bluuebunny/crossref_metadata_2025 huggingface dataset:
The Single Superoxide Dismutase of
Rhodobacter capsulatus
Is a Cambialistic, Manganese-Containing Enzyme
Authors: Tabares Leandro C. , Bittel Cristian , Carrillo Néstor , Bortolotti Ana , Cortez Néstor
Published: May 2003

The phototrophic bacterium
Rhodobacter capsulatus
contains a single, oxygen-responsive superoxide dismutase (SOD
Rc
) homologous to iron-containing superoxide dismutase enzymes. Recombinant SOD
Rc
, however, displayed higher activity after refolding with Mn
2+
, especially when the pH of the assay mixture was raised. SOD
Rc
isolated from
Rhodobacter
cells also preferentially contains manganese, but metal discrimination depends on the culture conditions, with iron fractions increasing from 7% in aerobic cultures up to 40% in photosynthetic cultures. Therefore, SOD
Rc
behaves as a Mn-containing dismutase with cambialistic properties.
The
Bacillus subtilis
Acyl Lipid Desaturase Is a Δ5 Desaturase
Authors: Altabe Silvia G. , Aguilar Pablo , Caballero Gerardo M. , de Mendoza Diego
Published: May 2003

Bacillus subtilis
was recently reported to synthesize unsaturated fatty acids (UFAs) with a double bond at positions Δ5, Δ7, and Δ9 (M. H. Weber, W. Klein, L. Muller, U. M. Niess, and M. A. Marahiel, Mol. Microbiol. 39:1321-1329, 2001). Since this finding would have considerable importance in the double-bond positional specificity displayed by the
B
.
subtilis
acyl lipid desaturase, we have attempted to confirm this observation. We report that the double bond of UFAs synthesized by
B
.
subtilis
is located exclusively at the Δ5 position, regardless of the growth temperature and the length chain of the fatty acids.
Development of a LightCycler PCR Assay for Detection and Quantification ofAspergillus fumigatusDNA in Clinical Samples from Neutropenic Patients
Authors: Spiess Birgit , Buchheidt Dieter , Baust Corinna , Skladny Heyko , Seifarth Wolfgang , Zeilfelder Udo , Leib-Mösch Christine , Mörz Handan , Hehlmann Rüdiger
Published: May 2003

The increasing incidence of invasive aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the diagnostic tools for this disease. We established a LightCycler-based real-time PCR assay to detect and quantify rapidly, specifically, and sensitivelyAspergillus fumigatusDNA in both bronchoalveolar lavage (BAL) and blood samples from high-risk patients. The primers and hybridization probes were derived from anA. fumigatus-specific sequence of the mitochondrial cytochromebgene. The assay is linear in the range between 13.2 fg and 1.3 ng ofA. fumigatusDNA, corresponding to 3 to 300,000 CFU per ml of BAL fluid or blood. No cross-amplification was observed with human DNA or with the DNA of fungal or bacterial pathogens. For clinical evaluation we investigated 10 BAL samples from nine neutropenic patients with malignant hematological diseases and 12 blood samples from seven neutropenic patients with malignant hematological diseases. Additionally, we tested one blood sample and one BAL sample from each of two neutropenic patients. In order to characterize the validity of the novel PCR assay, only samples that had shown positive results by a previously described sensitive and specific nested PCR assay were tested. Twelve of 12 BAL samples and 6 of 14 blood samples gave positive results by the LightCycler PCR assay. Eight of 14 blood samples gave negative results by the novel method. The LightCycler PCR-mediated quantification of the fungal burden showed 15 to 269,018 CFU per ml of BAL sample and 298 to 104,114 CFU per ml of blood sample. Twenty of 20 BAL samples and 50 of 50 blood samples from subjects without evidence of invasive pulmonary aspergillosis (IPA) were PCR negative. Compared to a previously described nested PCR assay, these preliminary data for the novel real-time PCR assay indicate a less sensitive rate of detection of IPA in high-risk patients, but the assay may be valuable for quantification of the fungal burden in individual clinical samples.
Characterization of a Tandem Repeat Polymorphism inLegionella pneumophilaand Its Use for Genotyping
Authors: Pourcel C. , Vidgop Y. , Ramisse F. , Vergnaud G. , Tram C.
Published: May 2003

We have analyzed the variability of minisatellite sequences (also called variable-number tandem repeats [VNTRs]) in the genome ofLegionella pneumophila. Based upon the genome sequence of the Philadelphia-1 strain (serogroup 1), 25 minisatellites were selected and their polymorphisms were analyzed by PCR with the DNA of serogroup 1 to 14 reference strains. For 22 markers, a PCR product of the expected size was found with the DNA of the Philadelphia-1 strain. Most of these markers did not amplify the DNA of otherLegionellaspecies or other bacteria used as controls. A polymorphism was observed for seven markers among theL. pneumophilastrains tested. To check whether these markers could be used to compare strains ofL. pneumophila, we analyzed two groups of isolates from clinical and environmental samples which had been independently genotyped by other methods. The results showed that, for the isolates in these two sets of samples, VNTR typing is as informative as pulsed-field gel electrophoresis for comparison of strains. Sequencing of one minisatellite from 14 reference strains was performed. Comparison of the sequences allowed a classification and confirmed the existence of subspecies ofL. pneumophila. We also tested the usefulness of one very polymorphic marker as a tool for the rapid screening of colonies grown from water samples. This allowed the rapid identification of theL. pneumophilacolonies and gave a first hint as to the presence of several strains in a single sample.
Virulence Markers of Enteroaggregative
Escherichia coli
Isolated from Children and Adults with Diarrhea in Brasília, Brazil
Authors: Piva Iriane C. , Pereira Alex L. , Ferraz Lúcia R. , Silva Rejane S. N. , Vieira Ataíza C. , Blanco Jesus E. , Blanco Miguel , Blanco Jorge , Giugliano Loreny G.
Published: May 2003

Escherichia coli
strains isolated from sporadic cases of acute diarrhea in children and adults and from children without diarrhea were investigated for the presence of the pAA plasmid. Strains harboring the pAA plasmid were isolated at similar frequencies from children with (19.6%) and without (10.8%) diarrhea and from adults with diarrhea (11.8%). The genotypic and phenotypic virulence markers of these strains were further analyzed. Most of the strains were positive for EAST1 (73%), and this toxin was detected significantly more frequently in strains from children with diarrhea than in strains from adults with diarrhea (
P
< 0.05). Likewise,
pic
sequences were detected significantly more frequently in strains from children with diarrhea than in strains from adults with diarrhea (
P
< 0.005) and controls (
P
< 0.025). Furthermore, the association of pAA positivity (pAA
+
) and
pic
positivity (
pic
+
) was more frequently found for strains from children with diarrhea than for strains from controls, indicating that pAA
+
pic
+
strains may represent a subset of pAA
+
strains associated with disease in children. Most of the strains (82.5%) adhered to cells presenting the typical aggregative pattern. The frequency of occurrence of enteropathogenic
E
.
coli
(EPEC) serogroups in the strains from children with diarrhea was very high (56%), while none of the strains from adults with diarrhea belonged to EPEC serogroups. Extraintestinal virulence markers were very commonly found in strains from adults with diarrhea. The frequencies of occurrence of the adhesins AFA and SFA were significantly higher in strains from adults with diarrhea than in strains from children with diarrhea. More than one extraintestinal virulence marker was found in 58% of the strains from adults with diarrhea but in only 7.7% of the strains from children with diarrhea. Our results show that pAA
+
strains isolated from children and adults with diarrhea present very different profiles when enteroaggregative
E. coli
virulence markers, serotypes, and extraintestinal virulence markers are considered.
Persistence of Oropharyngeal
Candida albicans
Strains with Reduced Susceptibilities to Fluconazole among Human Immunodeficiency Virus-Seropositive Children and Adults in a Long-Term Care Facility
Authors: Makarova Natalya U. , Pokrowsky V. V. , Kravchenko A. V. , Serebrovskaya L. V. , James Michael J. , McNeil Michael M. , Lasker Brent A. , Warnock David W. , Reiss Errol
Published: May 2003

Nineteen oropharyngeal
Candida albicans
isolates from six children and seven adults living with AIDS at the Russia AIDS Centre, Moscow, from 1990 to 1998 were selected for molecular typing. Two fluconazole-resistant
C. albicans
genotypes were identified from a child who contracted human immunodeficiency virus infection during the Elista Hospital outbreak in the Kalmyk Republic in 1989. Highly related strains were observed 4 years later in the oral lesions and colonization of two patients and a health care worker. There may be a tendency for persons who are living with AIDS in a long-term care facility and who receive fluconazole therapy for oropharyngeal candidiasis to harbor and spread fluconazole-resistant
C. albicans
strains.
Racial Distribution of
Candida dubliniensis
Colonization among South Africans
Authors: Blignaut Elaine , Pujol Claude , Joly Sophie , Soll David R.
Published: May 2003

Candida dubliniensis
is a yeast species that has only recently been differentiated from
Candida albicans. C. dubliniensis
colonization was initially associated with human immunodeficiency virus (HIV)-positive individuals. Because of the large proportion of AIDS patients in South Africa, we tested the generality of this association by assessing the prevalence of
C. dubliniensis
colonization among 253 black HIV-positive individuals, 66 healthy black individuals, 22 white HIV-positive individuals, and 55 healthy white individuals in South Africa carrying germ tube-positive yeasts in their oral cavities. Molecular fingerprinting with Ca3, a complex DNA fingerprinting probe specific for
C. albicans
, and Cd25, a complex DNA fingerprinting probe specific for
C. dubliniensis
, provides the first conclusive evidence of the existence of
C. dubliniensis
among South African clinical yeast isolates and reveals a higher relative prevalence of this species among white healthy individuals (16%) than among HIV-positive white individuals (9%), black healthy individuals (0%), and black HIV-positive individuals (1.5%). A cluster analysis separated South African
C. dubliniensis
isolates into two previously described groups, groups I and II, with the majority of isolates clustering in group I. Isolates from white healthy individuals exhibited a higher level of relatedness. A comparison of the
C. dubliniensis
isolates from South Africa with a general collection of
C. dubliniensis
isolates collected worldwide revealed no South Africa-specific clade, as has been demonstrated for
C. albicans
. These results suggest that in South Africa,
C. dubliniensis
carriage is influenced more by race than by HIV infection status.
Evaluation of Pulsed-Field Gel Electrophoresis as a Tool for Determining the Degree of Genetic Relatedness between Strains of
Escherichia coli
O157:H7
Authors: Davis Margaret A. , Hancock Dale D. , Besser Thomas E. , Call Douglas R.
Published: May 2003

Pulsed-field gel electrophoresis (PFGE) has been used extensively to investigate the epidemiology of
Escherichia coli
O157:H7, although it has not been evaluated as a tool for establishing genetic relationships. This is a critical issue when molecular genetic data are used to make inferences about pathogen dissemination. To evaluate this further, genomic DNAs from 62 isolates of
E. coli
O157:H7 from different cattle herds were digested with
Xba
I and
Bln
I and subjected to PFGE. The correlation between the similarity coefficients for these two enzymes was only 0.53. Four additional restriction enzymes (
Nhe
I,
Pac
I,
Sfi
I, and
Spe
I) were used with DNAs from a subset of 14 isolates. The average correlations between similarity coefficients using sets of one, two, and three enzymes were 0.405, 0.568, and 0.648, respectively. Probing with lambda DNA demonstrated that some DNA fragments migrated equal distances in the gel but were composed of nonhomologous genetic material. Genome sequence data from EDL933 indicated that 40 PFGE fragments would be expected from complete
Xba
I digestion, yet only 19 distinguishable fragments were visible. Two reasons that similarity coefficients from single-enzyme PFGE are poor measures of relatedness (and hence are poorly correlated with other enzymes) are evident from this study: (i) matching bands do not always represent homologous genetic material and (ii) there are limitations to the power of PFGE to resolve bands of nearly identical size. The findings of the present study indicate that if genetic relationships must be inferred in the absence of epidemiologic data, six or more restriction enzymes would be needed to provide a reasonable estimate using PFGE.
Rapid Detection of
Mycoplasma genitalium
,
Mycoplasma hominis
,
Ureaplasma parvum
, and
Ureaplasma urealyticum
Organisms in Genitourinary Samples by PCR-Microtiter Plate Hybridization Assay
Authors: Yoshida Takashi , Maeda Shin-Ichi , Deguchi Takashi , Miyazawa Takamaro , Ishiko Hiroaki
Published: May 2003

We present a method for detecting the presence of
Mycoplasma genitalium
,
Mycoplasma hominis
,
Ureaplasma parvum
, and
Ureaplasma urealyticum
organisms, which are thought to be associated with nongonococcal urethritis (NGU) and other genitourinary infections, in clinical samples. This method consists of PCR amplification of a part of the 16S rRNA gene followed by 96-well microtiter plate hybridization assay using four species-specific capture probes to detect the targets. To test the efficacy of this method, we applied it to the detection of the four species in the urine of patients with NGU. There were no cross-reactions with other human mycoplasmas or ureaplasmas, and the PCR-microtiter plate hybridization assay detected as few as 10 copies of the 16S rRNA gene of each of the four species. Based on these results, this PCR-microtiter plate hybridization assay can be considered an effective tool for the diagnosis of genitourinary infections with mycoplasmas or ureaplasmas.
Specific Delayed-Type Hypersensitivity Responses to ESAT-6 Identify Tuberculosis-Infected Cattle
Authors: Pollock J. M. , McNair J. , Bassett H. , Cassidy J. P. , Costello E. , Aggerbeck H. , Rosenkrands I. , Andersen P.
Published: May 2003

Human and bovine tuberculosis have long been detected by skin testing with purified protein derivative (PPD), a complex mix of partly denatured mycobacterial antigens with suboptimal specificity. In the present study, skin tests based on ESAT-6, a recombinantly produced antigen highly specific for tuberculosis infection, were investigated. Although ESAT-6 was strongly recognized in vitro and induced high levels of gamma interferon, initial investigations demonstrated that higher doses of ESAT-6 than of PPD were needed to induce substantial delayed-type hypersensitivity reactions. Also, the kinetics of the skin test response differed for the two reagents; PPD showed maximal response at 72 h, but the response to ESAT-6 often peaked later at 96 h. Tests based on an optimized strategy (400 μg of ESAT-6 measured between 72 and 96 h), in cattle infected with
Mycobacterium bovis
(
n
= 22) and animals sensitized by exposure to environmental mycobacteria showed ESAT-6 to have a promising diagnostic potential (sensitivity, 82%; specificity, 100%; optimal cutoff, 3 mm), compared with PPD (sensitivity, 86%; specificity, 90%; optimal cutoff, 4 mm). Larger investigations are required to refine cutoff points for any new diagnostic test, but the present results indicate great potential for skin tests based on specific antigens for accurate in vivo diagnosis of tuberculosis.
Evaluation and Utilization as a Public Health Tool of a National Molecular Epidemiological Tuberculosis Outbreak Database within the United Kingdom from 1997 to 2001
Authors: Drobniewski F. A. , Gibson A. , Ruddy M. , Yates M. D.
Published: May 2003

The aim of this study was to develop a national model and analyze the value of a molecular epidemiologicalMycobacterium tuberculosisDNA fingerprint-outbreak database. Incidents were investigated by the United Kingdom PHLS Mycobacterium Reference Unit (MRU) from June 1997 to December 2001, inclusive. A total of 124 incidents involving 972 tuberculosis cases, including 520 patient cultures from referred incidents and 452 patient cultures related to two population studies, were examined by using restriction fragment length polymorphism IS6110fingerprinting and rapid epidemiological typing. Investigations were divided into the following three categories, reflecting different operational strategies: retrospective passive analysis, retrospective active analysis, and retrospective prospective analysis. The majority of incidents were in the retrospective passive analysis category, i.e., the individual submitting isolates has a suspicion they may be linked. Outbreaks were examined in schools, hospitals, farms, prisons, and public houses, and laboratory cross-contamination events and unusual clinical presentations were investigated. Retrospective active analysis involved a major outbreak centered on a high school. Contact tracing of a teenager with smear-positive pulmonary tuberculosis matched 14 individuals, including members of his class, and another 60 cases were identified in schools clinically and radiologically and by skin testing. Retrospective prospective analysis involved an outbreak of 94 isoniazid-resistant tuberculosis cases in London, United Kingdom, that began after cases were identified at one hospital in January 2000. Contact tracing and comparison with MRU databases indicated that the earliest matched case had occurred in 1995. Subsequently, the MRU changed to an active prospective analysis targeting linked isoniazid-monoresistant isolates for follow up. The patients were multiethnic, born mainly in the United Kingdom, and included professionals, individuals from the music industry, intravenous drug abusers, and prisoners.
Evaluation ofCoxiella burnetiiAntibiotic Susceptibilities by Real-Time PCR Assay
Authors: Brennan Robert E. , Samuel James E.
Published: May 2003

Coxiella burnetiiis an obligate intracellular bacterium. The inability to cultivate this organism on axenic medium has made calculation of infectious units challenging and prevents the use of conventional antibiotic susceptibility assays. A rapid and reliable real-time PCR assay was developed to quantifyC. burnetiicells from J774.16 mouse macrophage cells and was applied to antibiotic susceptibility testing ofC. burnetiiNine Mile, phase I. For calculation of bacterial replication, real-time PCR performed equally as well as immunofluorescent-antibody (IFA) assay when J774.16 cells were infected with 10-fold serial dilutions ofC. burnetiiand was significantly (P< 0.05) more repeatable than IFA when 2-fold dilutions were used. Newly infected murine macrophage-like J774.16 cells were treated with 8 μg of chloramphenicol per ml, 4 μg of tetracycline per ml, 4 μg of rifampin per ml, 4 μg of ampicillin per ml, or 1 μg of ciprofloxacin per ml. After 6 days of treatment, tetracycline, rifampin, and ampicillin significantly (P< 0.01) inhibited the replication ofC. burnetii, while chloramphenicol and ciprofloxacin did not. In general, these results are consistent with those from prior reports on the efficacy of these antibiotics againstC. burnetiiNine Mile, phase I, and indicate that a real-time PCR-based assay is an appropriate alternative to the present methodology for evaluation of the antibiotic susceptibilities ofC. burnetii.
Evaluation of the Etest and Disk Diffusion Methods for Determining Susceptibilities of 235 Bloodstream Isolates of
Candida glabrata
to Fluconazole and Voriconazole
Authors: Pfaller M. A. , Diekema D. J. , Boyken L. , Messer S. A. , Tendolkar S. , Hollis R. J.
Published: May 2003

The performances of the Etest and the disk diffusion methods for testing of the susceptibilities of 235
Candida glabrata
isolates to fluconazole and voriconazole were compared with that of the National Committee for Clinical Laboratory Standards (NCCLS) approved standard broth microdilution (BMD) method. The NCCLS method used RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. Etest MICs were determined with RPMI 1640 agar containing 2% glucose (RPG agar) and with Mueller-Hinton agar containing 2% glucose and 0.5 μg of methylene blue per ml (MBE agar) and were read after incubation for 48 h at 35°C. Disk diffusion testing was performed with MBE agar, 25-μg fluconazole disks, and 1-μg voriconazole disks and by incubation at 35°C for 24 h. Overall agreements between the Etest and the BMD MICs obtained with RPG and MBE agars were 91 and 96%, respectively, for fluconazole and 93 and 95%, respectively, for voriconazole. Categorical agreements between the agar-based methods and BMD were 52.3 to 64.7% with fluconazole and 94.8 to 97.4% with voriconazole. The vast majority of the discrepancies by the disk diffusion and Etest methods with fluconazole were minor errors. The agar-based methods performed well in identifying isolates with resistance to fluconazole and decreased susceptibility to voriconazole.
DNA Fingerprinting of
Lactobacillus
crispatus
Strain CTV-05 by Repetitive Element Sequence-Based PCR Analysis in a Pilot Study of Vaginal Colonization
Authors: Antonio May A. D. , Hillier Sharon L.
Published: May 2003

Lactobacillus
crispatus
is one of the predominant hydrogen peroxide (H
2
O
2
)-producing species found in the vagina and is under development as a probiotic for the treatment of bacterial vaginosis. In this study, we assessed whether DNA fingerprinting by repetitive element sequence-based PCR (rep-PCR) can be used to distinguish the capsule strain of
L
.
crispatus
(CTV-05) from other endogenous strains as well as other species of vaginal lactobacilli. Vaginal and rectal lactobacilli were identified to the species level by using whole-chromosome probe DNA hybridization. The DNAs from
L
.
crispatus
,
L
.
jensenii
,
L
.
gasseri
, and an as-yet-unnamed H
2
O
2
-negative
Lactobacillus
species designated 1086V were subjected to rep-PCR. The results of gel electrophoresis and ethidium bromide staining of the DNA fingerprints obtained were compared.
L
.
crispatus
CTV-05 had a unique DNA fingerprint compared to all other lactobacilli. DNA fingerprints for 27 production lots of
L
.
crispatus
sampled from 1994 through 2001 were identical to that of the original strain isolated in 1993, suggesting strain stability. In a pilot study of nine women, this DNA fingerprinting method distinguished CTV-05 from other endogenous vaginal lactobacilli prior to and after vaginal capsule use. rep-PCR DNA fingerprinting is useful for strain typing and for evaluating longitudinal loss or acquisition of vaginal lactobacilli used as probiotics.
Multicenter Evaluation of Use of Dried Blood and Plasma Spot Specimens in Quantitative Assays for Human Immunodeficiency Virus RNA: Measurement, Precision, and RNA Stability
Authors: Brambilla Don , Jennings Cheryl , Aldrovandi Grace , Bremer James , Comeau Anne Marie , Cassol Sharon A. , Dickover Ruth , Jackson J. Brooks , Pitt Jane , Sullivan John L. , Butcher Ann , Grosso Lynell , Reichelderfer Patricia , Fiscus Susan A.
Published: May 2003

Eleven laboratories evaluated the use of dried blood and plasma spots for quantitation of human immunodeficiency virus (HIV) RNA by two commercially available RNA assays, the Roche Amplicor HIV-1 Monitor and the bioMerieux NucliSens HIV-1 QT assays. The recovery of HIV RNA was linear over a dynamic range extending from 4,000 to 500,000 HIV type 1 RNA copies/ml. The Monitor assay appeared to have a broader dynamic range and seemed more sensitive at lower concentrations. However, the NucliSens assay gave more consistent results and could be performed without modification of the kit. HIV RNA was stable in dried whole blood or plasma stored at room temperature or at −70°C for up to 1 year. Dried blood and dried plasma spots can be used as an easy and inexpensive means for the collection and storage of specimens under field conditions for the diagnosis of HIV infection and the monitoring of antiretroviral therapy.
Evaluation of a New Cellulose Sponge-Tipped Swab for Microbiological Sampling: a Laboratory and Clinical Investigation
Authors: Österblad Monica , Järvinen Helinä , Lönnqvist Kurt , Huikko Solja , Laippala Pekka , Viljanto Jouko , Arvilommi Heikki , Huovinen Pentti
Published: May 2003

A new type of swab (Cellswab; Cellomeda, Turku, Finland), utilizing a highly absorbent cellulose viscose sponge material, was compared to some traditional swabs. The survival of 14 aerobic and 10 anaerobic and microaerophilic bacterial species in the Cellswab, two commercial swab transport systems (Copan, Brescia, Italy, and Orion Diagnostica, Espoo, Finland), and one Dacron swab (Technical Service Consultants Ltd. [TSC], Heywood, United Kingdom) was evaluated. Bacteria were suspended in broth, into which the swabs were dipped. The Cellswab absorbed 1.3 times more fluid and released 3.5 times more fluid upon plating than the other swabs. Aerobic bacteria were stored in dry tubes, the others in transport medium, at 4°C and room temperature (RT), for up to 14 days. Swab samples were transferred to plates at 0, 1, 2, 4, 7, and 14 days. For 10 strains the Cellswab yielded ≥10% of the original CFU for longer than all the other swabs. In the clinical study, the ability of the Cellswab to detect beta-hemolytic streptococci from throat samples (
n
= 995) was compared to that of the TSC Dacron swab. The swabs performed equally, both when their samples were transferred to plates immediately and after storage for 1 day at 4°C or RT. The changes in normal microbiota after storage were also similar. The Cellswab was found to perform at least as well as ordinary swabs. It was better at storing fastidious strains, and at keeping bacteria viable for long storage times; it might well be a useful replacement or complement to ordinary swabs.
New Enzyme Immunoassay for Detection of Hepatitis B Virus Core Antigen (HBcAg) and Relation between Levels of HBcAg and HBV DNA
Authors: Kimura Tatsuji , Rokuhara Akinori , Matsumoto Akihiro , Yagi Shintaro , Tanaka Eiji , Kiyosawa Kendo , Maki Noboru
Published: May 2003

A new enzyme immunoassay specific for hepatitis B virus (HBV) core antigen (HBcAg) was developed. In order to detect HBcAg, specimens were pretreated with detergents to release HBcAg from the HBV virion and disassemble it to dimers, and simultaneously, the treatment inactivated anti-HBc antibodies. HBcAg detected by the assay peaked with HBV DNA in density gradient fractions of HBV-positive sera. The assay showed a wide detection range from 2 to 100,000 pg/ml. We observed no interference from anti-HBc antibody or blood components, but the assay was inhibited by very high concentrations (>1 μg/ml; corresponding to 80 signal/cutoff) of HBeAg. When the cutoff value was tentatively set at 4 pg/ml, all healthy control (HBsAg and HBV DNA negative,n= 160) and anti-hepatitis C virus-positive (n= 55) sera were identified as negative. HBcAg concentrations correlated very closely with HBV DNA (r= 0.946,n= 145) in 216 samples from 72 hepatitis B patients. In seroconversion panels, HBcAg concentrations changed in parallel with HBV DNA levels. The assay, therefore, offers a simple method for monitoring hepatitis B patients. With a series of sera during lamivudine therapy, HBV DNA levels fell sharply and the HBcAg concentration also decreased, but the change in HBcAg was smaller and more gradual. The supposed mechanism of these changes and their clinical significance are discussed.
Candida dubliniensis
at a University Hospital in Saudi Arabia
Authors: Fotedar R. , Al Hedaithy S. S. A.
Published: May 2003

Candida dubliniensis
is a newly described yeast species that is a close phylogenetic relative of
C. albicans
. Although it has been reported from different parts of the world, no detailed investigation of this species has been done in Saudi Arabia. The purpose of the present study was to identify
C. dubliniensis
isolates recovered from clinical specimens at a tertiary-care hospital in Riyadh, Saudi Arabia, and to determine the drug susceptibility profiles of those isolates. Over a period of 8 months, 823 germ tube- and chlamydospore-positive yeasts identified as
C. albicans
and recovered from different clinical specimens were screened for their ability to grow at 45°C on Sabouraud dextrose agar. Isolates which failed to grow at 45°C were presumptively identified as
C. dubliniensis.
The species identities were further confirmed by the production of pseudohyphae and chlamydospores on Staib agar and their inability to assimilate
d
-xylose and α-methyl-
d
-glucoside by using the API 20C AUX system. A total of 27 (3.3%) isolates were identified as
C. dubliniensis
. They were all recovered from 23 human immunodeficiency virus-negative patients. The prevalence of
C. dublinensis
in bronchoalveolar lavage (33.3%), oral (16.7%), and blood (16.7%) specimens was high. In addition, 33 isolates previously identified as
C. albicans
and preserved among our stock blood culture isolates were also recruited for the study. Of these, 5 isolates were found to be
C. dubliniensis
, thus making the total number of isolates identified as this species 32. Antifungal susceptibility testing of the
C. dubliniensis
isolates showed 100% sensitivity to amphotericin B, 97% sensitivity to each of fluconazole and ketoconazole, and 87.5% sensitivity to itraconazole. However, in contrast to other studies, the majority of the isolates (65.6%) showed high levels of resistance to flucytosine (MIC > 64 μg/ml). Further studies are warranted to investigate the cause of this unusually high rate of resistance to flucytosine of the
C. dubliniensis
isolates in this region.
Quality Control for β-Lactam Susceptibility Testing with a Well-Defined Collection of
Enterobacteriaceae
and
Pseudomonas aeruginosa
Strains in Spain
Authors: Cantón Rafael , Loza Elena , Del Carmen Conejo María , Baquero Fernando , Martínez-Martínez Luis
Published: May 2003

Eighteen
Enterobacteriaceae
and
Pseudomonas aeruginosa
strains, 16 of them with well-defined β-lactam re sistance mechanisms, were sent to 52 Spanish microbiology laboratories. Interpretative categories for 8 extended-spectrum β-lactams were collected. Participating laboratories used their own routine susceptibility testing procedures (88% automatic systems, 10% disk diffusion, and 2% agar dilution). Control results were established by two independent reference laboratories by applying the NCCLS microdilution method and interpretative criteria. Interpretative discrepancies were observed in 16% of the results (4.4% for cefepime, 3.0% for aztreonam, 2.8% for piperacillin-tazobactam, 1.7% for cefotaxime [CTX] and ceftazidime, 1.1% for ceftriaxone, 0.9% for meropenem, and 0.3% for imipenem). High consistency with reference values (<5% of major plus very major errors) was observed with (i) American Type Culture Collection quality control strains; (ii) strains with low-efficiency mechanisms inactivating extended-spectrum β-lactams, such as OXA-1-producing
Escherichiacoli
or SHV-1-hyperproducing
Klebsiella pneumoniae
; (iii) strains with highly efficient mechanisms, such as SHV-5 porin-deficient
K. pneumoniae
, CTX-M-10 in
Enterobacter cloacae
hyperproducing AmpC, and
P. aeruginosa
with the MexAB OprM efflux phenotype or hyperproducing AmpC. Low consistency (>30% major plus very major errors) was detected in K1-producing
Klebsiella oxytoca
, CTX-M-9-producing
E. coli
, and in OprD
−
P. aeruginosa
strains. Extended-spectrum β-lactamase (ESBL)-producing strains accounted for 86% of very major errors. Recognition of the ESBL phenotype was particularly low in
Enterobacter cloacae
strains (<35%), due to the lack of NCCLS-specific rules in this genus. A K1-producing
K. oxytoca
was misidentified by 10% of laboratories as an ESBL producer. The use of well-defined resistant strains is useful for improving proficiency in susceptibility testing in clinical laboratories.
Antimicrobial Resistance and Genetic Diversity of
Shigella sonnei
Isolates from Western Ireland, an Area of Low Incidence of Infection
Authors: DeLappe Niall , O'Halloran Fiona , Fanning Seamus , Corbett-Feeney G. , Cheasty T. , Cormican M.
Published: May 2003

Shigella sonnei
is a significant cause of gastroenteritis in both developing and industrialized countries. Definition of the diversity and antimicrobial susceptibility of
S. sonnei
isolates may be helpful in the management of individual cases and outbreaks. Antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) were performed with 67 isolates of
S. sonnei
predominantly (
n
= 59) from three counties in the west of Ireland. Phage typing (
n
= 17), plasmid profiling (
n
= 28), and integron analysis (
n
= 24) were performed with subsets of strains. PFGE typing permitted recognition of two major clusters: PFGE type A (
n
= 53) and PFGE type B (
n
= 14). PFGE type A was associated with resistance to ampicillin, streptomycin, and sulfonamides (51 of 53 isolates), and those that were phage typed (
n
= 6) were phage type 3. PFGE type B was associated with resistance to streptomycin, sulfonamides, tetracycline, and trimethoprim (11 of 14 isolates) and phage type 6 (9 of 11 isolates). Fifteen different plasmid profiles were identified among the 28 isolates analyzed. A class 2 integron was present in all 14 PFGE type B isolates. One of these isolates also contained a class 1 integron and showed a unique variant of the PFGE type B pattern. Sequence analysis of the gene cassette structures contained within these integrons identified distinct open reading frames that encoded determinants of resistance to trimethoprim, streptomycin, and streptothricin. Our data demonstrate two predominant PFGE types among
S. sonnei
isolates circulating in this region. The limited diversity of the
S. sonnei
isolates in this region means that detection of isolates indistinguishable by PFGE and according to their antibiograms in two or more patients is not persuasive evidence of a common-source food- or waterborne outbreak. Indistinguishable plasmid profiles in addition to indistinguishable PFGE and antibiogram types may be more suggestive of an epidemiologically relevant link between cases.
BVBlue Test for Diagnosis of Bacterial Vaginosis
Authors: Myziuk Linda , Romanowski Barbara , Johnson Stephen C.
Published: May 2003

Bacterial vaginosis (BV) is a disorder of the vaginal ecosystem characterized by a shift in the vaginal flora from the normally predominant
Lactobacillus
to one dominated by sialidase enzyme-producing mixed flora. It is the most common cause of abnormal vaginal discharge in adult women. The BVBlue system (Gryphus Diagnostics, L.L.C.) is a chromogenic diagnostic test based on the presence of elevated sialidase enzyme in vaginal fluid samples. BVBlue was compared to the standard method for diagnosing BV (Amsel criteria and Nugent score). Fifty-seven nonmenstruating women of ≥16 years of age who presented for a pelvic examination were recruited. Demographic features were collected via a self-administered questionnaire. The Amsel criteria were assessed based on three of four of the following characteristics of vaginal discharge: consistency, odor, pH, and presence of clue cells on Gram stain. BVBlue was compared to the Gram stain and Amsel criteria. The sensitivity, specificity, positive predictive value, and negative predictive value for BVBlue versus the Gram stain and Amsel criteria were 91.7, 97.8, 91.7, and 97.8% and 50.0, 100, 100, and 88.2%, respectively. A significantly greater proportion of patients with a vaginal pH of >4.5, a positive amine test, or with clue cells on vaginal Gram smear were found to have a positive BVBlue test (
P
< 0.001). Women previously treated for BV were 2.98 times more likely to have another episode of BV. BVBlue is a useful point-of-care diagnostic tool to provide a presumptive diagnosis of BV, especially in situations where microscopic capabilities are unavailable.
Escherichia coli
Strains from Pregnant Women and Neonates: Intraspecies Genetic Distribution and Prevalence of Virulence Factors
Authors: Watt Stéphane , Lanotte Philippe , Mereghetti Laurent , Moulin-Schouleur Maryvonne , Picard Bertrand , Quentin Roland
Published: May 2003

To determine the extent to which the vagina, endocervix, and amniotic fluid screen the
Escherichia coli
strains responsible for neonatal infections, we studied the genetic relationships among 105
E. coli
strains isolated from all of the ecosystems involved in this infectious process. Twenty-four strains were isolated from the intestinal flora, and 25 strains were isolated from the vaginas of pregnant women. Twenty-seven strains were isolated from the amniotic fluid, blood, and cerebrospinal fluid (CSF) of infected neonates. The intraspecies genetic characteristics of all of the isolates were determined by random amplified polymorphic DNA (RAPD) analysis, PCR ECOR (E. coli reference) grouping, and PCR virulence genotyping. A correlation was found between the intraspecies distributions of the strains in the A, B1, B2, and D ECOR groups and in the two major RAPD groups (I and II). Nevertheless, the distribution of the
E. coli
strains in the RAPD groups according to their anatomical origins was more significant than their distribution in the ECOR groups. This may be explained by the existence of an
E. coli
subpopulation, defined by the RAPD I group, within the ECOR B2 group. This RAPD I group presents a major risk for neonates: 75% of the strains isolated from patients with meningitis and 100% of the strains isolated from patients with bacteremia were in this group. The vagina and the amniotic fluid are two barriers that favor colonization by highly infectious strains. Indeed, only 17% of fecal strains belonged to the RAPD I group, whereas 52% of vaginal strains and 67% of amniotic fluid strains belonged to this subpopulation. The
ibeA
and
iucC
genes were significantly associated with CSF strains, whereas the
hly
and
sfa/foc
genes were more frequent in blood strains. These findings could serve as a basis for developing tools to recognize vaginal strains, which present a high risk for neonates, for use in prophylaxis programs.
Comparison of the Pathogenesis of Two Genetically Different H3N2 Influenza A Viruses in Pigs
Authors: Landolt Gabriele A. , Karasin Alexander I. , Phillips Lynette , Olsen Christopher W.
Published: May 2003

In 1997 and 1998, H3N2 influenza A viruses emerged among pigs in North America. Genetic analyses of the H3N2 isolates demonstrated that they had distinctly different genotypes. The most commonly isolated viruses in the United States have a triple-reassortant genotype, with the hemagglutinin, neuraminidase, and PB1 polymerase genes being of human influenza virus origin, the nucleoprotein, matrix, and nonstructural genes being of classical swine influenza virus origin, and the PA and PB2 polymerase genes being of avian influenza virus origin. In contrast, a wholly human H3N2 virus was isolated from a single baby pig in Ontario, Canada, in 1997, but it did not spread within the swine population. Genetic differences between this wholly human virus and the triple-reassortant viruses may affect their replication efficiencies in pigs. In the present study we compared the pathogenicities and replication kinetics of the wholly human virus and a triple-reassortant virus in 7-week-old pigs that were infected intranasally with 2 × 10
3
to 2 × 10
6
50% tissue culture infective doses of virus. Our results demonstrate that the wholly human virus replicated to significantly lower titers and that the onset of virus shedding was delayed compared to the replication titers and the time of onset of virus shedding in triple-reassortant viruses. In addition, infection with the triple-reassortant virus was associated with moderate to severe gross pathological and histological pulmonary lesions, while infection with the wholly human virus induced only mild pulmonary changes.
Regional Variation amongvacAAlleles ofHelicobacter pyloriin China
Authors: Wang Jingtong , van Doorn Leen-Jan , Robinson Philip A. , Ji Xuhuai , Wang Dong , Wang Yu , Ge Lianying , Telford John L. , Crabtree Jean E.
Published: May 2003

Allelic variation amongHelicobacter pylori vacAoccurs in the signal and middle region of the gene. The aim of the study was to investigate alleleic variation ofvacAamongH. pyloristrains from three regional areas of China and the relationship betweenvacAalleles and PUD. DNA extracted from 88 clinical isolates ofH. pyloriwas analyzed by type-specific PCR and reverse hybridization line probe assay (LiPA) to determine the genotype ofvacAand presence ofcagA.In 87 isolates, all of thevacAalleles could be classified as either type s1c or type s1a, and all could be classified as m1, m2a, or m2b. One strain could not be typed. In all, 41% of patients were infected with multiplevacAgenotypes, with the highest level being observed in Shanghai (63%). In strains from Beijing, s1a was dominant; by contrast, s1c was dominant in Guangxi and Shanghai. The prevalence of m2b strains in Shanghai (63%) was significantly higher than that in Beijing (83%) or Guangxi (0%). Thirty of the 87 patients had peptic ulcers. However, there was no association betweenvacAgenotype and PUD. This study demonstrates that there is significant geographic diversity of genotype ofvacAwithin China. The absence ofvacAs2 genotypes precluded analysis of an association ofvacAs genotypes and clinical disease.
Genomic Diversity of Clinical and Environmental
Vibrio cholerae
Strains Isolated in Brazil between 1991 and 2001 as Revealed by Fluorescent Amplified Fragment Length Polymorphism Analysis
Authors: Thompson Fabiano L. , Thompson Cristiane C. , Vicente Ana C. P. , Theophilo Grace N. D. , Hofer Ernesto , Swings Jean
Published: May 2003

Vibrio cholerae
is a ubiquitous and abundant organism in aquatic environments, particularly in coastal areas, estuaries, and rivers. This organism was the cause of a considerable number of deaths in Brazil during the last decade. In this study we applied the genomic fingerprinting technique fluorescent amplified fragment length polymorphism (FAFLP) to analyze 106
V. cholerae
O1 and non-O1 and non-O139 strains isolated from clinical specimens and the environment between 1991 and 2001. Numerical analysis of the FAFLP patterns disclosed seven main groups of genomes, all of them originated from a variety of different places in different years, suggesting that
V. cholerae
is a very diverse species. O1 and non-O1 and non-O139 strains were distinguishable by FAFLP, although clinical and environmental strains clustered together in a few cases. The persistence of some strains of highly related genomes during several years and in completely different geographical regions suggests that these strains are highly successful in adapting to changing environmental conditions.
Validation of the Automated Reading and Incubation System with Sensititre Plates for Antimicrobial Susceptibility Testing
Authors: Chapin Kimberle C. , Musgnug Michael C.
Published: May 2003

The present study compared the antimicrobial susceptibility testing (AST) results generated by the Automated Incubation and Reading System (ARIS) with custom Sensititre plates (TREK Diagnostic Systems, Cleveland, Ohio) and MicroScan PC10 GP and NUMIC10 GN plates interpreted with the WalkAway-96 system (Dade Behring, West Sacramento, Calif.) for gram-positive (GP) and gram-negative (GN) organisms as part of an in-house validation. A total of 326 isolates (3,689 antimicrobial agent-organism combinations) were evaluated. Sensititre plates were inoculated according to the instructions of the manufacturer with a suspension adjusted to a 0.5 McFarland standard, while the Prompt Inoculation System was used for the MicroScan plates. ARIS and the WalkAway system were used for automated reading of the Sensititre and MicroScan plates, respectively, at 18 to 24 h. The results were analyzed for essential (±1 twofold dilution) and categorical (sensitive, intermediate, or resistant) agreements. Plates that resulted in ARIS interpretations with major (falsely resistant) or very major (falsely susceptible) errors compared to the results obtained with the WalkAway system were read manually to corroborate instrument readings. Isolates for which very major or major errors were obtained and for which the results were not resolved by manual reading were retested in parallel. Isolates for which very major or major errors were obtained and for which the results were not resolved upon repeat testing were tested by the National Committee for Clinical Laboratory Standards M7-A5 frozen reference microdilution method. Essential agreement was 95.8% for 246 GN isolates. The following categorical error rates were obtained for the GN isolates: 1.3% minor errors, 0% major errors, and 0.4% very major errors. For 95 GP isolates, there was 93.5% essential agreement. Categorical error rates for GP isolates were 0.9% minor errors, 0.6% major errors, and 0.4% very major errors. ARIS-Sensititre is a diagnostic system feasible for use for automated AST in a clinical laboratory.
Detection ofChlamydia pneumoniae-Specific Antibodies Binding to the VD2 and VD3 Regions of the Major Outer Membrane Protein
Authors: Klein Marcus , Kötz Arne , Bernardo Katussevani , Krönke Martin
Published: May 2003

AlthoughChlamydia pneumoniaeis an important human pathogen, the antigens eliciting a specific humoral immune response remain elusive. We scrutinized several recombinant chlamydial surface proteins for species-specific recognition by a panel of human sera previously tested for the presence of anti-C. pneumoniaeand anti-C. trachomatisantibodies by microimmunofluorescence and enzyme-linked immunosorbent assay. The 15-kDa cysteine-rich protein (CrpA), porin-b (PorB), 9-kDa outer membrane protein (OMP3), 60-kDa outer membrane protein (OMP2), and four fragments of the major outer membrane protein (MOMP) representing each variable domain (VD) were overexpressed inEscherichia coli, affinity purified, and employed for Western blot analysis. None of the sera tested contained antibodies recognizing PorB and OMP3 ofC. pneumoniae. Sera fromC. pneumoniae-immune patients cross-reacted with OMP2 ofC. trachomatis, and sera fromC. trachomatis-immune patients cross-reacted with CrpA ofC. pneumoniae, indicating that some of chlamydial surface molecules share antigenic epitopes. In contrast, the VD2, as well as the VD3, regions of the MOMP ofC. pneumoniaewere only recognized byC. pneumoniae-positive sera, suggesting the existence of species-specific epitopes. The identification of such epitopes of cell surface molecules provides new insights intoC. pneumoniae-specific immune responses and may be of value for the improvement ofC. pneumoniae-specific diagnostic assay systems based on defined recombinant antigens.