass microscope slides. The correct genotype of
Cryptosporidium parvum
was amplified by PCR from 89 (85%) of 105 smears following conventional staining but not from negative controls. This technique may have applications for analysis of other infectious agents.
Increased Prevalence of Class I Integrons in
Escherichia coli
,
Klebsiella
Species, and
Enterobacter
Species Isolates over a 7-Year Period in a German University Hospital
Authors: Schmitz Franz-Josef , Hafner Dieter , Geisel Roland , Follmann Patrick , Kirschke Christian , Verhoef Jan , Köhrer Karl , Fluit Ad C. 
Published: July 2002

ABSTRACT

The prevalence of integrons in five enterobacterial species was analyzed in 900 blood culture isolates from 1993, 1996, and 1999. Remarkably, the prevalence increased from 4.7% in 1993 to 9.7% in 1996 and finally to 17.4% in 1999 (
P
< 0.01). Within 7 years the combined percentage of P1 strong promoters and P1 weak plus P2 active promoters with high transcription efficacies has increased from 23.1 to 33.3 and finally 60% (
P
< 0.05).
Enzyme-Linked Immunosorbent Assay for Serological Diagnosis of Chagas' Disease Employing a
Trypanosoma cruzi
Recombinant Antigen That Consists of Four Different Peptides
Authors: Ferreira A. W. , Belem Z. R. , Lemos E. A. , Reed S. G. , Campos-Neto A. 
Published: July 2002

ABSTRACT

Serological tests to detect
Trypanosoma cruzi
antibodies have been used for screening blood donors, for epidemic studies, and for diagnosis of probably infected persons. Among different tests, the enzyme-linked immunosorbent assay (ELISA) with total, semipurified, or synthetic antigens has been widely used, mainly due to its easy automation. Aiming to improve serological studies concerning Chagas' disease, we have developed and evaluated a new test, the TcF-ELISA, using an artificially engineered recombinant antigen, which contains tandem sequences of different
T. cruzi
-specific peptides. The sensibility of the TcF-ELISA was determined with 101 serum samples from chagasic patients well-defined by clinical and epidemiological criteria. The specificity was determined with 39 serum samples from leishmaniasis or kala-azar patients and 150 serum samples from nonchagasic blood donors from Sao Paulo, Brazil. The TcF-ELISA showed 100% sensitivity and 98.94% of specificity. Compared with conventional ELISA (with semipurified
T. cruzi
epimastigote antigens), the TcF-ELISA showed advantages; for example, it distinguishes better between reagent and nonreagent serum and provides better precision and a lower occurrence of leishmaniasis cross-reactions. Our studies demonstrate high reproducibility between two different lots of the TcF ELISA and its applicability for the serological diagnosis of Chagas' disease.
Quantitative Real-Time PCR Detection of Rift Valley Fever Virus and Its Application to Evaluation of Antiviral Compounds
Authors: Garcia Stephan , Crance Jean Marc , Billecocq Agnes , Peinnequin Andre , Jouan Alain , Bouloy Michele , Garin Daniel 
Published: July 2002

ABSTRACT

The Rift Valley fever virus (RVFV), a member of the genus
Phlebovirus
(family
Bunyaviridae)
is an enveloped negative-strand RNA virus with a tripartite genome. Until 2000, RVFV circulation was limited to the African continent, but the recent deadly outbreak in the Arabian Peninsula dramatically illustrated the need for rapid diagnostic methods, effective treatments, and prophylaxis. A method for quantifying the small RNA segment by a real-time detection reverse transcription (RT)-PCR using TaqMan technology and targeting the nonstructural protein-coding region was developed, and primers and a probe were designed. After optimization of the amplification reaction and establishment of a calibration curve with synthetic RNA transcribed in vitro from a plasmid containing the gene of interest, real-time RT-PCR was assessed with samples consisting of RVFV from infected Vero cells. The method was found to be specific for RVFV, and it was successfully applied to the detection of the RVFV genome in animal sera infected with RVFV as well as to the assessment of the efficiency of various drugs (ribavirin, alpha interferon, 6-azauridine, and glycyrrhizin) for antiviral activity. Altogether, the results indicated a strong correlation between the infectious virus titer and the amount of viral genome assayed by real time RT-PCR. This novel method could be of great interest for the rapid diagnosis and screening of new antiviral compounds, as it is sensitive and time saving and does not require manipulation of infectious material.
Differentiation of
Ehrlichia platys
and
E. equi
Infections in Dogs by Using 16S Ribosomal DNA-Based PCR
Authors: Hancock Susan I. , Breitschwerdt Edward B. , Pitulle Christian 
Published: July 2002

ABSTRACT

We have encountered a previously unrecognized specificity problem when using the small-subunit ribosomal DNA (16S rDNA)-based PCR primers recommended for use in the identification of
Ehrlichia equi
in clinical samples. These PCR primers annealed to
E. platys
16S rDNA in blood samples containing high levels of
E. platys
organisms. Therefore, we designed and tested new PCR primers for the identification of
E. equi
.
Isolation of
Helicobacter cinaedi
from the Colon, Liver, and Mesenteric Lymph Node of a Rhesus Monkey with Chronic Colitis and Hepatitis
Authors: Fox J. G. , Handt L. , Sheppard B. J. , Xu S. , Dewhirst F. E. , Motzel S. , Klein H. 
Published: July 2002

ABSTRACT

On the basis of biochemical, phenotypic, and 16S rRNA analyses,
Helicobacter cinaedi
was isolated from the colon, liver, and mesenteric lymph nodes of a 2-year-old rhesus monkey with chronic diarrhea. Histologically, the liver had mild to moderate biliary hyperplasia and hypertrophy with periportal inflammation and fibrosis. Colonic and cecal lesions consisted of diffuse chronic inflammation and glandular hyperplasia extending the length of the crypts. This is the first observation of
H. cinaedi
associated with active hepatitis and colitis in a nonhuman primate.
Vertebral Osteomyelitis Caused by
Enterococcus raffinosus
Authors: Sandoe Jonathan A. T. , Witherden Ian R. , Settle Christopher 
Published: July 2002

ABSTRACT

Enterococcus raffinosus
is a rare isolate in clinical specimens. A case of vertebral osteomyelitis caused by
E. raffinosus
in an elderly patient is described and confirms this organism to be an opportunistic human pathogen.
Molecular Detection of Human Calicivirus in Young Children Hospitalized with Acute Gastroenteritis in Melbourne, Australia, during 1999
Authors: Kirkwood Carl D. , Bishop Ruth F. 
Published: July 2002

ABSTRACT
Reverse transcription-PCR and sequence analysis identified calciviruses in 32 of 60 stool specimens (negative for other enteric pathogens) obtained from children admitted to our hospital with acute gastroenteritis. The overall annual incidence rate for calcivirus was 9% (32 of 354 children). Molecular analysis identified 30 “Norwalk-like virus” genogroup II (predominantly Lordsdale cluster) and 2 “Sapporo-like virus” strains.
Limited Level of Accuracy Provided by Available Rapid Diagnosis Tests for Malaria Enhances the Need for PCR-Based Reference Laboratories
Authors: Rubio J. M. , Buhigas I. , Subirats M. , Baquero M. , Puente S. , Benito A. 
Published: July 2002

ABSTRACT
The rise of imported malaria cases and the high fatality rate in Europe make the search for new and easy diagnostic methods necessary. Rapid diagnosis tests (RDTs) are, in part, developed to cover the lack of diagnosis experience. Unfortunately, our data suggest that the accuracy of RDTs is insufficient and could increase the number of incorrect malaria diagnoses.
Detection of Dengue Viral RNA Using a Nucleic Acid Sequence-Based Amplification Assay
Authors: Wu Shuenn-Jue L. , Lee Eun Mi , Putvatana Ravithat , Shurtliff Roxanne N. , Porter Kevin R. , Suharyono Wuryadi , Watts Douglas M. , King Chwan-Chuen , Murphy Gerald S. , Hayes Curtis G. , Romano Joseph W. 
Published: July 2002

ABSTRACT
Faster techniques are needed for the early diagnosis of dengue fever and dengue hemorrhagic fever during the acute viremic phase of infection. An isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes. The NASBA assay involved the use of silica to extract viral nucleic acid, which was amplified without thermocycling. The amplified product was detected by a probe-hybridization method that utilized electrochemiluminescence. Using normal human plasma spiked with dengue viruses, the NASBA assay had a detection threshold of 1 to 10 PFU/ml. The sensitivity and specificity of the assay were determined by testing 67 dengue virus-positive and 21 dengue virus-negative human serum or plasma samples. The “gold standard” used for comparison and evaluation was the mosquito C6/36 cell culture assay followed by an immunofluorescent assay. Viral infectivity titers in test samples were also determined by a direct plaque assay in Vero cells. The NASBA assay was able to detect dengue viral RNA in the clinical samples at plaque titers below 25 PFU/ml (the detection limit of the plaque assay). Of the 67 samples found positive by the C6/36 assay, 66 were found positive by the NASBA assay, for a sensitivity of 98.5%. The NASBA assay had a specificity of 100% based on the negative test results for the 21 normal human serum or plasma samples. These results indicate that the NASBA assay is a promising assay for the early diagnosis of dengue infections.
Rapid and Accurate Identification of Coagulase-Negative Staphylococci by Real-Time PCR
Authors: Edwards K. J. , Kaufmann M. E. , Saunders N. A. 
Published: July 2002

ABSTRACT
Biprobe identification assays based on real-time PCR were designed for 15 species of coagulase-negative staphylococci (CNS). Three sets of primers and four biprobes were designed from two variable regions of the 16S rRNA gene. An identification scheme was developed based on the pattern of melting peaks observed with the four biprobes that had been tested on 24 type strains. This scheme was then tested on 100 previously identified clinical isolates and 42 blindly tested isolates. For 125 of the 142 clinical isolates there was a perfect correlation between the biprobe identification and the result of the ID 32 Staph phenotypic tests and PCR. For 12 of the other isolates a 300-bp portion of the 16S rRNA gene was sequenced to determine identity. The remaining five isolates could not be fully identified. LightCycler real-time PCR allowed rapid and accurate identification of the important CNS implicated in infection.
Multiplex PCR Protocol for the Diagnosis of Staphylococcal Infection
Authors: Mason William J. , Blevins Jon S. , Beenken Karen , Wibowo Noroyono , Ojha Neelum , Smeltzer Mark S. 
Published: July 2002

ABSTRACT

We report the development of a multiplex PCR protocol for the diagnosis of staphylococcal infection. The protocol was designed to (i) detect any staphylococcal species to the exclusion of other bacterial pathogens (based on primers corresponding to
Staphylococcus
-specific regions of the 16S rRNA genes), (ii) distinguish between
S. aureus
and the coagulase-negative staphylococci (CNS) (based on amplification of the
S. aureus
-specific
clfA
gene), and (iii) provide an indication of the likelihood that the staphylococci present in the specimen are resistant to oxacillin (based on amplification of the
mecA
gene). The expected fragments were amplified from each of 60 staphylococcal isolates (13 oxacillin-resistant
S. aureus
isolates, 23 oxacillin-sensitive
S. aureus
isolates, 17 oxacillin-resistant CNS, and 7 oxacillin-sensitive CNS). No amplification products were observed with template DNA from nonstaphylococcal species, and the efficiency of amplification of staphylococcal targets was not adversely affected by the presence of DNA from other bacterial species in the same sample. The utility of the protocol for the analysis of clinical samples was verified by analysis of aliquots taken directly from BacT/Alert blood culture bottles. Of 77 blood cultures tested, only 7 yielded results inconsistent with those of conventional methods of diagnosis and susceptibility testing. Of those, one was identified as a CNS species by PCR and
S. aureus
by conventional methods. We also identified two isolates that were
mecA
positive but were oxacillin sensitive according to conventional methods. The other four samples failed to yield any amplification product even with a control set of primers corresponding to a conserved region of the eubacterial rRNA genes.
Evaluation of Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test by Using Respiratory and Nonrespiratory Specimens in a Tertiary Care Center Laboratory
Authors: O'Sullivan Cathal E. , Miller David R. , Schneider Peggy S. , Roberts Glenn D. 
Published: July 2002

ABSTRACT
The performance of the Amplified Mycobacterium Tuberculosis Direct (AMTD) test (Gen-Probe Inc., San Diego, Calif.) was assessed in a large tertiary care mycobacteriology laboratory. Both acid-fast smear-positive and smear-negative respiratory and nonrespiratory clinical specimens were analyzed. From February 1998 to 4 October 2001, AMTD assays were performed on 391 respiratory specimens and 164 nonrespiratory specimens. The AMTD assay was compared to the “gold standard” of combined culture and clinical diagnosis. The overall sensitivity for all specimens, including those for which no smear result was available, was 91.2%. The overall sensitivities of the assay, including acid-fast smear-positive and -negative specimens, were 97.8 and 77.3% for respiratory and nonrespiratory specimens, respectively. The corresponding specificities for respiratory and nonrespiratory specimens were 99.1 and 98.5%, respectively. The overall specificity for all specimens was 98.9%. Positive and negative predictive values were 93.9 and 99.7% and 91.7 and 96.4% for respiratory and nonrespiratory specimens, respectively. The time saved by using the AMTD test for making a diagnosis of tuberculosis instead of using culture was 8.99 days. Inhibitors to the AMTD assay were found in 3.1% of respiratory specimens and 3.1% of nonrespiratory specimens. The assay, used in a general mycobacteriology laboratory setting, represents an important advance in improving the speed and accuracy of diagnosis in the management of patients with tuberculosis.
Molecular Characterization of Pigmented and Nonpigmented Isolates of
Mycobacterium avium
subsp.
paratuberculosis
Authors: Stevenson Karen , Hughes Valerie M. , de Juan Lucía , Inglis Neil F. , Wright Frank , Sharp J. Michael 
Published: July 2002

ABSTRACT

Five pigmented isolates of
Mycobacterium avium
subsp.
paratuberculosis
were examined by pulsed-field gel electrophoresis (PFGE), IS
900
restriction fragment length polymorphism (IS
900
-RFLP), and IS
1311
polymorphism analysis using PCR. All of the pigmented isolates exhibited one of three distinct PFGE profiles with
Sna
BI, designated 9, 10, and 11, and with
Spe
I, designated 7, 8, and 9, which generated three multiplex profiles designated [9-7], [10-8], and [11-9]. All of the pigmented isolates had the same IS
900
-RFLP
Bst
EII and
Pvu
II profiles. The IS
900
-RFLP
Bst
EII profile was new, but the IS
900
-RFLP
Pvu
II profile corresponded to
Pvu
II type 6 of a sheep strain described by Cousins and colleagues (D. V. Cousins, S. N. Williams, A. Hope, and G. J. Eamens, Aust. Vet. J. 78:184-190, 2000). IS
1311
-PCR analysis typed all of the pigmented isolates as sheep (S) strains. The genetic relationship between pigmented and nonpigmented isolates was investigated by using multiplex PFGE data from the analysis of both the 5 pigmented isolates and 88 nonpigmented isolates of
M. avium
subsp.
paratuberculosis
from a variety of host species and geographic locations. It was possible to classify the isolates into two distinct types designated type I, comprising the pigmented isolates, and type II, comprising the nonpigmented isolates, which exhibit a very broad host range.
3′-End Stem-Loops of the Subviral RNAs Associated with Turnip Crinkle Virus Are Involved in Symptom Modulation and Coat Protein Binding
Authors: Wang Jianlong , Simon Anne E. 
Published: July 2002

ABSTRACT

Many plant RNA viruses are associated with one or more subviral RNAs. Two subviral RNAs, satellite RNA C (satC) and defective interfering RNA G (diG) intensify the symptoms of their helper, turnip crinkle virus (TCV). However, when the coat protein (CP) of TCV was replaced with that of the related Cardamine chlorotic fleck virus (CCFV), both subviral RNAs attenuated symptoms of the hybrid virus TCV-CP
CCFV
. In contrast, when the translation initiation codon of the TCV CP was altered to ACG and reduced levels of CP were synthesized, satC attenuated symptoms while diG neither intensified nor attenuated symptoms. The determinants for this differential symptom modulation were previously localized to the 3′-terminal 100 bases of the subviral RNAs, which contain six positional differences (Q. Kong, J.-W. Oh, C. D. Carpenter, and A. E. Simon, Virology 238:478–485, 1997). In the current study, we have determined that certain sequences within the 3′-terminal stem-loop structures of satC and diG, which also serve as promoters for complementary strand synthesis, are critical for symptom modulation. Furthermore, the ability to attenuate symptoms was correlated with weakened binding of TCV CP to the hairpin structure.
Cell Type-Specific Enhancement of Hepatitis C Virus Internal Ribosome Entry Site-Directed Translation due to 5′ Nontranslated Region Substitutions Selected during Passage of Virus in Lymphoblastoid Cells
Authors: Lerat Hervé , Shimizu Yoko K. , Lemon Stanley M. 
Published: July 2002

ABSTRACT

Low-level replication of hepatitis C virus (HCV) in cultured lymphoblastoid cells inoculated with H77 serum inoculum led to the appearance of new virus variants containing identical substitutions at three sites within the viral 5′ nontranslated RNA (5′NTR): G
107
→A, C
204
→A, and G
243
→A (N. Nakajima, M. Hijikata, H. Yoshikura, and Y. K. Shimizu, J. Virol. 70:3325–3329, 1996). These results suggest that virus with this 5′NTR sequence may have a greater capacity for replication in such cells, possibly due to more efficient cap-independent translation, since these nucleotide substitutions reside within the viral internal ribosome entry site (IRES). To test this hypothesis, we examined the translation of dicistronic RNAs containing upstream and downstream reporter sequences (
Renilla
and firefly luciferases, respectively) separated by IRES sequences containing different combinations of these substitutions. The activity of the IRES was assessed by determining the relative firefly and
Renilla
luciferase activities expressed in transfected cells. Compared with the IRES present in the dominant H77 quasispecies, an IRES containing all three nucleotide substitutions had significantly greater translational activity in three of five human lymphoblastoid cell lines (Raji, Bjab, and Molt4 but not Jurkat or HPBMa10-2 cells). In contrast, these substitutions did not enhance IRES activity in cell lines derived from monocytes or granulocytes (HL-60, KG-1, or THP-1) or hepatocytes (Huh-7) or in cell-free translation assays carried out with rabbit reticulocyte lysates. Each of the three substitutions was required for maximally increased translational activity in the lymphoblastoid cells. The 2- to 2.5-fold increase in translation observed with the modified IRES sequence may facilitate the replication of HCV, possibly accounting for differences in quasispecies variants recovered from liver tissue and peripheral blood mononuclear cells of the same patient.