bluuebunny/crossref_metadata_2025 huggingface dataset:
“
Actinobaculum massiliae
,” a New Species Causing Chronic Urinary Tract Infection
Authors: Greub Gilbert , Raoult Didier
Published: October 2002

We report on a new
Actinobaculum
species, “
Actinobaculum massiliae
,” isolated from the urine of an elderly woman with recurrent cystitis. Its phenotypic pattern was similar to those of both of the other
Actinobaculum
species described to date. On 16S rRNA sequencing, the Marseille isolate shared 95% homology with
Actinobaculum suis
, 92 to 93% homology with
Actinobaculum schaalii
, 91 to 92% homology with
Arcanobacterium
spp., and 87 to 90% homology with
Actinomyces
species. A bootstrap value of 99% supports the node separating the
Actinobaculum
sp. from its closest neighbor (
A. suis
). In conclusion, on the basis of phenotypic, genotypic, and phylogenetic assessments, we show that the Marseille isolate is a previously unrecognized organism within the
Actinobaculum
genus, and we propose placement of the organism in the taxon “
Actinobaculum massiliae
.”
Dissemination of
Streptococcus pneumoniae
Clone Colombia
5
-19 in Latin America
Authors: Gamboa Liliana , Camou Teresa , Hortal María , Castañeda Elizabeth
Published: October 2002

Streptococcus pneumoniae
serotype 5 is the third most common capsular type causing invasive diseases in children younger than 5 years in Latin America. Preliminary data on Colombian serotype 5 isolates indicated a common clonal origin associated with resistance to tetracycline (TET) and chloramphenicol (CHL). We studied 172
S. pneumoniae
serotype 5 invasive isolates from Argentina, Brazil, Colombia, Guatemala, Mexico, and Uruguay and confirmed the presence of the Colombia
5
-19 clone throughout Latin America. Fifteen subtypes of a pulsed-field gel electrophoresis pattern and 4 electrophoretic types (ET) were obtained. Most of the isolates from different geographical regions belonged to pattern A (34.3%), subtype A5 (41.9%), and ET1 (91.1%). The A pattern (
n
= 59) was resistant to TET and had variable resistance to CHL; it was present in Brazil (10.2%), Colombia (78%), Guatemala (8.5%), and Mexico (3.4%). Subtype A5 with variable susceptibility to TET and sensitive to CHL was found in Argentina (29.2%), Mexico (8.3%), and Uruguay (62.5%). Subtypes A1-A4, A7-A8, and A9-A11 (closely related to A) also shared ET1, while subtype A6 was assigned to ET1, ET2, and ET3. Eleven subtypes (
n
= 21) were found to be specific for one country each. In summary, the
S. pneumoniae
serotype 5 isolates from Latin American are genetically closely related but show different patterns of antibiotic resistance, probably as a result of horizontal transfer.
Both Urinary and Rectal
Escherichia coli
Isolates Are Dominated by Strains of Phylogenetic Group B2
Authors: Zhang Lixin , Foxman Betsy , Marrs Carl
Published: October 2002

To compare the genetic structures of uropathogenic and commensal
Escherichia coli
populations, a total of 181 urinary and rectal
E. coli
isolates were classified into intraspecies phylogenetic groups by PCR amplifications of phylogenetic markers. The genetic variability of these isolates within phylogenetic groups was further assessed by enterobacterial repetitive intergenic consensus (ERIC) typing. The distributions of 10 known virulence factors were also examined. In contrast with most reports, phylogenetic group B2 not only accounted for the majority of urinary isolates from young women with urinary tract infections (69%) but also was the dominant group among the rectal isolates from healthy young women (48%). Such difference may be explained by geographic variation, difference in host population characteristics, or differences in sampling method, or a combination of the three. Strains with known virulence factors most frequently belonged to phylogenetic groups B2 and D. Additionally, group B2 and D rectal isolates were more heterogeneous than urinary isolates. Two subclusters existed within group B2 strains by ERIC typing. These subclusters were not evenly distributed between rectal and urine isolates and differed in virulence gene distribution.
Protocol for Real-Time PCR Identification of Anthrax Spores from Nasal Swabs after Broth Enrichment
Authors: Oggioni Marco R. , Meacci Francesca , Carattoli Alessandra , Ciervo Alessandra , Orru Germano , Cassone Antonio , Pozzi Gianni
Published: October 2002

A mass-screening protocol for the diagnosis of anthrax from nasal swabs based on an enrichment step in liquid medium was devised. Incubation for growth was performed in autoclavable vials and racks which allow real-time PCR analysis of sterilized cultures. A dual-color PCR was set up with primers and probes for the chromosomal marker
rpoB
and the plasmid marker
lef
. Specific primer and probe sets were designed for the differentiation of
Bacillus anthracis
from
B. cereus
and for the differentiation of the Sterne vaccine strain from field isolates and the Ames strain, which was used in the recent anthrax bioterrorist attack. The present protocol thus combines the high specificity and sensitivity of real-time PCR with excellent biosafety and the low hands-on time necessary for the processing of large numbers of samples, which is extremely important during control programs involving the processing of large numbers of samples.
Extrapulmonary and Pulmonary Tuberculosis in Antananarivo (Madagascar): High Clustering Rate in Female Patients
Authors: Rasolofo Razanamparany Voahangy , Ménard Didier , Aurégan Guy , Gicquel Brigitte , Chanteau Suzanne
Published: October 2002

Antananarivo, the capital city of Madagascar, has an endemic focus of tuberculosis (TB). We specifically studied patients with extrapulmonary TB (EPTB) and grouped patients according to infected body site. The strains were characterized by IS
6110
fingerprinting and compared with those isolated from patients with pulmonary TB (PTB) during the same period in order to determine the possible association between the genotype and the clinical expression of TB. A total of 316 TB patients were included in this study: 151 individuals with EPTB, 10 with both PTB and EPTB, and 155 with PTB alone. Pleural TB was the major EPTB localization (77%) and was found more often in older patients, while PTB or EPTB in which the localization was other than pleural (other EPTB) was found in younger patients. The male-to-female ratio was slightly higher in pleural TB patients (3.06:1) than in patients with other EPTB (1.35:1). There was no significant difference in the BCG status among patients with PTB, pleural TB, and other EPTB. Analysis of IS
6110
patterns showed that 167 patients (52.8%) were assigned to 37 clusters of 2 to 34 patients. Analysis of the IS
6110
clusters and the IS
6110
families did not show any association with a particular clinical expression of the disease. Patients with PTB or other EPTB were more likely to have strains with one IS
6110
copy than patients with pleural TB. The clustering rate was found to be significantly higher in female patients (62%) than in male patients (48%) (
P
= 0.029), suggesting that Malagasy women were more likely to progress to disease after infection than men.
mecA
Gene Is Widely Disseminated in
Staphylococcus aureus
Population
Authors: Wielders C. L. C. , Fluit A. C. , Brisse S. , Verhoef J. , Schmitz F. J.
Published: October 2002

Methicillin-resistant
Staphylococcus aureus
(MRSA) is one of the most important causes of hospital infections worldwide. High-level resistance to methicillin is caused by the
mecA
gene, which encodes an alternative penicillin-binding protein, PBP 2a. To determine the clonal relationships between methicillin-susceptible
S. aureus
(MSSA) and MRSA, we typed 1,069
S. aureus
isolates (493 MSSA isolates and 576 MRSA isolates), collected mainly in North American and European hospitals between the 1960s and the year 2000, using pulsed-field gel electrophoresis and ribotyping. Of 10 widespread
S. aureus
lineages recognized, 8 had corresponding
mecA
-positive strains. Multiresistant MRSA strains are found in hospitals worldwide, while unrelated and more susceptible strains represent less than 1% of the MRSA population. This supports the hypothesis that horizontal transfer plays an important role in the dissemination of the
mecA
gene in the
S. aureus
population.
Multicenter Evaluation of Ethambutol Susceptibility Testing of
Mycobacterium tuberculosis
by Agar Proportion and Radiometric Methods
Authors: Madison B. , Robinson-Dunn B. , George I. , Gross W. , Lipman H. , Metchock B. , Sloutsky A. , Washabaugh G. , Mazurek G. , Ridderhof J.
Published: October 2002

Reproducibility of ethambutol (EMB) susceptibility test results for
Mycobacterium tuberculosis
has always been difficult for a variety of reasons, including the narrow range between the critical breakpoint for EMB resistance and the MIC for susceptible strains, borderline results obtained with the BACTEC 460TB method, the presence of microcolonies determined using the agar proportion (AP) method, and a lack of agreement between these two testing methods. To assess the frequency of these problems,
M. tuberculosis
drug susceptibility data were collected in a multicenter study involving four laboratories. Resistant, borderline, and susceptible isolates were shared among the laboratories to measure interlaboratory test agreement. Half of isolates determined by BACTEC 460TB to be resistant were determined to be susceptible by the AP method. Isolates determined to be resistant to EMB by both BACTEC 460TB and AP methods were almost always resistant to isoniazid. Results from isolates tested by the BACTEC 460TB method with an EMB concentration of 3.75 μg/ml in addition to the standard 2.5 μg/ml did not show improved agreement by the AP method. While these results do not indicate that the AP method is more accurate than the BACTEC 460TB method, laboratories should not report EMB monoresistance based on BACTEC 460TB results alone. Monoresistance to EMB should only be reported following confirmation by the AP method. Microcolonies could not be confirmed as resistant by the BACTEC 460TB method or by repeat testing with the AP method and do not appear to be indicative of resistance.
Serotype and Phage Type Distribution of
Salmonella
Strains Isolated from Humans, Cattle, Pigs, and Chickens in The Netherlands from 1984 to 2001
Authors: van Duijkeren E. , Wannet W. J. B. , Houwers D. J. , van Pelt W.
Published: October 2002

We studied serotypes and phage types of
Salmonella
strains isolated from humans and animals in The Netherlands over the period 1984 to 2001. All human strains (
n
= 59,168) were clinical isolates. The animal strains (
n
= 65,567) were from clinical and nonclinical infections. All isolates were serotyped, and
Salmonella enterica
serovar Typhimurium and serovar Enteritidis strains were further phage typed. The most prevalent serotypes were as follows: in humans, serovars Typhimurium and Enteritidis; in cattle, serovars Typhimurium and Dublin; in pigs, serovar Typhimurium; and in chickens, serovars Enteritidis, Infantis, and Typhimurium. Serovar Enteritidis phage type 4 (pt 4) was the most common phage type in humans and chickens. Serovar Typhimurium pt 510 was the most prevalent serovar Typhimurium phage type in humans and pigs, pt 200 was the most prevalent serovar Typhimurium phage type in cattle, and pt 150 was the most prevalent serovar Typhimurium phage type in chickens. Analysis of the distribution of sero- and phage types during the study period indicated that types shifted over time in humans and animals. Serovar Typhimurium DT 104 emerged in 1991 in humans, cattle, pigs, and chickens and became the most common serovar Typhimurium phage type in 2001. In general, similar sero- and phage types were found in humans and animals, although distinct types were more common in animals. Between the animal species, the sero- and phage type distributions varied considerably.
Comparison of Quantitative Competitive PCR with LightCycler-Based PCR for Measuring Epstein-Barr Virus DNA Load in Clinical Specimens
Authors: Stevens Servi J. C. , Verkuijlen Sandra A. W. M. , Brule Adriaan J. C. van den , Middeldorp Jaap M.
Published: October 2002

The aim of this study was to develop a LightCycler-based real-time PCR assay for monitoring the Epstein-Barr virus (EBV) DNA load in unfractionated whole blood. This assay was compared with quantitative competitive PCR (Q-PCR) for EBV. The LightCycler-based assay was highly sensitive and reproducible when quantifying plasmid DNA in either the presence or absence of healthy donor blood DNA. Amplifying plasmid DNA in DNA backgrounds from different donors slightly increased the variation of quantification, indicating that clinical specimen DNA has an influence on quantification. In most transplant recipients, a good correlation was observed between EBV DNA load dynamics determined by LightCycler and Q-PCR in follow-up samples, although the correlation between absolute values of EBV DNA loads was weak and occasional samples were false negative in the LightCycler assay. In 253 cross-sectional blood samples from patients with Burkitt's lymphoma, infectious mononucleosis, or human immunodeficiency virus infection, a weak but significant correlation between the two methods was found (
r
2
= 0.37,
P
< 0.001). Our results indicate that the clinical specimen DNA background may influence the absolute values of EBV DNA load in LightCycler analyses but that this effect is rare. LightCycler PCR is very well suited for monitoring of EBV DNA load dynamics, and its diagnostic value is comparable to that of Q-PCR. To avoid false negativity or underestimation of viral load, future internal calibration of the LightCycler is recommended. This would also enhance EBV load assay standardization and interinstitute comparisons.
Virologic and Serologic Identification of Minute Virus of Canines (Canine Parvovirus Type 1) from Dogs in Japan
Authors: Mochizuki Masami , Hashimoto Michiru , Hajima Takayuki , Takiguchi Mitsuyoshi , Hashimoto Akira , Une Yumi , Roerink Frank , Ohshima Takahisa , Parrish Colin R. , Carmichael Leland E.
Published: October 2002

Minute virus of canines (MVC), also known as canine parvovirus type 1, was initially believed to be a nonpathogenic agent, since it was first isolated from canine fecal specimens in the late 1960s. However, subsequent pathological as well as epidemiological studies suggested that MVC is a pathogen of neonatal puppies and is widely distributed among domestic dogs in the United States. The virus also has been shown to cause fetal deaths. Nevertheless, the virus was not detected in dogs outside the United States until recently, presumably because of a lack of widespread availability of the only susceptible canine cell line, WRCC/3873D, used for MVC isolation. We examined 470 clinical specimens from 346 dogs by PCR and detected MVC-specific gene fragments from four diseased puppies (positive rate, 1.2%). Viruses were recovered from three PCR-positive rectal specimens by using WRCC/3873D and MDCK cells. The isolates possessed antigenic and genomic properties similar to those of the U.S. reference strain GA3 and were identified as MVC. In addition, seroepidemiological evidence that 5.0% of dogs possessed anti-MVC antibodies also indicated the presence of MVC infection among dogs in Japan. From this study and several recent European reports describing MVC field cases, it is evident that MVC is distributed among domestic dogs worldwide.
Collaborative Evaluation of Optimal Antifungal Susceptibility Testing Conditions for Dermatophytes
Authors: Fernández-Torres Belkys , Cabañes Francisco J. , Carrillo-Muñoz Alfonso J. , Esteban Alexandre , Inza Isabel , Abarca Lourdes , Guarro Josep
Published: October 2002

A multicenter study was conducted to define the most suitable testing conditions for antifungal susceptibility of dermatophytes. Broth microdilution MICs of clotrimazole, itraconazole, and terbinafine were determined in three centers against 60 strains of dermatophytes. The effects of inoculum density (ca. 10
3
and 10
4
CFU/ml), incubation time (3, 7, and 14 days), endpoint criteria for MIC determination (complete [MIC-0] and prominent [MIC-2] growth inhibition), and incubation temperature (28 and 37°C) on intra- and interlaboratory agreement were analyzed. The optimal testing conditions identified were an inoculum of 10
4
CFU/ml, a temperature of incubation of 28°C, an incubation period of 7 days, and MIC-0.
False-Positive
Mycobacterium tuberculosis
Cultures in 44 Laboratories in The Netherlands (1993 to 2000): Incidence, Risk Factors, and Consequences
Authors: de Boer Annette S. , Blommerde Barbara , de Haas Petra E. W. , Sebek Maruschka M. G. G. , Lambregts-van Weezenbeek Kitty S. B. , Dessens Mirjam , van Soolingen Dick
Published: October 2002

False-positive
Mycobacterium tuberculosis
cultures are a benchmark for the quality of laboratory processes and patient care. We studied the incidence of false-positive cultures, risk factors, and consequences for patients during the period from 1993 to 2000 in 44 peripheral laboratories in The Netherlands. The national reference laboratory tested 8,889
M. tuberculosis
isolates submitted by these laboratories. By definition, a culture was false positive (i) if the DNA fingerprint of the isolate was identical to that of an isolate from another patient processed within 7 days in the same laboratory, (ii) if the isolate was taken from a patient without clinical signs of tuberculosis, and/or (iii) if the false-positive test result was confirmed by the peripheral laboratory and/or the public health tuberculosis officer. We identified 213 false-positive cultures (2.4%). The overall incidence of false-positive cultures decreased over the years, from 3.9% in 1993 to 1.1% in 2000. Laboratories with false-positive cultures more often processed less than 3,000 samples per year (
P
< 0.05). Among 110 patients for whom a false-positive culture was identified from 1995 to 1999, we found that for 36% of the patients an official tuberculosis notification had been provided to the appropriate public health services, 31% of the patients were treated, 14% of the patients were hospitalized, and a contact investigation had been initiated for 16% of the patients. The application of DNA fingerprinting to identify false-positive
M. tuberculosis
cultures and the provision of feedback to peripheral laboratories are useful instruments to improve the quality of laboratory processes and the quality of medical care.
Molecular Detection and Seroepidemiology of the
Chlamydia pneumoniae
Bacteriophage (ΦCpn1)
Authors: Karunakaran Karuna P. , Blanchard James F. , Raudonikiene Ausra , Shen Caixia , Murdin Andrew D. , Brunham Robert C.
Published: October 2002

Recent whole-genome analysis has demonstrated limited genetic variation in
Chlamydia pneumoniae
, with one strain (AR39) containing a 4,524 nucleotide single-stranded DNA bacteriophage, ΦCpn1. Using PCR, reverse transcription (RT)-PCR, and Western blotting, we confirmed the presence and functional expression of ΦCpn1 in
C. pneumoniae
strain AR39 and its absence in strain CWL029. Six additional epidemiologically distinct clinical isolates of
C. pneumoniae
also did not contain ΦCpn1. We generated recombinant viral protein 1 (Vp1) from ΦCpn1 in
Escherichia coli
and showed that Vp1 antigen is highly immunogenic in mice and that murine antisera readily recognize native Vp1 from
C. pneumoniae
strain AR39 elementary bodies (EB). We developed an enzyme-linked immunosorbent assay (ELISA) to measure antibodies to recombinant Vp1 in human sera collected from 32 patients with abdominal aortic aneurysm (AAA) and 40 controls. Among the 72 subjects, 61 had
C. pneumoniae
EB antibodies shown by ELISA. Antibodies to Vp1 were found in 39 of the 61 (64%) seropositive individuals and were significantly correlated with AAA (adjusted odds ratio, 13.9; 95% confidence interval, 1.1 to 175). Our studies indicate that phage-containing strains of
C. pneumoniae
are uncommonly found by isolation but may commonly infect individuals with vascular disease.
Precision and Accuracy of a Procedure for Detecting Recent Human Immunodeficiency Virus Infections by Calculating the Antibody Avidity Index by an Automated Immunoassay-Based Method
Authors: Suligoi Barbara , Galli Claudio , Massi Mario , Di Sora Fiorella , Sciandra Mauro , Pezzotti Patrizio , Recchia Olga , Montella Francesco , Sinicco Alessandro , Rezza Giovanni
Published: October 2002

We evaluated the precision and accuracy of a procedure for detecting recent human immunodeficiency virus (HIV) infections, specifically, the avidity index (AI) calculated using a method based on an automated AxSYM HIV 1/2gO assay (Abbott). To evaluate precision, we performed multiple replicates on eight HIV-positive serum samples. To evaluate the accuracy in identifying recent infections (i.e., within 6 months of seroconversion), we used 216 serum samples from 47 persons whose dates of seroconversion were known. To evaluate the sensitivity and specificity of the procedure for different AI cutoff values, we performed receiver operating characteristic (ROC) analysis. To determine the effects of antiretroviral treatment, advanced stage of the disease (i.e., low CD4-cell count), and low HIV viral load on the AI, we analyzed 15 serum samples from 15 persons whose dates of seroconversion were unknown. The precision study showed that the procedure was robust (i.e., the total variance of the AI was lower than 10%). Regarding accuracy, the mean AI was significantly lower for samples collected within 6 months of seroconversion, compared to those collected afterwards (0.68 ± 0.16 versus 0.99 ± 0.10;
P
< 0.0001), with no overlap of the 95% confidence intervals. The ROC analysis revealed that an AI lower than 0.6 had a sensitivity of 33.3% and a specificity of 98.4%, compared to 87.9 and 86.3%, respectively, for an AI lower than 0.9. Antiretroviral treatment, low CD4-cell count, and low viral load had no apparent effect on the AI. In conclusion, this procedure is reproducible and accurate in identifying recent infections; it is automated, inexpensive, and easy to perform, and it provides a quantitative result with different levels of sensitivity and specificity depending on the selected cutoff.
Genetic Characterization and Sequence Heterogeneity of a Canadian Isolate of Swine Hepatitis E Virus
Authors: Pei Yanlong , Yoo Dongwan
Published: October 2002

Swine hepatitis E virus (HEV) is a newly identified potentially zoonotic agent that is possibly transmitted to humans from pigs. Swine HEV is prevalent in pig populations and does not cause abnormal clinical symptoms in infected pigs, further implicating a likelihood of a risk of transmission to humans by normal contact. To date in North America, only one strain of swine HEV (strain US swine) has been fully sequenced. In the present study, we identified a swine HEV isolate from pigs in Canada, designated the Arkell strain, and determined the full length of the genomic sequence. The genome of Canadian strain Arkell consisted of 7,242 nucleotides, excluding the poly(A) tail of at least 15 A residues. The genome contained three open reading frames (ORFs), ORF1, ORF2, and ORF3, which had coding capacities for proteins of 1,708, 660, and 122 amino acids, respectively. Comparative analysis of the full-length genomic sequence indicated that the sequence of strain Arkell was distinct from those of all other known HEV isolates by 13 to 27% and shared the highest degrees of identity with human HEV isolates US-1 and US-2, HEV isolate US swine, and the human and swine HEV isolates recently isolated in Japan. On the basis of sequence similarities and phylogenetic analyses, HEV strain Arkell was grouped into genotype 3. The sequence of the Arkell swine HEV isolate differed from those of HEV isolate US swine and HEV isolate Japan swine by 13 and 14%, respectively. To date, two isolates of swine HEV (isolates Arkell and SK3 [D. Yoo et al., Clin. Diagn. Lab. Immunol. 8:1213-1219, 2001]) have been identified in Canadian pigs, and their sequences also differ from each other by 11.8%. Our studies indicate that, as with human HEV strains, swine HEV isolates exhibit extensive genetic heterogeneity.
Identification and Broad Dissemination of the CTX-M-14 β-Lactamase in Different
Escherichia coli
Strains in the Northwest Area of Spain
Authors: Bou German , Cartelle Monica , Tomas Maria , Canle Delia , Molina Francisca , Moure Rita , Eiros Jose Maria , Guerrero Antonio
Published: October 2002

During the course of a molecular epidemiology study of mechanisms of antibiotic resistance in the area served by our hospital (516,000 inhabitants), we isolated the gene encoding CTX-M-14 β-lactamase. Thirty clinical strains (27
Escherichia coli
and 3
Klebsiella pneumoniae
isolates) with a phenotype of extended-spectrum β-lactamase were collected from January to October 2001 and studied for the presence of the CTX-M-14 β- lactamase gene. By isoelectric point determination, PCR, and nucleotide sequencing, we detected the presence of this gene in 17
E. coli
strains belonging to 15 different genotypes (REP-PCR) causing infections in 17 different patients. Epidemiological studies based on medical records did not suggest any relationship between the patients infected with these
E. coli
strains and, interestingly, 7 of 30 patients harboring strains with extended-spectrum β-lactamases never had contact with the hospital environment before the clinical
E. coli
isolation. Conjugation experiments revealed that this gene was plasmid mediated in the 17
E. coli
strains, and plasmid restriction fragment length polymorphisms showed 9 different patterns in the 17
E. coli
strains. By PCR, the sequence of the
tnpA
transposase gene of the insert sequence ISEcp-1 was detected in all the plasmids harboring the CTX-M-14 gene. These results strongly suggest that plasmid dissemination between different
E. coli
strains in addition to a mobile element (transposon) around the β-lactamase gene may be involved in the spreading of the CTX-M-14 gene. This study reinforces the hypothesis that the epidemiology of the prevalence of the β-lactamase genes is changing and should alert the medical community to the increase in the emergence of the CTX-M β-lactamases worldwide.
Severe Acquired Toxoplasmosis in Immunocompetent Adult Patients in French Guiana
Authors: Carme B. , Bissuel F. , Ajzenberg D. , Bouyne R. , Aznar C. , Demar M. , Bichat S. , Louvel D. , Bourbigot A. M. , Peneau C. , Neron P. , Dardé M. L.
Published: October 2002

The most common presentation of symptomatic postnatally acquired toxoplasmosis in immunocompetent patients is painless cervical adenopathy. Acute visceral manifestations are associated in rare cases. We report 16 cases of severe primary toxoplasmosis diagnosed in French Guiana during a 6.5-year period. All of the subjects were immunocompetent adults hospitalized with clinical presentations consisting of a marked, nonspecific infectious syndrome accompanied by an altered general status with at least one visceral localization, mainly pulmonary involvement (14 cases). Acute toxoplasmosis was diagnosed according to the results of serological tests suggestive of recent primary infection and the absence of an alternative etiology. Recovery was rapid following specific antitoxoplasmosis treatment. Thirteen of the 16 patients had consumed game in the 2 weeks before the onset of the symptoms, and in eight cases the game was considered to have been undercooked.
Toxoplasma
strains, which were virulent in mice, were isolated from three patients. Microsatellite analysis showed that all of these isolates exhibited an atypical multilocus genotype, with one allele found only for isolates of this region.
Use of Epitope Mapping To Identify a PCR Template for Protein Amplification and Detection by Enzyme-Linked Immunosorbent Assay of Bovine Herpesvirus Type 1 Glycoprotein D
Authors: Joseph Tomy , Lyaku Japhet , Fredrickson Robert A. , Cepica Arnost , Kibenge Frederick S. B.
Published: October 2002

Infection with bovine herpesvirus type 1 (BHV-1) occurs worldwide and causes serious economic losses due to the deaths of animals, abortions, decreased milk production, and loss of body weight. BHV-1 is frequently found in bovine semen and is transmitted through natural service and artificial insemination. The detection of BHV-1 in bovine semen is a long-standing problem in veterinary virology which is important in disease control schemes. In the present study, ordered deletions of the full-length BHV-1 glycoprotein open reading frame were used to identify an epitope recognized by a specific monoclonal antibody (MAb). A glycoprotein D fragment containing this epitope was then amplified using an in vitro protein amplification assay developed previously (J. Zhou, J. Lyaku, R. A. Fredrickson, and F. S. Kibenge, J. Virol. Methods 79:181-189, 1999), and the resulting peptide was detected by indirect enzyme-linked immunosorbent assay (ELISA) with the specific MAb. This method detected 0.0395 50% tissue culture infective dose of BHV-1 in raw bovine semen, which was 1,000-fold more sensitive than traditional PCR. We therefore conclude that this in vitro protein amplification assay combined with ELISA has superior sensitivity for direct virus detection in clinical samples.
Nosocomial Infections Caused by Multidrug-Resistant Isolates of
Pseudomonas putida
Producing VIM-1 Metallo-β-Lactamase
Authors: Lombardi Gianluigi , Luzzaro Francesco , Docquier Jean-Denis , Riccio Maria Letizia , Perilli Mariagrazia , Colì Alessandra , Amicosante Gianfranco , Rossolini Gian Maria , Toniolo Antonio
Published: October 2002

Successful carbapenem-based chemotherapy for the treatment of
Pseudomonas
infections has been seriously hindered by the recent appearance of IMP- and VIM-type metallo-β-lactamases, which confer high-level resistance to carbapenems and most other β-lactams. Recently, multidrug-resistant
Pseudomonas putida
isolates for which carbapenem MICs were ≥32 μg/ml were recovered from cultures of urine from three inpatients in the general intensive care unit of the Ospedale di Circolo, Varese, Italy. Enzyme assays revealed production of a metallo-β-lactamase activity, while molecular analysis detected in each isolate a
bla
VIM-1
determinant carried by an apparently identical medium-sized plasmid. Conjugation experiments were unsuccessful in transferring the β-lactamase determinant to
Escherichia coli
or
Pseudomonas aeruginosa
. Macrorestriction analysis by pulsed-field gel electrophoresis demonstrated that the isolates were of clonal origin. PCR mapping and sequencing of the variable region of the plasmid-borne class 1 integron carrying the
bla
VIM-1
determinant (named In110) showed that the
bla
VIM-1
-containing cassette was identical to that previously found in strains of different species from other Italian hospitals and that the cassette array of In110 was not identical but clearly related to that of In70 (a
bla
VIM-1
-containing plasmid-borne integron from an
Achromobacter xylosoxidans
isolate), pointing to a common origin of this cassette and to a related evolutionary history of their cognate integrons.
Confirmation by 16S rRNA PCR of the COBAS AMPLICOR CT/NG Test for Diagnosis of
Neisseria gonorrhoeae
Infection in a Low-Prevalence Population
Authors: Diemert David J. , Libman Michael D. , Lebel Pierre
Published: October 2002

The COBAS AMPLICOR CT/NG test is widely used for the diagnosis of
Neisseria gonorrhoeae
infection using genital swabs or urine samples. Although highly specific, cross-reactivity occurs with some nonpathogenic strains of
Neisseria
and
Lactobacillus
species. In low-prevalence populations, even highly specific assays may require confirmatory testing of positive results. We assessed the positive predictive value (PPV) of this test in a low-prevalence (0.5%) setting. Genital and urine specimens testing positive using the COBAS AMPLICOR NG test were retested using an investigational 16S rRNA PCR assay. Additionally, 737 specimens were tested in parallel by both culture and the above PCR protocol. Of 9,772 specimens tested in-house, 168 were positive by the AMPLICOR test; in addition, 62 AMPLICOR-positive specimens were referred to our laboratory for confirmatory testing, yielding 230 positive specimens. Of these, 72 were confirmed positive by 16S rRNA PCR, yielding a specificity of 98.7% and a PPV of 31.3%. Specificity was similar for all specimen types, whereas PPV varied with prevalence: specimens from males, females, urine specimens, and genital swabs had PPVs of 70.8, 13.3, 51.9, and 20.1%, respectively. The PPV was higher when the initial AMPLICOR optical density (OD) was ≥3.5 versus initial and repeat OD readings in an equivocal zone of ≥0.2 to <3.5 (65.1 versus 10.1%;
P
< 0.001). On repeat testing of specimens with ODs in the equivocal zone, 54 gave ODs of ≥0.2 and <2.0, 35 gave ODs of ≥2.0 and <3.5, and 12 gave ODs of ≥3.5, with 3.7, 20, and 33.3% confirmed positive, respectively (
P
= 0.004). Comparing PCR to culture as the “gold standard,” specificity increased from 96.8 to 99.9% when 16S rRNA PCR was performed on specimens positive by the COBAS AMPLICOR NG test. Confirmatory testing with a more specific method such as 16S rRNA PCR should be considered in low-prevalence populations, especially for specimens with an OD in the equivocal zone.
Analysis of the Genetic Variability of Genes Encoding the RNA III-Activating Components Agr and TRAP in a Population of
Staphylococcus aureus
Strains Isolated from Cows with Mastitis
Authors: Gilot Philippe , Lina Gérard , Cochard Thierry , Poutrel Bernard
Published: October 2002

The expression of
Staphylococcus aureus
virulence proteins is under the control of RNA III, a central pleiotropic regulator transcribed from the
agr
locus. RNA III is activated by at least two two-component systems, one encoded by the
agr
locus (AgrC-AgrA) and another encoded outside of this locus (TRAP-RAP). In this work, we developed new typing methods based on genes encoding these two systems, which we used to characterize a nonclonal population of
S. aureus
bovine mastitis isolates. Twelve
agr
restriction types were identified in this population, but the majority of strains (56.3%) were grouped in the R III-A1 type. No strain isolated from humans, whose
agr
sequence is available from GenBank, was found to belong to this major type. Restriction maps constructed for all of those
agr
variants allowed the linking of all types in an evolution scheme and their grouping in one of the four
agr
interference groups. This analysis indicates that groups 2, 3, and 4 probably evolved from the more frequently encountered type, which belongs to group 1.
agr
group 1 was also found to be the most prevalent (69.0% of the strains) and the most polymorphic interference group. By developing an
agr
group-specific multiplex PCR, we confirmed the above classification of strains in the
agr
interference groups. Four allelic variants of
trap
were also identified, indicating that this two-component system is also polymorphic. The majority of strains was grouped in the
trap
1 type (71.8%). Whereas no relationships between
agr
group and
trap
types were found, strains of similar
agr
restriction type were also of similar
trap
type (with the exception of strains belonging to the
agr
R IV-A5 and R VI-A8 types). Our analysis indicates that
S. aureus
isolated from cows has predominantly a clonal structure and that the highly prevalent
agr
R III-A1,
trap
1 type (56.3% of the strains) probably possesses a genetic background which endows it with superior ability to infect the bovine mammary gland.
Occult Hepatitis B Virus Infection and Clinical Outcomes of Patients with Chronic Hepatitis C
Authors: Kao Jia-Horng , Chen Pei-Jer , Lai Ming-Yang , Chen Ding-Shinn
Published: October 2002

Although occult hepatitis B virus (HBV) infections in individuals without detectable hepatitis B surface antigen (HBsAg) may occur and have been reported to be common in patients with chronic hepatitis C, the clinical relevance remains controversial. We searched for serum HBV DNA in 210 HBsAg-negative patients with hepatitis C virus (HCV)-related liver disease (110 patients with chronic hepatitis, 50 patients with cirrhosis, and 50 patients with hepatocellular carcinoma) by PCR. Most of the patients had detectable antibodies to HBsAg or HBV core antigen. All of the 110 chronic hepatitis C patients were treated with a combination therapy consisting of interferon plus ribavirin. In addition, 100 HBsAg-negative healthy adults served as controls. Thirty-one of the 210 patients (14.8%) had HBV DNA in their sera, as did 15 of the 100 healthy controls (15%). HBV DNA was not detected in the sera of those negative for serological markers of HBV infection. In patients with chronic HCV infection, the prevalence of occult HBV infection did not parallel the severity of liver disease (14.5% in patients with chronic hepatitis, 8% in patients with liver cirrhosis, and 22% in patients with hepatocellular carcinoma). In addition, the sustained response to combination therapy against hepatitis C was comparable between patients with and without occult HBV infection (38 versus 39%). In conclusion, these data suggest that occult HBV infection does not have clinical significance in chronic hepatitis C patients residing in areas where HBV infection is endemic.
Experimental versus In Silico Fluorescent Amplified Fragment Length Polymorphism Analysis of
Mycobacterium tuberculosis
: Improved Typing with an Extended Fragment Range
Authors: Sims Elizabeth J. , Goyal Madhu , Arnold Catherine
Published: October 2002

Whole-genome fingerprinting fluorescent amplified fragment length polymorphism (FAFLP) data were compared with in silico data for the sequenced strains of
Mycobacterium tuberculosis
(H37Rv and CDC1551). For this G+C-rich genome, many predicted fragments were not detected experimentally. For H37Rv, only 108 (66%) of the 163 predicted
Eco
RI-
Mse
I fragments between 100 and 500 bp were visualized in vitro. FAFLP was also used to identify polymorphism in 10 clinical isolates of
M. tuberculosis
characterized previously by IS
6110
typing, examining fragments of up to 1,000 bp in size rather than up to 500 bp as was done previously. Five isolates had unique IS
6110
profiles and were not known to be epidemiologically related, two isolates were the same single-band IS
6110
type but were not known to be epidemiologically related, and the remaining three isolates were epidemiologically related with identical IS
6110
profiles. Analysis of fragments in the 500- to 1,000-bp range using nonselective primers differentiated better between strains than analysis of fragments in the 50- to 500-bp range using a set of four selective primers. Seventeen polymorphic fragments were identified between 500 and 1,000 bp in size compared with nine polymorphic fragments between 50 and 500 bp. Using the 500- to 1,000-bp analysis, a level of discrimination similar to that of IS
6110
typing was achieved which, unlike the IS
6110
typing, was able to differentiate the two
M. tuberculosis
strains, each of which had only a single copy of IS
6110
.
Method for Inactivating and Fixing Unstained Smear Preparations of
Mycobacterium tuberculosis
for Improved Laboratory Safety
Authors: Chedore Pamela , Th'ng Cecelia , Nolan Dennis H. , Churchwell George M. , Sieffert David E. , Hale Yvonne M. , Jamieson Frances
Published: October 2002

The inactivation of smears that contain
Mycobacterium tuberculosis
for microscopy before removal of the material from a biosafety cabinet is an important safety factor in preventing the potential transmission of tuberculosis to laboratory workers. The fixing and inactivating properties of heat flaming, 70% ethanol, and 1, 3, and 5% phenol in ethanol for smears containing
M. tuberculosis
were investigated. Heat flaming failed to inactivate the smear material, whereas 5% phenol in ethanol successfully fixed and inactivated all smears containing
M. tuberculosis
both from concentrated sputum samples and from culture material.
Remarkable Genetic Polymorphism among
Entamoeba histolytica
Isolates from a Limited Geographic Area
Authors: Haghighi Ali , Kobayashi Seiki , Takeuchi Tsutomu , Masuda Gohta , Nozaki Tomoyoshi
Published: October 2002

In order to understand genetic polymorphisms among
Entamoeba histolytica
strains in a limited geographic area and among restricted social populations, we studied nucleotide polymorphism in DNA regions that do not encode proteins (locus 1-2 and locus 5-6) and in genes coding for chitinase and for serine-rich
E. histolytica
protein. Thirty
E. histolytica
isolates from domestically infected Japanese amebiasis patients (male homosexuals and residents in institutions for the mentally handicapped) and four reference strains were examined. PCR revealed remarkable polymorphisms in both the number and size of the PCR fragments containing these loci. Polymorphisms in lengths, types, and numbers of internal repeat units were observed in locus 1-2 and the repeat-containing region of serine-rich
E. histolytica
protein among the Japanese isolates. In contrast, polymorphism at locus 5-6 was observed almost exclusively in the number of repeats of a 16-nucleotide unit. The repeat-containing region of chitinase appeared to be the least polymorphic among the four loci with a single dominant genotype representing 66% (20 out of 30) of all of the isolates. Isolates obtained from male homosexuals showed a more complex genetic polymorphism than those from residents in institutions. Considering all four polymorphic loci together, all 19 Japanese isolates from male homosexuals were distinct. In contrast, all isolates obtained from mass-infection cases at a single institution had an identical genotype, suggesting that these cases were caused by a single
E. histolytica
strain. No significant correlation was found between genotypes and zymodemes or between genotypes and clinical presentations, e.g., colitis or liver abscess. Certain genotypes were observed with higher frequencies in male homosexuals or residents of institutions. These data indicate that genotyping of the
E. histolytica
isolates by using these four polymorphic loci could serve as a tool to fingerprint individual isolates. We propose that genotyping of ameba isolates should help to determine geographic origins of isolates and routes of transmission.
Sensitive Detection of RNA Viruses Associated with Gastroenteritis by a Hanging-Drop Single-Tube Nested Reverse Transcription-PCR Method
Authors: Ratcliff Rodney Mark , Doherty James Christopher , Higgins Geoffrey David
Published: October 2002

The detection of the human RNA viruses, calicivirus and astrovirus, requires high sensitivity and broad reactivity. A novel single-tube nested reverse transcription-PCR (RT-PCR) method is described here, in which all of the required reagents are included in the one tube; however, those required for the nested amplification are separated in a “hanging drop” in the cap to be introduced by centrifugation after the RT and first-round cDNA amplification steps. Broad reactivity was obtained by using primer cocktails covering the published sequence variation in the primer targets. The method was evaluated with clinical fecal samples from outbreak and sporadic cases. Norwalk-like virus types 1 and 2 and rotavirus were the causal agents in 10 of 12 outbreaks. A viral agent was detected in 44% of 197 samples from sporadic infections in patients presenting to community health centers and a children's hospital. Interestingly, whereas rotavirus was more common than astrovirus in patients presenting to the hospital (33 and 7.6%, respectively), the reverse was true for patients presenting to community health centers (4.2 and 34%, respectively).
Estimation of the Rate of Unrecognized Cross-Contamination with
Mycobacterium tuberculosis
in London Microbiology Laboratories
Authors: Ruddy M. , McHugh T. D. , Dale J. W. , Banerjee D. , Maguire H. , Wilson P. , Drobniewski F. , Butcher P. , Gillespie S. H.
Published: October 2002

Isolates from patients with confirmed tuberculosis from London were collected over 2.5 years between 1995 and 1997. Restriction fragment length polymorphism (RFLP) analysis was performed by the international standard technique as part of a multicenter epidemiological study. A total of 2,779 samples representing 2,500 individual patients from 56 laboratories were examined. Analysis of these samples revealed a laboratory cross-contamination rate of between 0.54%, when only presumed cases of cross-contamination were considered, and 0.93%, when presumed and possible cases were counted. Previous studies suggest an extremely wide range of laboratory cross-contamination rates of between 0.1 and 65%. These data indicate that laboratory cross-contamination has not been a common problem in routine practice in the London area, but in several incidents patients did receive full courses of therapy that were probably unnecessary.
Human Cytomegalovirus DNA in Plasma and Serum Specimens of Renal Transplant Recipients Is Highly Fragmented
Authors: Boom René , Sol Cees J. A. , Schuurman Tim , van Breda Alex , Weel Jan F. L. , Beld Marcel , ten Berge Ineke J. M. , Wertheim-van Dillen Pauline M. E , de Jong Menno D.
Published: October 2002

Quantitation of cytomegalovirus (CMV) DNA in plasma and serum by PCR is increasingly used to identify patients at risk for developing CMV disease and to monitor the efficacy of antiviral therapy. Although CMV DNA levels are generally interpreted as viral loads, the exact nature of the viral DNA in these specimens is unknown. We studied the state of CMV DNA in plasma and serum specimens obtained from three renal transplant recipients at peak viral DNA levels during primary CMV infection. For this purpose, DNA isolated from these specimens was fractionated by size, and CMV DNA levels in the resulting DNA fractions were measured by quantitative PCR targeted at large (578-bp) and small (134-bp) amplicons. These experiments showed that the molecular sizes of DNA fragments from which CMV DNA is amplified were small (<2,000 bp), indicating that CMV DNA in plasma and serum is highly fragmented. Furthermore, CMV DNA levels were consistently higher when targeted at the smaller amplicon, providing additional evidence for the fragmentation of viral DNA. In conclusion, the first results with three patients have shown that CMV DNA in plasma and serum is highly fragmented and does not necessarily reflect the amount of infectious virus. These observations have potential consequences for understanding CMV pathogenesis and interpreting CMV DNA levels in individual patient management.
Incidence of Human Astrovirus in Central Australia (1995 to 1998) and Comparison of Deduced Serotypes Detected from 1981 to 1998
Authors: Schnagl Roger D. , Belfrage Kate , Farrington Rachel , Hutchinson Kylie , Lewis Victoria , Erlich John , Morey Fran
Published: October 2002

The incidence of astrovirus infection was determined among infants and young children admitted to Alice Springs Hospital (central Australia) with gastroenteritis from 1995 to 1998. Astrovirus was detected by reverse transcription-PCR in 33 of 495 stool samples, and this represented 4.3% of a total of 774 stool samples tested for astrovirus, rotavirus, and Norwalk-like viruses. Astrovirus incidence was substantially lower than that of rotavirus but higher than that of Norwalk-like viruses both overall and in each of the 4 years individually. Over the period from 1981 to 1998, including the period from 1981 to 1994 during which astrovirus was identified only by electron microscopy, astrovirus serotypes (deduced from genotypes) 1, 2, 3, and 4 were identified. Deduced serotypes 1, 3, and 4 all appeared regularly over this 18-year period. Also over this period, nucleotide variation (in some cases substantial) in a section of the capsid protein precursor region of the virus genome was evident among strains of all four of the deduced central Australian serotypes. Consequent amino acid changes were, however, only evident among deduced serotype 3 strains. Geographic variation at both the genome and the resultant amino acid levels was evident among strains of all four of the deduced central Australian serotypes and their respective prototypes isolated in the United Kingdom.