bluuebunny/crossref_metadata_2025 huggingface dataset:
Nested PCR Assays for Detection of
Blastomyces dermatitidis
DNA in Paraffin-Embedded Canine Tissue
Authors: Bialek Ralf , Cascante Cirera Anna , Herrmann Tanja , Aepinus Christian , Shearn-Bochsler Valerie I. , Legendre Alfred M.
Published: January 2003

A
Blastomyces dermatitidis
nested PCR assay targeting the gene encoding the Wisconsin 1 (WI-1) adhesin was developed and compared with a nested PCR targeting the 18S rRNA gene (rDNA) of members of the family
Onygenaceae
. We examined 73 paraffin-embedded tissue samples obtained from nine dogs which died of blastomycosis and nine dogs which succumbed to lymphosarcoma according to autopsy findings; amplifiable canine DNA was extracted from 25 and 33 specimens from the two groups, respectively. The
B. dermatitidis
PCR amplified DNA from 8 of 13 tissue samples in which yeast cells were detected by microscopy. Sequencing revealed that all PCR products were homologous to the
B. dermatitidis
WI-1 adhesin gene. No PCR product was amplified from 12 microscopically negative biopsy specimens from dogs with blastomycosis or from 33 biopsy specimens from dogs with lymphosarcoma. The 18S rDNA PCR amplified DNA from 10 and 9 tissue samples taken from dogs which died of blastomycosis and lymphosarcoma, respectively. Only six products were identified as being identical to
B. dermatitidis
18S rDNA; they were exclusively obtained from specimens positive by the
B. dermatitidis
nested PCR. For specificity testing, 20 human biopsy specimens proven to have histoplasmosis were examined, and a specific
H. capsulatum
product was amplified by the 18S rDNA PCR from all specimens, whereas no product was obtained from any of the 20 samples by the
B. dermatitidis
PCR assay. In conclusion, the PCR targeting a gene encoding the unique WI-1 adhesin is as sensitive as but more specific than the PCR targeting the 18S rDNA for detection of
B. dermatitidis
in canine tissue.
Evaluation of an Enzyme Immunoassay Technique for Detection of Antibodies against
Treponema pallidum
Authors: Castro Rita , Prieto Emília S. , Santo Irene , Azevedo Jacinta , Exposto Filomena da L.
Published: January 2003

In the present study, the performance of an enzyme-linked immunosorbent assay (ELISA) technique (Eti-syphilis-G and Eti-syphilis-M; DiaSorin) for detection of
Treponema pallidum
immunoglobulin M (IgM) and IgG antibodies for the laboratory diagnosis of syphilis was evaluated. Four hundred forty-one samples were studied. The sensitivity and specificity of the ELISA were 100 and 93%, respectively, compared with the results of a microhemagglutination assay for
Treponema pallidum
(MHA-TP) and 99.4 and 100%, respectively, compared with the results of the fluorescent treponemal antibody absorption (FTA-Abs) test. The results of the ELISA technique were concordant with those of MHA-TP for 98% of the samples tested, while the rate of concordance with the FTA-Abs test was 99.5%. The sensitivities of the rapid plasma reagin (RPR) test, MHA-TP, and the ELISA in the different phases of syphilis compared with the results of the FTA-Abs test were 92, 88, and 100%, respectively, for patients with primary syphilis; 100% for all tests evaluated for patients with secondary syphilis; 97.2, 99.4, and 100%, respectively, for patients with latent syphilis; and 57.9, 92.6, and 97.9%, respectively, for patients with past treated syphilis. The RPR test was reactive with 12 samples that were negative by all the specific tests. IgM antibodies were most frequently detected by the ELISA for IgM antibodies (32.8%) than by the FTA-Abs for IgM antibodies (28.4%). Detection of these antibodies by the FTA-Abs test and the ELISA for IgM antibodies decreased with the stage of disease (72 and 88%, respectively, for patients with primary syphilis to 17 and 19%, respectively, for patients with early latent syphilis). The high sensitivity and specificity of this ELISA technique during all stages of syphilis, together with the fact that it is a simple, objective, and easily automated method, lead us to believe that it could be used as a screening test for syphilis.
Rapid Detection of Methicillin-Resistant
Staphylococcus aureus
Directly from Sterile or Nonsterile Clinical Samples by a New Molecular Assay
Authors: Francois Patrice , Pittet Didier , Bento Manuela , Pepey Béatrice , Vaudaux Pierre , Lew Daniel , Schrenzel Jacques
Published: January 2003

A rapid procedure was developed for detection and identification of methicillin-resistant
Staphylococcus aureus
(MRSA) directly from sterile sites or mixed flora samples (e.g., nose or inguinal swabs). After a rapid conditioning of samples, the method consists of two main steps: (i) immunomagnetic enrichment in
S. aureus
and (ii) amplification-detection profile on DNA extracts using multiplex quantitative PCR (5′-exonuclease qPCR, TaqMan). The triplex qPCR assay measures simultaneously the following targets: (i)
mecA
gene, conferring methicillin resistance, common to both
S. aureus
and
Staphylococcus epidermidis
; (ii)
femA
gene from
S. aureus
; and (iii)
femA
gene from
S. epidermidis.
This quantitative approach allows discrimination of the origin of the measured
mecA
signal. qPCR data were calibrated using two reference strains (MRSA and methicillin-resistant
S. epidermidis
) processed in parallel to clinical samples. This 96-well format assay allowed analysis of 30 swab samples per run and detection of the presence of MRSA with exquisite sensitivity compared to optimal culture-based techniques. The complete protocol may provide results in less than 6 h (while standard procedure needs 2 to 3 days), thus allowing prompt and cost-effective implementation of contact precautions.
Characterization of Heteroresistance to Fluconazole among Clinical Isolates of
Cryptococcus neoformans
Authors: Yamazumi T. , Pfaller M. A. , Messer S. A. , Houston A. K. , Boyken L. , Hollis R. J. , Furuta I. , Jones R. N.
Published: January 2003

Strains of
Cryptococcus neoformans
expressing heteroresistance to fluconazole have been described previously. The present study was conducted to investigate the prevalence of heteroresistance among clinical isolates of
C. neoformans
and to characterize the heteroresistant phenotypes. A total of 107 clinical isolates of
C. neoformans
for which the MICs of fluconazole ranged from 0.25 to 32 μg/ml were selected. The isolates were chosen to represent a broad geographic distribution. Of the 107
C. neoformans
isolates tested, 4 grew on medium containing fluconazole at concentrations that were four to eight times higher than the MICs for each strain. A fifth isolate, for which the fluconazole MIC was 32 μg/ml, grew on agar with 64 μg of fluconazole per ml. These five isolates (4.7% of the total number) were confirmed to exhibit heteroresistant compositions by population analysis. The degree and frequency of resistance varied among the isolates. Stepwise selection by exposure to fluconazole resulted in subclones of all five strains for which the fluconazole MIC was >64 μg/ml. Subclones of three strains demonstrated a homogenous population of resistant cells on medium containing 64 μg of fluconazole/ml. The resistance was sensitive to incubation temperature, that is, heteroresistance was demonstrable only at 30°C by agar-based tests, and was reversible through serial transfers on fluconazole-free medium over a period of 8 days. These results suggest that the fluconazole-heteroresistant phenotype of
C. neoformans
exists in a significant proportion of clinical isolates and that fluconazole resistance can be developed by selection from heteroresistant clones and induction by exposure to fluconazole.
Nonencapsulated
Neisseria meningitidis
Strain Produces Amylopectin from Sucrose: Altering the Concept for Differentiation between
N. meningitidis
and
N. polysaccharea
Authors: Zhu Peixuan , Tsang Raymond S. W. , Tsai Chao-Ming
Published: January 2003

Neisseria meningitidis
is the causative agent of meningococcal sepsis and meningitis.
Neisseria polysaccharea
is a nonpathogenic species.
N. polysaccharea
is able to use sucrose to produce amylopectin, a starch-like polysaccharide, which distinguishes it biochemically from the pathogenic species
N. meningitidis
. The data presented here indicate that this may be an insufficient criterion to distinguish between these two species. The nonencapsulated
Neisseria
strain 93246 expressed a phenotype of amylopectin production similar to that of
N. polysaccharea
. However, strain 93246 reacted with
N. meningitidis
serotype 4 and serosubtype P1.14 monoclonal antibodies and showed the
N. meningitidis
L1(8) lipo-oligosaccharide immunotype. Further analyses were performed on four genetic loci in strain 93246, and the results were compared with 7
N. meningitidis
strains, 13
N. polysaccharea
strains, and 2
N. gonorrhoeae
strains. Three genetic loci,
opcA
,
siaD
, and
lgt-1
in strain 93246, were the same as in
N. meningitidis
. Particularly, the
siaD
gene encoding polysialyltransferase responsible for biosynthesis of
N. meningitidis
group B capsule was detected in strain 93246. This
siaD
gene was inactivated by a frameshift mutation at the poly(C) tract, which makes strain 93246 identical to other nonencapsulated
N. meningitidis
strains. As expected, the
ams
gene encoding amylosucrase, responsible for production of amylopectin from sucrose, was detected in strain 93246 and all 13
N. polysaccharea
strains but not in
N. meningitidis
and
N. gonorrhoeae
strains. These data suggest that strain 93246 is nonencapsulated
N. meningitidis
but has the ability to produce extracellular amylopectin from sucrose. The gene for amylopectin production in strain 93246 was likely imported from
N. polysaccharea
by horizontal genetic exchange. Therefore, we conclude that genetic analysis is required to complement the traditional phenotypic classification for the nonencapsulated
Neisseria
strains.
Performance Evaluation of the VERSANT HCV RNA Qualitative Assay by Using Transcription-Mediated Amplification
Authors: Gorrin Gregg , Friesenhahn Michel , Lin Patsy , Sanders Marla , Pollner Reinhold , Eguchi Brandon , Pham Jimmykim , Roma Gianluca , Spidle Joseph , Nicol Susann , Wong Carol , Bhade Suvarna , Comanor Lorraine
Published: January 2003

A preclinical evaluation of a qualitative assay for the detection of hepatitis C virus (HCV) RNA by transcription-mediated amplification (TMA) was conducted according to the guidelines of the National Committee for Clinical Laboratory Standards and the U.S. Food and Drug Administration. Our results showed that this assay, HCV TMA, detected 95% of samples with HCV RNA concentrations of 5.3 IU/ml and 29 copies/ml. HCV TMA showed an overall specificity of 99.6% and was highly reproducible, detecting 99.3% of samples with HCV RNA concentrations of 50 copies/ml across seven different lots of reagents. Experiments with clinical samples showed that HCV TMA detected all HCV genotypes with similar efficiencies, detecting ≥95% of samples at 50 HCV RNA copies/ml from patients infected with HCV genotypes 1a, 2b, 3a, 4a, 5a, and 6a. In experiments with RNA transcripts, HCV TMA detected ≥96.6% of transcripts derived from HCV genotypes 1a, 1b, 2a, 2c, 3a, 4a, 5a, and 6a at 50 HCV RNA copies/ml. Detection of transcripts derived from HCV genotype 2b was slightly lower (88.4%) at 50 copies/ml but was 97.0% at 75 copies/ml. In addition, HCV TMA exhibited robust performance in detecting HCV RNA in samples subjected to various conditions commonly encountered in a clinical laboratory, including long-term storage, multiple freeze-thaw cycles, different collection tubes, and the presence of endogenous substances, commonly prescribed drugs, or other microorganisms and viruses. With its high sensitivity, specificity, reproducibility, and equivalent genotype reactivity, HCV TMA may provide an attractive alternative for routine qualitative HCV RNA testing in clinical laboratories.
Rapid Enterovirus RNA Detection in Clinical Specimens by Using Nucleic Acid Sequence-Based Amplification
Authors: Landry Marie L. , Garner Robin , Ferguson David
Published: January 2003

Enterovirus (EV) detection by nucleic acid sequence-based amplification was compared with EV isolation in cell culture. The NucliSens Basic kit (bioMerieux) was utilized for RNA detection. For virus isolation, samples were inoculated into MRC-5, primary rhesus monkey kidney, A549, rhabdomyosarcoma, and/or Buffalo green monkey kidney cells.
Detection of North American Eastern and Western Equine Encephalitis Viruses by Nucleic Acid Amplification Assays
Authors: Lambert Amy J. , Martin Denise A. , Lanciotti Robert S.
Published: January 2003

We have developed nucleic acid sequence-based amplification (NASBA), standard reverse transcription PCR (RT-PCR), and TaqMan nucleic acid amplification assays for the rapid detection of North American eastern equine encephalitis (EEE) and western equine encephalitis (WEE) viral RNAs from samples collected in the field and clinical samples. The sensitivities of these assays have been compared to that of virus isolation. While all three types of nucleic acid amplification assays provide rapid detection of viral RNAs comparable to the isolation of viruses in Vero cells, the TaqMan assays for North American EEE and WEE viral RNAs are the most sensitive. We have shown these assays to be specific for North American EEE and WEE viral RNAs by testing geographically and temporally distinct strains of EEE and WEE viruses along with a battery of related and unrelated arthropodborne viruses. In addition, all three types of nucleic acid amplification assays have been used to detect North American EEE and WEE viral RNAs from mosquito and vertebrate tissue samples. The sensitivity, specificity, and rapidity of nucleic acid amplification demonstrate the usefulness of NASBA, standard RT-PCR, and TaqMan assays, in both research and diagnostic settings, to detect North American EEE and WEE viral RNAs.
Stability of Serotypes during Nasopharyngeal Carriage of
Streptococcus pneumoniae
Authors: Meats Emma , Brueggemann Angela B. , Enright Mark C. , Sleeman Karen , Griffiths David T. , Crook Derrick W. , Spratt Brian G.
Published: January 2003

Serotype changes among natural isolates of
Streptococcus pneumoniae
are well documented and occur by recombinational exchanges at the capsular biosynthetic locus. However, the frequency with which this phenomenon occurs within the nasopharynx of children is not clear and is likely to be highest in the nasopharynx of children, who have high rates of pneumococcal carriage. A birth cohort of 100 infants was studied, and pneumococci were recovered from nasopharyngeal samples taken at monthly intervals during the first 6 months of life and then at 2-monthly intervals until the age of 2 years. Among the 1,353 nasopharyngeal samples were 523 that contained presumptive pneumococci, and three colonies from each were serotyped. A total of 333 isolates, including all isolates of differing serotypes from the same child, were characterized by multilocus sequence typing. Sixty-eight children carried multiple serotypes during the first 2 years of life. Two children carried a typeable and a nonserotypeable pneumococcus of identical genotype, and five children carried genetically indistinguishable isolates of serotypes 15B and 15C. These isolates were considered, respectively, to be due to loss of capsule expression and the known ability of serotype 15B and 15C pneumococci to interconvert by loss or gain of an acetyl group on the capsular polysaccharide. In all other cases, isolates from the same children that differed in serotype also differed in genotype, indicating the acquisition of a different pneumococcal strain rather than a change in capsular type. There was therefore no evidence in this study for any change of serotype due to recombinational replacements at the capsular locus among the pneumococci carried within the nasopharynges of the children.
Real-Time PCR Assay for Rapid and Accurate Detection of Point Mutations Conferring Resistance to Clarithromycin in
Helicobacter pylori
Authors: Oleastro Monica , Ménard Armelle , Santos Adriana , Lamouliatte Hervé , Monteiro Lurdes , Barthélémy Philippe , Mégraud Francis
Published: January 2003

The main cause of failure of
Helicobacter pylori
eradication therapy is resistance to clarithromycin. The resistance is due to three point mutations in two positions on the 23S rRNA (A2142C, A2142G, and A2143G). Our aim was to develop a rapid and accurate method to detect these mutations directly on biopsy specimens. We developed a real-time PCR that included a simultaneous detection of the amplicons by hybridization of two probes labeled with LC-Red and fluorescein by using the fluorescence resonance energy transfer (FRET) technology and melting curve analysis with the LightCycler thermocycler. The assay was first applied successfully on reference strains, reference plasmids, and
H. pylori
-negative biopsies. Biopsies from 200 patients having failed a first eradication attempt and for whom the
H. pylori
strain was available were then tested with the new assay. A result was obtained in 199 cases; a single genotype was detected in 157 cases, two genotypes were detected in 41 cases, and three genotypes were detected in one case. There were, in total, seven discrepancies between the real-time PCR and the phenotypic method of determination of clarithromycin susceptibility, and in an additional four cases the two phenotypic methods were in disagreement. PCR-restriction fragment length polymorphism was applied to a sampling of biopsies, including all of the cases with multiple genotypes and all the cases with discrepant results. Finally, in four cases with discrepant results, the real-time PCR detected the resistant population at a concentration so low that it could not be detected by the phenotypic method, while in three cases other mutations could be involved. This assay had an accuracy at least as satisfactory as that of the phenotypic tests and could be performed within 2 h, allowing it to be used before the administration of therapy in the case of a first
H. pylori
eradication.
Determination of Immunoglobulin A against
Gardnerella vaginalis
Hemolysin, Sialidase, and Prolidase Activities in Vaginal Fluid: Implications for Adverse Pregnancy Outcomes
Authors: Cauci Sabina , Thorsen Poul , Schendel Diana E. , Bremmelgaard Annie , Quadrifoglio Franco , Guaschino Secondo
Published: January 2003

A nested case-control study of low birth weight and preterm delivery was performed with singleton women. Immunoglobulin A (IgA) against the
Gardnerella vaginalis
hemolysin (anti-Gvh IgA) and sialidase and prolidase activities were determined in vaginal fluid at 17 weeks of gestation. Sialidase positivity and bacterial vaginosis with high prolidase activity were associated with 2- and 11-fold increased risks for low birth weight, respectively. No woman with bacterial vaginosis plus a strong anti-Gvh IgA response had an adverse outcome.
Use of Real-Time PCR To Detect and QuantifyMycoplasma haemofelisand “CandidatusMycoplasma haemominutum” DNA
Authors: Tasker Séverine , Helps Chris R. , Day Michael J. , Gruffydd-Jones Tim J. , Harbour Dave A.
Published: January 2003

A real-time PCR assay using Taqman probes was developed to detect and quantifyMycoplasma haemofelisand “CandidatusMycoplasma haemominutum” in feline blood samples. The assay was rapid and sensitive and was successfully used to monitor the in vivo kinetics of cats experimentally infected with each species.
Sequence Polymorphism of the 16S rRNA Gene of
Vibrio vulnificus
Is a Possible Indicator of Strain Virulence
Authors: Nilsson William B. , Paranjype Rohinee N. , DePaola Angelo , Strom Mark S.
Published: January 2003

Vibrio vulnificus
exhibits considerable strain-to-strain variation in virulence. Attempts to associate phenotypic or genotypic characteristics with strain virulence have been largely unsuccessful. Based on a 17-nucleotide difference throughout the sequence of the small subunit 16S rRNA gene, there are two major groups of
V. vulnificus
designated types A and B. In a survey of the 16S rRNA genotype in 67
V. vulnificus
human clinical and nonclinical strains, we determined that the majority of nonclinical isolates are type A (31 of 33) and that there is a statistically significant association between the type B genotype and human clinical strains (26 of 34).
Identification of
Balamuthia mandrillaris
by PCR Assay Using the Mitochondrial 16S rRNA Gene as a Target
Authors: Booton Gregory C. , Carmichael Jennifer R. , Visvesvara Govinda S. , Byers Thomas J. , Fuerst Paul A.
Published: January 2003

Balamuthia mandrillaris
is an opportunistic pathogen that causes granulomatous amebic meningoencephalitis in animals, including humans. Based on sequence analysis of mitochondrial small-subunit-rRNA genes, we developed primers that amplify a
Balamuthia
-specific PCR product. These primers will be useful for retrospective analyses of fixed tissues and possible identification of
Balamuthia
in vivo.
Evaluation of New Medium for Identification of Dermatophytes and Primary Dimorphic Pathogens
Authors: Gromadzki Sally , Ramani Rama , Chaturvedi Vishnu
Published: January 2003

Only 25 of 77 dermatophytic isolates caused dermatophyte identification medium (DIM) to turn purple after incubation at the recommended temperature (37°C); the accuracy of the results was improved at 30°C (71 of 77 isolates yielded positive results). Many dimorphic pathogenic fungi also tested positive at both incubation temperatures. Thus, DIM has limited usefulness for presumptive identification of dermatophytes.
Detecting
Candida albicans
in Human Milk
Authors: Morrill Jimi Francis , Pappagianis Demosthenes , Heinig M. Jane , Lönnerdal Bo , Dewey Kathryn G.
Published: January 2003

Procedures for diagnosis of mammary candidosis, including laboratory confirmation, are not well defined. Lactoferrin present in human milk can inhibit growth of
Candida albicans
, thereby limiting the ability to detect yeast infections. The inhibitory effect of various lactoferrin concentrations on the growth of
C. albicans
in whole human milk was studied. The addition of iron to the milk led to a two- to threefold increase in cell counts when milk contained 3.0 mg of lactoferrin/ml and markedly reduced the likelihood of false-negative culture results. This method may provide the necessary objective support needed for diagnosis of mammary candidosis.
Trichosporon loubieri
Infection in a Patient with Adult Polycystic Kidney Disease
Authors: Padhye Arvind A. , Verghese Susan , Ravichandran P. , Balamurugan G. , Hall Leslie , Padmaja P. , Fernandez Maria C.
Published: January 2003

A 45-year-old man from Nepal with a 13-year history of polycystic kidney disease was diagnosed as suffering from chronic renal failure with end-stage renal disease. After receiving empirical antituberculosis treatment, he was treated with broad-spectrum antibiotics. A left nephrectomy was performed, and after 4 months, he received a kidney transplant. The left kidney was grossly enlarged, with multiple cystic spaces filled with blackish material. Histologic examination of the excised left kidney tissue stained with hematoxylin and eosin and Gomori's methenamine silver stains showed numerous hyaline, septate, fungal hyphae of various lengths, many broken into rectangular arthroconidia in the cystic spaces. Culture of the kidney tissue yielded white, glabrous, yeast-like colonies. Based on its micromorphology, growth at 42°C, and ribosomal DNA (rDNA) sequence analysis, and also sequence analysis of the internal-transcribed-spacer and D1/D2 rDNA regions, the yeast was identified as
Trichosporon loubieri.
Postsurgically, the patient was treated with amphotericin B and oral itraconazole, followed by maintenance therapy with fluconazole. He remained afebrile and asymptomatic. At the final follow-up, all parameters were found normal and the patient was doing well, with normal renal function reports. This paper presents the first known case of human infection caused by
T. loubieri.
Antimicrobial Activities against 84
Listeria monocytogenes
Isolates from Patients with Systemic Listeriosis at a Comprehensive Cancer Center (1955-1997)
Authors: Safdar Amar , Armstrong Donald
Published: January 2003

Listeriosis is a serious complication in patients undergoing treatment for cancer. We present antimicrobial susceptibility profiles of 84 clinical
Listeria monocytogenes
isolates. During 1955 to 1997, in vitro susceptibility for penicillin (97.6%), ampicillin (90.7%), erythromycin (98.8%), tetracycline (96.9%), and gentamicin (98.0%) remained unchanged. All isolates were susceptible to amikacin, ciprofloxacin, imipenem, rifampin, trimethoprim-sulfamethoxazole (TMP-SMX), and vancomycin. High prevalence of clindamycin resistance (96.2%) was unexpected. Ampicillin plus gentamicin is standard therapy for systemic listerosis, and TMP-SMX may be used for patients with beta-lactam intolerance. In vitro susceptibility profiles for carbapenem and fluoronated quinolone are promising, although clinical validation is critically needed before routine use is advocated, especially for listeric patients with severe cellular immune defects.
Clarithromycin Resistance among
Helicobacter pylori
Strains Isolated from Children: Prevalence and Study of Mechanism of Resistance by PCR-Restriction Fragment Length Polymorphism Analysis
Authors: Alarcón Teresa , Vega Alba E. , Domingo Diego , Martínez Maria Josefa , López-Brea Manuel
Published: January 2003

Clarithromycin and metronidazole resistance was 29.1 and 23.9%, respectively, in 96
Helicobacter pylori
strains obtained from pediatric patients. No resistance to amoxicillin was observed. Resistance according to patients' ages to clarithromycin and metronidazole was 45.4 and 18.2% in 22 patients from 4 to 8 years old, 30.2 and 20.7% in 53 patients from 9 to 13 years old, and 9.5 and 38.1% in 21 patients from 14 to 18 years old, respectively. The A2143G mutation was the most prevalent (82.1%) among clarithromycin-resistant strains.
Cell Wall Thickening Is a Common Feature of Vancomycin Resistance in
Staphylococcus aureus
Authors: Cui Longzhu , Ma Xiaoxue , Sato Katsuhiro , Okuma Keiko , Tenover Fred C. , Mamizuka Elsa M. , Gemmell Curtis G. , Kim Mi-Na , Ploy Marie-Cecile , El Solh N. , Ferraz Vivian , Hiramatsu Keiichi
Published: January 2003

We have previously shown that a thickened cell wall is responsible for the vancomycin resistance of vancomycin-resistant
Staphylococcus aureus
(VRSA) (equivalent to vancomycin-intermediate
S. aureus
and glycopeptide-intermediate
S. aureus
) strain Mu50 (L. Cui, H. Murakami, K. Kuwahara-Arai, H. Hanaki, and K. Hiramatsu, Antimicrob. Agents Chemother. 44:2276-2285, 2000). However, the mechanism of vancomycin resistance in other VRSA strains remained unclear. In this study, 16 clinical VRSA strains from seven countries were subjected to serial daily passage in drug-free medium. After 10 to 84 days of passage in the nonselective medium, passage-derived strains with decreased MICs of vancomycin (MIC, <4 mg/liter) were obtained. However, all of the passage-derived strains except one (15 of 16) still possessed subpopulations that were resistant to vancomycin as judged by population analysis, and vancomycin-resistant mutant strains were selected from the passage-derived strains by one-step vancomycin selection with a frequency of 4.25 × 10
−6
to 1.64 × 10
−3
. The data indicated that vancomycin-resistant cells are frequently generated from the passage-derived strains even after vancomycin selective pressure is lifted. Cell wall thicknesses and MICs of glycopeptides (vancomycin and teicoplanin) and beta-lactams (imipenem and oxacillin) were determined for a total of 48 strains, including 15 sets of three strains: the clinical VRSA strain, the passage-derived strain, and the vancomycin-resistant mutant strain obtained from the passage-derived strain. No simple correlation between glycopeptide and beta-lactam MICs was seen, while significant correlations between MICs of vancomycin and teicoplanin (
r
= 0.679;
P
< 0.001) and between MICs of imipenem and oxacillin (
r
= 0.787;
P
< 0.001) were recognized. Moreover, all of the VRSA strains had significantly thickened cell walls, which became thinner with the loss of vancomycin resistance during drug-free passages and again became thick in the resistant mutant strains. The data showed that cell wall thickness had high correlation with the MICs of the two glycopeptides (correlation coefficients, 0.908 for vancomycin and 0.655 for teicoplanin) but not with those of the beta-lactam antibiotics tested. These results together with coupled changes of cell wall thickness and vancomycin MICs in 16 isogenic sets of strains indicate that thickening of the cell wall is a common phenotype of clinical VRSA strains and may be a phenotypic determinant for vancomycin resistance in
S. aureus.
Uses and Limitations of the XTT Assay in Studies of
Candida
Growth and Metabolism
Authors: Kuhn D. M. , Balkis M. , Chandra J. , Mukherjee P. K. , Ghannoum M. A.
Published: January 2003

Colorimetric tetrazolium assays are used increasingly in studies of fungi, often in the absence of standardization or correlation with other methods. We examined species- and strain-related tetrazolium metabolism in
Candida albicans
and
Candida parapsilosis
by using XTT {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide} and WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphonyl)-2H-tetrazolium] and found marked variations. Also, significant signal was often missed in the absence of dimethyl sulfoxide extraction.
Clostridium difficile
Brain Empyema after Prolonged Intestinal Carriage
Authors: Gravisse J. , Barnaud G. , Hanau-Berçot B. , Raskine L. , Riahi J. , Gaillard J. L. , Sanson-Le-Pors M. J.
Published: January 2003

Clostridium difficile
, the most common cause of antibiotic-associated diarrhea, is occasionally isolated from extraintestinal sites and is usually found as part of a polymicrobial flora. We report a case of brain empyema that occurred after the recurrent intestinal carriage of a nontoxigenic strain of
C. difficile
. Brain abscess cultures contained both toxigenic and nontoxigenic isolates. Pulsed-field gel electrophoresis showed that nontoxigenic isolates from the intestine and from the brain were identical.
Introduction of Human Immunodeficiency Virus Type 2 Infection in the Philippines
Authors: Leaño Prisca Susan , Kageyama Seiji , Espantaleon Adelfa , Maniar Janak , Iwasaki Masaomi , Saple Dattatray , Yoshihara Namiko , Kurimura Takashi , Agdamag Dorothy May
Published: January 2003

The aim of this study was to describe and document the first case of human immunodeficiency virus type 2 (HIV-2) in the Philippines by using serological and molecular techniques and to compare the diversity of this strain to that of strains from other countries. With the introduction of HIV-2 into the country and the presence of diversified strains of HIV-1, the use of highly sensitive assays to detect all these strains is recommended.
Serotyping of 467
Cryptococcus neoformans
Isolates from Clinical and Environmental Sources in Brazil: Analysis of Host and Regional Patterns
Authors: Nishikawa Marília M. , Lazera Márcia S. , Barbosa Glaucia G. , Trilles Luciana , Balassiano Beatriz R. , Macedo Regina C. L. , Bezerra Cláudia C. F. , Pérez Maurício A. , Cardarelli Paola , Wanke Bodo
Published: January 2003

Cryptococcus neoformans
is an important zoopathogen, and it is one of the most prevalent lethal mycotic agents. Its polysaccharide capsule, synthesized in vivo and in vitro, is a virulence factor, contains predominantly glucuronoxylomannan, and is responsible for the antigenic differentiation of serotypes A, B, C, D, and AD. A total of 467 isolates of
C. neoformans
obtained from clinical and environmental sources from Brazilian regions were studied serologically by using the Crypto Check Iatron RM 304-K kit. Serotyping of the clinical isolates showed the following prevalences of the serotypes: A (77.95%), followed by B (18.2%), AD (1.3%), D (0.4%), C (0.2%), and untypeable (1.93%). The epidemiology of serotype A in the Brazilian southern and southeastern regions reproduces the picture observed worldwide. In contrast, serotype B was the most frequent agent of cryptococcosis in the northeastern region, occurring nearly equally in male and female healthy hosts. Among the isolates from environmental sources, serotypes A and B were found to occur in the hollows of tropical trees of the genera
Cassia
,
Ficus
, and
Moquillea
. The few isolates from
Eucalyptus camaldulensis
debris were serotypes A and B and untypeable. Overall, no association with a specific host tree was identified for these serotypes, denoting a distinct ecoepidemiological regional pattern. The one serotype C isolate was recovered from a human immunodeficiency virus-negative host. Serotype AD predominated over serotype D among both clinical and environmental isolates.
In Vitro Activities of Voriconazole, Posaconazole, and Four Licensed Systemic Antifungal Agents against
Candida
Species Infrequently Isolated from Blood
Authors: Pfaller M. A. , Diekema D. J. , Messer S. A. , Boyken L. , Hollis R. J. , Jones R. N.
Published: January 2003

We determined the in vitro susceptibilities of 314 strains of
Candida
spp., representing 13 species rarely isolated from blood, to posaconazole and voriconazole as well as four licensed systemic antifungal agents (amphotericin B, flucytosine, fluconazole, and itraconazole). The organisms included 153 isolates of
C. krusei
, 67 isolates of
C. lusitaniae
, 48 isolates of
C. guilliermondii
, 10 isolates of
C. famata
, 10 isolates of
C. kefyr
, 6 isolates of
C. pelliculosa
, 5 isolates of
C. rugosa
, 4 isolates of
C. lipolytica
, 3 isolates of
C. dubliniensis
, 3 isolates of
C. inconspicua
, 2 isolates of
C. sake
, and 1 isolate each of
C. lambica
,
C. norvegensis
, and
C. zeylanoides
. MIC determinations were made by the National Committee for Clinical Laboratory Standards reference broth microdilution method and Etest (amphotericin B). Resistance to both amphotericin B and fluconazole was observed in strains of
C. krusei
,
C. lusitaniae
,
C. guilliermondii
,
C. inconspicua
, and
C. sake
. Resistance to amphotericin B, but not to fluconazole, was also observed among isolates of
C. kefyr
and
C. rugosa
. Posaconazole and voriconazole were active (MIC, ≤1 μg/ml) against 94 to 100% of these isolates. In contrast to the more common species of
Candida
causing bloodstream infection, these rare species appear to be less susceptible to the currently licensed systemic antifungal agents, with the exception of voriconazole. Continued surveillance will be necessary to detect the emergence of these species as more prevalent, resistant pathogens. The new triazoles appear to offer acceptable coverage of uncommon
Candida
sp. bloodstream infections.
Regional Distribution of Antibodies to Herpes Simplex Virus Type 1 (HSV-1) and HSV-2 in Men and Women in Ontario, Canada
Authors: Howard Michelle , Sellors John W. , Jang Dan , Robinson Noah J. , Fearon Margaret , Kaczorowski Janusz , Chernesky Max
Published: January 2003

This study estimated the regional and age- and gender-specific seroprevalences of herpes simplex virus type 1 (HSV-1) and HSV-2 in Ontario, Canada. Stored serum specimens from subjects aged 15 to 44 years, including men (
n
= 979), women not under prenatal care (
n
= 638), and women under prenatal care (
n
= 701) submitted for routine viral serology were randomly selected according to regional population size from public health laboratories. HSV-1 and HSV-2 testing was done with the MRL enzyme immunoassay (EIA) (Focus Technologies), and HSV-2 was also tested by the Gull/Meridian EIA. Specimens discordant for HSV-2 antibodies between the two EIAs were resolved by a recombinant immunoblot assay (Focus Technologies). The overall age- and gender-standardized seroprevalences of HSV-1 and HSV-2 were 51.1% (95% confidence interval [CI], 50.1 to 52.1) and 9.1% (95% CI, 8.6 to 9.7), respectively. The seroprevalence of HSV-1 antibodies increased from 26.9 to 54.7% in men between 15 to 16 and 40 to 44 years of age, from 32.0 to 88.7% in women not under prenatal care, and from 55.2 to 69.2% in women under prenatal care. The seroprevalence of HSV-2 increased from 3.8 to 21.3% in men between 15 to 16 and 40 to 44 years of age, from 0 to 18.9% in women not under prenatal care, and from 3.4 to 23.1% in women under prenatal care. HSV-2 results were discordant for 3.3% (76 of 2,318) of specimens. Both types of HSV antibodies appeared to be acquired earlier among women under prenatal care than among men and women not under prenatal care. Antibodies were more prevalent among people in northern Ontario (72.9% of subjects [range, 68.4 to 77.4%] for HSV-1 and 13.7% of subjects [95% CI, 10.2 to 17.2%] for HSV-2) than elsewhere.
Evaluation of Etest Method for Determining Voriconazole and Amphotericin B MICs for 162 Clinical Isolates of
Cryptococcus neoformans
Authors: Maxwell M. J. , Messer S. A. , Hollis R. J. , Diekema D. J. , Pfaller M. A.
Published: January 2003

The performance of the Etest for voriconazole and amphotericin B susceptibility testing of 162 isolates of
Cryptococcus neoformans
was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 72 h at 35°C. MICs were determined by Etest for all 162 isolates with RPMI 1640 agar containing 2% glucose (RPG agar) and were read after incubation for 72 h at 35°C. The Etest results for both voriconazole and amphotericin B correlated well with reference MICs. Agreement was 94% for voriconazole and 99% for amphotericin B. When discrepancy was noted between the results obtained by Etest and broth microdilution for voriconazole, the Etest generally provided a higher MIC. The Etest method using RPG agar appears to be a useful method for determining the susceptibility of
C. neoformans
to voriconazole and amphotericin B.
Programme sensitivity and effectiveness of mammography service screening in Helsinki, Finland
Authors: Anttila A. , Koskela J. , Hakama M.
Published: January 2003

OBJECTIVES: To evaluate the effectiveness of mammography service screening. SETTING: Helsinki (population 0.5 million), the capital of Finland, the breast cancer service screening programme was started gradually in 1986. MATERIALS AND METHODS: The study was based on the data from the breast cancer screening programme in Helsinki. All incident cases of breast cancer and rates in the female population in Helsinki during the period 1970–97, and information on the subsequent breast cancer mortality were derived from the files of the Finnish Cancer Registry. To estimate the sensitivity of the programme, the number of screen detected cancers were compared with the overall number of breast cancers at the targeted age groups. Effects of screening were estimated for cumulative incidence and for the refined mortalities among the birth cohorts screened. RESULTS: Altogether 144 400 invitations had been sent during 1986–97 among the 50–9 year old women as targeted in Helsinki. The average attendance rate was 82%; and 545 breast cancers were detected by screening, with a detection rate of 0.46%. In the birth cohort of women born between the start of 1935 and the end of 1939—namely, those subjected to most complete screening and long follow up time since the onset of the programme—there was an increase in the cumulative incidence of breast cancer (relative risk (RR) 1.18, 95% confidence interval (95% CI) 1.09 to 1.27). The estimated sensitivity of the screening programme was 58%; and 53% if corrected for overdiagnosis. There was also a decrease of 19% (RR 0.81, 95% CI 0.62 to 1.05) in the refined mortality within this screened birth cohort, compared with the death rate of women of comparable ages in the non-screened reference cohort. CONCLUSION: The study provides further support to the hypothesis that service screening with mammography reduces the risk of breast cancer mortality.
Routine urinalysis of patients in hospital in Lebanon: how worthwhile is it?
Authors: Pashayan N. , Khogali M. , Major S.C.
Published: January 2003

OBJECTIVES: To examine the impact of routine urinalysis at admission on inpatient care at a hospital in Lebanon, where physicians perceive it to be a valuable diagnostic tool, in a country where preventive services are underdeveloped and where the epidemiology of kidney diseases possibly differs from that of the western world. SETTING: American University Hospital, a tertiary teaching hospital in Beirut, Lebanon. METHODS: A retrospective medical record review of all adult patients admitted over 2 weeks to the medicine and surgery wards of the American University Hospital. Outcomes measured were frequency of routine urinalysis versus urinalysis for a clinical indication, investigation of abnormal test results, and implications of test results on clinical management. RESULTS: 367 (79%) of 462 study patients underwent urinalysis. 266 (73%) patients had routine urinalysis. Abnormal results were found in 97(37%) routine tests and 67 (66%) of those clinically indicated urinalysis (p<0.001). Abnormalities were investigated in 21 (22%) of the abnormal routine urinalyses and 45 (67%) of the abnormal clinically indicated urinalyses (p<0.001). Logistic regression analysis showed no factors to correlate positively with investigation of abnormal urinalysis. Treatment was given to two (1%) patients who had had routine urinalysis and 26 (26%) of all those tested because of a clinical indication (p<0.001). CONCLUSIONS: Clinical response to any abnormal urinalysis is more likely when a urine test is done for a clinical indication. In this study setting, impact of routine admission urinalysis on patient care was negligible. Despite physicians’ perception of routine urinalysis being a valuable case finding tool, in this study its true value remains questionable.
Changing times, spaces, and faces: tests and implications of adaptive morphological plasticity in the fishes of northern postglacial lakes
Authors: Robinson Beren W , Parsons Kevin J
Published: January 2003

The phenotypic diversity exhibited within and among populations of freshwater fishes in postglacial lakes has intrigued biologists for two reasons: (i) their high phenotypic variation and (ii) the apparently recent and rapid divergence of forms. Genetic and ecological studies of these taxa are shedding new light on mechanisms of divergence and species formation. Surprisingly, the roles of phenotypic plasticity in the origins, maintenance, and generation of phenotypic diversity in this system are rarely directly addressed. We synthesize the available literature on morphological plasticity in these fishes and, using a meta-analysis, test for adaptive plasticity. We conclude that (i) morphological plasticity is common in at least six families of northern freshwater fishes, (ii) plastic responses can often be induced by conditions related to littoral and pelagic lake environments, (iii) plasticity often represents adaptive responses to conditions in these habitats, and (iv) that although rarely tested, heritable variation in morphological plasticity is present. The rich amount of phenotypic plasticity has not constrained recent adaptive divergence and species formation in postglacial fishes, and instead plasticity may play a role in the notably high rates of divergence observed in these and other fishes currently undergoing adaptive radiation.
Hierarchical Bayesian analysis of capturemarkrecapture data
Authors: Rivot E , Prévost E
Published: January 2003

We present a hierarchical Bayesian model (HBM) for capturemarkrecapture (CMR) data analysis. It aims at estimating the probability of capture (θi) and the total population size (Ni) in a series of I years i = 1,...,I. The HBM assumes that the θis and Nis are sampled from a common probability distribution with unknown parameters. It is compared with the model assuming independence between years in the θis and Nis (ABM). We show how a transfer of information between years is organized by the HBM. We compare the merits of HBM vs. ABM to estimate the spawning run and smolt run of an Atlantic salmon (Salmo salar) population of the River Oir (France) over a period of 17 years. In the spawners case, yearly data are poorly informative. Consequently, the HBM greatly improves posterior inferences compared with the ABM in terms of dispersion and robustness to the choice of prior. In the smolts case, the HBM does not significantly improve inferences compared with the ABM because data are more informative. We discuss why hierarchical modeling should be recommended in any ecological study where the data are collected on several sampling units that share some common features.
A size-based model of the impacts of bottom trawling on benthic community structure
Authors: Duplisea Daniel E , Jennings Simon , Warr Karema J , Dinmore Tracy A
Published: January 2003

Bottom trawling causes widespread disturbance to the sediments in shallow-shelf seas. The resultant mortality of benthic fauna is strongly size dependent. We empirically demonstrate that beam trawling frequency in the central North Sea had a greater effect on fauna size distribution in a soft sediment benthic community than variables such as sediment particle size and water depth. Accordingly, we simulated the impacts of trawling disturbance on benthos using a model consisting of 37 organism size classes between 1 μg and 140 g wet weight. The model produced a productionbiomass versus size relationship consistent with published studies and allowed us to predict the impacts of trawling frequency on benthos size distributions. Outputs were consistent with empirical data; however, at high yet realistic trawling frequencies, the model predicted an extirpation of most macrofauna. Empirical data show that macrofauna persist in many heavily trawled regions; therefore, we suggest that trawling by real fisheries is sufficiently heterogeneous to provide spatial refuges less impacted by trawling. If correct, our analyses suggest that fishery management measures that do not reduce total effort but do lead to effort displacement and spatial homogenization (e.g., temporarily closed areas) may have adverse effects on the systemic persistence of intermediate- and large-sized macrofauna.
Diapause in parasitic nematodes: a review
Authors: Sommerville R I , Davey K G
Published: January 2003

This review considers in a selective way the literature on diapause in parasitic nematodes, concentrating on four species of animal parasites and three species of plant parasites. We define diapause as a developmental arrest which is temporarily irreversible, so development will not resume, even under favourable conditions, until some intrinsic changes have been completed. Our analysis recognises four stages in diapause. The first is induction, typically brought about by environmental signals (although diapause may be genetically programmed independently of the environment). These environmental signals typically do not have an immediate effect on development, but we recognise a second phase, which we call the diapause pathway, in which worms have been induced to enter diapause at a later developmental stage. Surprisingly, entry into the diapause pathway may under some circumstances be reversible. The third stage is diapause development, a period during which development is suspended, but some ill-understood process must be completed prior to the fourth stage, emergence from diapause. Although diapause development is complete, resumption of development may be further delayed because of conditions in the host or in the environment: the worm is once more capable of development, but development is prevented by unfavourable conditions extrinsic to the worm. These may include the immune state of the host or the total parasite burden in animal hosts.