bluuebunny/crossref_metadata_2025 huggingface dataset:
Genotypes of Invasive Pneumococcal Isolates Recently Recovered from Italian Patients
Authors: Dicuonzo Giordano , Gherardi Giovanni , Gertz Robert E. , D'Ambrosio Fabio , Goglio Antonio , Lorino Giulia , Recchia Simona , Pantosti Annalisa , Beall Bernard
Published: September 2002

We examined 73 recent invasive pneumococcal isolates within selected areas of Italy for genotypic variability. Thirty-three genomic macrorestriction types were found, three of which represented multiple serotypes. Restriction fragment patterns of
pbp2b
,
pbp2x
, and
pspA
were conserved within the majority of isolates that shared macrorestriction types. Of the nine macrorestriction types found among the 22 penicillin-nonsusceptible
Streptococus pneumoniae
(PNSP) isolates, seven comprised isolates with allelic profiles showing five to seven allelic matches to profiles in the multilocus sequence typing database (
www.mlst.net
); however, three of the seven profiles represented serotypes not previously associated with these clonal clusters. Two PNSP macrorestriction types represented new clones with unique allelic profiles. Allelic profiles obtained from isolates of 3 of the 25 macrorestriction types found among the 51 penicillin-susceptible
S. pneumoniae
(PSSP) isolates were closely related to previously described profiles. One PSSP isolate was a novel type 24F isolate related to the multiresistant clone France
9V
-3. This work reports new PNSP strains and new serotype-clone associations.
Rapid and Sensitive Routine Detection of All Members of the Genus
Enterovirus
in Different Clinical Specimens by Real-Time PCR
Authors: Nijhuis Monique , van Maarseveen Noortje , Schuurman Rob , Verkuijlen Sandra , de Vos Machiel , Hendriksen Karin , van Loon Anton M.
Published: September 2002

We developed a rapid and sensitive method for the routine detection of all members of the enterovirus genus in different clinical specimens by using real-time TaqMan quantitative PCR. Multiple primer and probe sets were selected in the highly conserved 5′-untranslated region of the enterovirus genome. Our assay detected all 60 different enterovirus species tested, whereas no reactivity was observed with the viruses from the other genera of the picornaviridae family, e.g., hepatovirus and parechovirus. Weak cross-reactivity was observed with 7 of the 90 different high-titer rhinovirus stocks but not with rhinovirus-positive clinical isolates. Analysis of a well-characterized reference panel containing different enteroviruses at various concentrations demon-strated that the enterovirus real-time TaqMan PCR is as sensitive as most of the currently used molecular detection assays. Evaluation of clinical isolates demonstrated that the assay is more sensitive than the “gold standard” method, i.e., viral culture. Moreover, the PCR assay can be used on different clinical specimens, such as plasma, serum, nose and throat swabs, cerebrospinal fluid, and bronchoalveolar lavage, without apparent inhibition. Our data demonstrate that the real-time TaqMan PCR is a rapid and sensitive assay for the detection of enterovirus infection. The assay has a robust character and is easily standardized, which makes it an excellent alternative for the conventional time-consuming viral culture.
Development of a Multilocus Sequence Typing Scheme for the Pig Pathogen
Streptococcus suis
: Identification of Virulent Clones and Potential Capsular Serotype Exchange
Authors: King Samantha J. , Leigh James A. , Heath Peter J. , Luque Inmaculada , Tarradas Carmen , Dowson Christopher G. , Whatmore Adrian M.
Published: September 2002

Streptococcus suis
is an important pathogen of pigs and occasionally causes serious human disease. However, little is known about the
S. suis
population structure, the clonal relationships between strains, the potential of particular clones to cause disease, and the relevance of serotype as a marker for epidemiology. Here we describe a multilocus sequence typing (MLST) scheme for
S. suis
developed in order to begin to address these issues. Seven housekeeping gene fragments from each of 294
S. suis
isolates obtained from various
S. suis
diseases and from asymptomatic carriage representing 28 serotypes and nine distinct countries of origin were sequenced. Between 32 and 46 alleles per locus were identified, giving the ability to distinguish >1.6 × 10
11
sequence types (STs). However only 92 STs were identified in this study. Of the 92 STs 18 contained multiple isolates, the most common of which, ST1, was identified on 141 occasions from six countries. Assignment of the STs to lineages resulted in 37 being identified as unique and unrelated STs while the remaining 55 were assigned to 10 complexes. ST complexes ST1, ST27, and ST87 dominate the population; while the ST1 complex was strongly associated with isolates from septicemia, meningitis, and arthritis, the ST87 and ST27 complexes were found to contain significantly higher numbers of lung isolates. In agreement with the observed distribution of disease-causing isolates of
S. suis
, most isolates previously characterized as of high virulence in porcine infection models belong to ST1, while isolates belonging to other STs appear to be less virulent in general. Finally nine STs were found to contain isolates of multiple serotypes, and many isolates belonging to the same serotypes were found to have very disparate genetic backgrounds. As well as highlighting that the serotype can often be a poor indicator of genetic relatedness between
S. suis
isolates, these findings suggest that capsular genes may be moving horizontally through the
S. suis
population.
Improved Detection by DNA Amplification of
Trichomonas vaginalis
in Males
Authors: Schwebke Jane R. , Lawing Lisa F.
Published: September 2002

Trichomoniasis is a sexually transmitted infection that is highly prevalent worldwide and has been linked to preterm birth and human immunodeficiency virus acquisition. In females, trichomoniasis causes vaginitis, while in males, it is frequently asymptomatic but can be a cause of urethritis. Control efforts have been hampered by the lack of a sensitive diagnostic technique for this infection in males. Men attending a sexually transmitted disease (STD) clinic for a new complaint were screened for
Trichomonas vaginalis
by culture and by PCR analysis of urine and urethral-swab specimens. The prevalence of
Trichomonas
determined by culture was 5% (15 of 300 specimens), compared to 17% (52 of 300) determined by PCR. Urine specimens yielded a greater number of positive results by PCR than did urethral-swab specimens. The sensitivity of PCR analysis of urine specimens in comparison to that of culture was 100%. The use of PCR techniques in urine specimen-based detection of
T. vaginalis
was highly sensitive and revealed a prevalence of infection more than three times that revealed by culture for men at high risk for STDs.
Analysis of Intratypic Variation Evident in an Ibaraki Virus Strain and Its Epizootic Hemorrhagic Disease Virus Serogroup
Authors: Ohashi Seiichi , Yoshida Kazuo , Yanase Tohru , Tsuda Tomoyuki
Published: September 2002

A new strain of Ibaraki virus (IBAV) was isolated from cattle showing atypical symptoms of Ibaraki disease. The isolate was genetically characterized, and the genetic diversity and evolution of the capsid proteins of viruses in the epizootic hemorrhagic disease virus (EHDV) serogroup were investigated. The nucleotide sequences of the isolate's viral RNA segments 2, 3, 6, and 7, which encode the viral structural proteins VP2, VP3, VP5, and VP7, respectively, were determined and were then compared against those of the existing strains of IBAV and EHDV, to which IBAV belongs serologically. The nucleotide sequences of segments 3 and 7 were conserved within the EHDV serogroup, particularly well among the strains of IBAV and Australian EHDV. The similarity of the sequence of segment 6 of the isolate to sequences of corresponding segments of the other strains of IBAV and EHDV was found to be about 93%. The similarity of segment 2 of the isolate to segments 2 of the other strains of IBAV and EHDV was less than 70%. Phylogenetic analysis based on the deduced amino acid sequences of segments 3 and 7 revealed that the viruses differed according to their geographical distributions. However, the new isolate of IBAV was categorized as having a distinct lineage in the phylogenetic tree of VP2. These results suggest that the isolate was modified by a reassortment of segment 2 and that it exhibits unique genetic and antigenic characteristics.
Evaluation of a Cytomegalovirus Glycoprotein B Recombinant Enzyme Immunoassay To Discriminate between a Recent and a Past Infection
Authors: Sipewa M. J. , Goubau P. , Bodéus M.
Published: September 2002

Fetal damage following cytomegalovirus (CMV) intrauterine infection is mostly linked to primary infection. To differentiate primary infection from nonprimary infection, immunoglobulin M (IgM) tests are not reliable enough, and measurement of the IgG avidity appears to be the method that is the most widely used at present. In the present study the performance of the Vidas (bioMérieux) avidity assay was compared with that of a new enzyme immunoassay based on the use of a recombinant CMV glycoprotein B protein (Biotest).
Development of a Rapid Immunodiagnostic Test for
Haemophilus ducreyi
Authors: Patterson Kristine , Olsen Bonnie , Thomas Christopher , Norn Dora , Tam Milton , Elkins Christopher
Published: September 2002

Haemophilus ducreyi
is the etiologic agent of chancroid, a sexually transmitted disease that increases the rate of transmission of human immunodeficiency virus. Chancroid ulcerations are difficult to distinguish from those produced by syphilis and herpes. Diagnosis based solely on clinical grounds is inaccurate, and culture is insensitive. Highly sensitive PCR has largely superseded culture as the preferred method of laboratory diagnosis; however, neither culture nor PCR is feasible where chancroid is endemic. We developed a rapid (15-min) diagnostic test based on monoclonal antibodies (MAbs) to the hemoglobin receptor of
H. ducreyi
, HgbA. This outer membrane protein is conserved in all strains of
H. ducreyi
tested and is required for the establishment of experimental human infection. MAbs to HgbA were generated and tested for cross-reactivity against a panel of geographically diverse strains. Three MAbs were found to be unique and noncompetitive and bound to all strains of
H. ducreyi
tested. Using an immunochromatography format, we evaluated the sensitivity and specificity of the test using geographically diverse strains of
H. ducreyi
, other
Haemophilus
strains, and other bacteria known to superinfect genital ulcers. All
H. ducreyi
strains were positive, and all other bacteria were negative, resulting in a specificity of 100%. The minimum number of CFU of
H. ducreyi
detected was 2 × 10
6
CFU, and the minimum amount of purified HgbA protein detected was 8.5 ng. Although this level of sensitivity may not be sufficient to detect
H. ducreyi
in all clinical specimens, further work to increase the sensitivity could potentially make this a valuable bedside tool in areas where chancroid is endemic.
Evaluation of the Etest ESBL and the BD Phoenix, VITEK 1, and VITEK 2 Automated Instruments for Detection of Extended-Spectrum Beta-Lactamases in Multiresistant
Escherichia coli
and
Klebsiella
spp
Authors: Leverstein-van Hall Maurine A. , Fluit Ad C. , Paauw Armand , Box Adrienne T. A. , Brisse Sylvain , Verhoef Jan
Published: September 2002

Seventy-four isolates of multiresistant
Escherichia coli
and
Klebsiella
spp. recovered during a 3-year period and 17 control strains with genotypically identified beta-lactamases were tested for the production of extended-spectrum beta-lactamases (ESBLs) by using the Etest and the VITEK 1, VITEK 2, and Phoenix automated instruments. The use of the Etest was evaluated by investigating its accuracy in detecting the ESBLs of the control strains and by comparing interpretation results of laboratory technicians and experts. The accuracy of the Etest was 94%. With the Etest as the reference for the clinical strains and the genotype as the reference for the control strains, the automated instruments detected the ESBLs with accuracies of 78% (VITEK 2), 83% (VITEK 1), and 89% (Phoenix). No significant difference between the systems with regard to the control strains was detected. The VITEK 2 did, however, perform less well than the Phoenix (
P
= 0.03) on the collection of clinical isolates, mainly because of its high percentage of indeterminate test results (11%). No significant difference between the performances of the VITEK 1 and either the VITEK 2 or the Phoenix was found. However, because of its associated BDXpert system the Phoenix showed the best performance. The Etest was found to be an accurate test but was limited by its indeterminate results (4%), its inability to differentiate between K1 hyperproduction and ESBLs, questionable guidelines concerning mutants inside the inhibition zones, and the inability of the technicians to recognize subtle zone deformations.
IS
1311
and IS
1245
Restriction Fragment Length Polymorphism Analyses, Serotypes, and Drug Susceptibilities of
Mycobacterium avium
Complex Isolates Obtained from a Human Immunodeficiency Virus-Negative Patient
Authors: Dvorska Lenka , Bartos Milan , Ostadal Oldrich , Kaustova Jarmila , Matlova Ludmila , Pavlik Ivo
Published: September 2002

Six isolates of
Mycobacterium avium
of genotype
dnaJ
+
IS
901
−
IS
1311
+
IS
1245
+
and serotypes 6 (
n
= 1), 6/9, (
n
= 2), and 9 (
n
= 3) were obtained within a 5-month period from a human immunodeficiency virus-negative patient treated for tuberculosis. The isolates were identified with
Pvu
II restriction fragment length polymorphism (RFLP) analysis as a single IS
1311
RFLP type and six different IS
1245
RFLP types. Six separate colonies/clones obtained by subculture from each of the six isolates were tested for MICs of a set of 10 drugs. This report documents the appearance of isolates that are resistant to antimycobacterial drugs as the duration of therapy increases. Because isolates recovered from the patient following longer duration of treatment were more likely to be resistant to more antimycobacterial drugs, we would conclude that there was selection for antimycobacterial drug-resistant isolates. Analyses of all 36 clones identified three IS
1311
and 22 IS
1245
types forming three clusters. Tests of 105 environmental samples collected in the home and the work place of the patient yielded 16 mycobacterial isolates, of which one
M. avium
from soil was of genotype
dnaJ
+
IS
901
+
IS
1311
+
IS
1245
+
and serotype 2, and the second
M. avium
from a vacuum cleaner was of genotype
dnaJ
+
IS
901
−
IS
1311
+
IS
1245
+
and serotype 9. Overall analyses of the results did not reveal any relation between serotype, RFLP type, and drug susceptibility. Based on the course of the disease in the patient and different serotypes, IS
1311
and IS
1245
RFLP types of isolates of
M. avium
we suppose represent polyclonal infection.
Real-Time Quantitative PCR for Detection ofHelicobacter pylori
Authors: He Qiang , Wang Jian-Ping , Osato Michael , Lachman Lawrence B.
Published: September 2002

Helicobacter pyloriis one of the most common chronic infections in humans, in whom it is a key etiological factor in peptic ulcer disease, gastric mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma. Humans are the bacterium's only host. Here we report the development of a real-time quantitative (Q) PCR-based assay to measureureCgene copy number to detectH. pylori,based on the fact that there is only one copy of theureCgene per bacterium. Upon optimization of LightCycler Q-PCR conditions, we obtained a standard curve with a linear range (correlation coefficient = 1) across six logs of DNA concentration. We were able to accurately quantify as few as 1,000 bacteria in our assay. Analysis of variance on 15 randomly selected clinical samples showed good reproducibility of this assay. Comparison of Q-PCR results with bacterial culture and histopathological results from an additional 85 clinical biopsy samples showed a significant difference for the presence ofH. pylori. Many samples that were negative forH. pyloriby culture and histopathology were positive by Q-PCR. Contamination of PCR byH. pyloriorH. pylorigenetic material could not be ruled out. In summary, we developed a rapid, sensitive, and real-time Q-PCR method for detectingH. pylori. This technique offers a significant improvement over other available methods for detectingH. pyloriin clinical and research samples.
Monitoring Drug Resistance in Chronic Hepatitis B Virus (HBV)-Infected Patients during Lamivudine Therapy: Evaluation of Performance of INNO-LiPA HBV DR Assay
Authors: Lok Anna S. F. , Zoulim Fabien , Locarnini Stephen , Mangia Alessandra , Niro Grazia , Decraemer Hilde , Maertens Geert , Hulstaert Frank , De Vreese Karen , Sablon Erwin
Published: September 2002

Sensitive and early detection of emerging hepatitis B virus (HBV) drug resistance may not only help monitor the viral dynamics associated with lamivudine treatment but could also improve therapeutic decision making. This is especially important when new antivirals effective against lamivudine-resistant HBV become available. A total of 159 serum samples from 33 chronic HBV patients receiving lamivudine treatment were analyzed at four centers for the presence of lamivudine-resistant mutations at codons 528 [180] (proposed revised nomenclature according to Stuyver et al. [Hepatology33:751-757, 2001] shown in brackets), 552 [204], and 555 [207] of the HBV polymerase. Sequencing data were compared with results generated by the INNO-LiPA HBV DR line probe assay (LiPA), an assay based on reverse hybridization of amplified HBV DNA fragments with specific nucleotide probes immobilized on nitrocellulose strips. LiPA provided at least the same information as sequencing for 97.5% of all codons analyzed for codon 528 [180], 95% for codon 552 [204], and 100% for codon 555 [207]. The most common reason for discrepant or indeterminate results (0.4% and 1.5%, respectively) in a small percentage of the population tested could be attributed to polymorphisms not yet covered by LiPA probes. In at least five patients, a mutant could be detected earlier by LiPA than by sequencing. In 15 patients, LiPA detected mixed wild-type and mutant virus populations before viral breakthrough. These results demonstrate that INNO-LiPA HBV DR is a highly sensitive and easily applicable assay for the detection and monitoring of lamivudine-resistant mutations in chronic hepatitis B patients and that the assay is more sensitive than sequencing in detecting mixed mutant and wild-type sequences.
Trypanosoma cruziRecombinant Complement Regulatory Protein: a Novel Antigen for Use in an Enzyme-Linked Immunosorbent Assay for Diagnosis of Chagas' Disease
Authors: Meira Wendell S. F. , Galvão Lúcia M. C. , Gontijo Eliane D. , Machado-Coelho George L. L. , Norris Karen A. , Chiari Egler
Published: September 2002

Currently, diagnosis of Chagas' disease is based on serological methods, but due to the high occurrence of inconclusive results, more reliable methods are needed. The use of recombinant antigens for serodiagnosis of Chagas' disease is recommended in order to increase the sensitivity and specificity of the serological tests. TheTrypanosoma cruzicomplement regulatory protein (CRP) is a surface glycoprotein present on the trypomastigote forms of the parasite, and the recombinant CRP (rCRP) was cloned in a mammalian expression system and purified by affinity chromatography. The purified recombinant protein was used as an antigen in an enzyme-linked immunosorbent assay (rCRP ELISA) in order to verify its sensitivity and specificity compared with other established methods. In this evaluation, a panel of 184 serum samples distributed among chronic chagasic patients (n= 65), blood bank donors (n= 100), and patients infected withLeishmaniaspp. (n= 19) was used. The sensitivity and specificity of the rCRP ELISA were 100% when compared to conventional serology and complement-mediated lysis tests from these groups. When hemoculture and PCR tests were evaluated for diagnosis of chronic chagasic patients, using the rCRP ELISA as a reference test, the positivities were found to be 64.62 and 81.54%, respectively, showing a higher degree of sensitivity of the test. The data demonstrate that rCRP ELISA was able to discriminate between chronic chagasic patients and nonchagasic individuals, such as blood donors and patients with leishmaniasis. Thus, the rCRP is an excellent antigen for use in Chagas' disease diagnosis, due to the absence of false-negative or false-positive results.
Molecular Epidemiology of
Neisseria gonorrhoeae
: Sequence Analysis of the
porB
Gene Confirms Presence of Two Circulating Strains
Authors: Unemo Magnus , Olcén Per , Berglund Torsten , Albert Jan , Fredlund Hans
Published: September 2002

The phenotypic and genotypic characteristics of
Neisseria gonorrhoeae
strains fluctuate over time both locally and globally, and highly discriminative and precise characterization of the strains is essential. Conventional characterization of
N. gonorrhoeae
strains for epidemiological purposes is mostly based on phenotypic methods, which have some inherent limitations. In the present study sequence analysis of
porB1b
gene sequences was used for examination of the genetic relationships among
N. gonorrhoeae
strains. Substantial genetic heterogeneity was identified in the
porB
genes of serovar IB-2 isolates (8.1% of the nucleotide sites were polymorphic) and serovar IB-3 isolates (5.2% of the nucleotide sites were polymorphic) as well as between isolates of different serovars. The highest degree of diversity was identified in the gene segments encoding the surface-exposed loops of the mature PorB protein. Phylogenetic analysis of the
porB1b
gene sequences confirmed previous findings that have indicated the circulation of one
N. gonorrhoeae
strain each of serovar IB-2 and serovar IB-3 in the Swedish community. These strains caused the majority of the cases in two domestic core groups comprising homosexual men and young heterosexuals, respectively, and were also detected in other patients. The phylogenetic analyses of
porB
gene sequences in the present study showed congruence, but not complete identity, with previous results obtained by pulsed-field gel electrophoresis of the same isolates. In conclusion,
porB
gene sequencing can be used as a molecular epidemiological tool for examination of genetic relationships among emerging and circulating
N. gonorrhoeae
strains, as well as for confirmation or discrimination of clusters of gonorrhea cases.
Ability of the MicroScan Rapid Gram-Negative ID Type 3 Panel To Identify Nonenteric Glucose-Fermenting and Nonfermenting Gram-Negative Bacilli
Authors: O'Hara Caroline M. , Miller J. Michael
Published: September 2002

The MicroScan Rapid Neg ID3 panel is designed for the identification of
Enterobacteriaceae
and nonenteric glucose-fermenting and nonfermenting gram-negative bacilli. We evaluated this panel for its ability to identify gram-negative non-
Enterobacteriaceae
bacteria. A total of 134 strains, representing 26 genera and 42 species, were taken from storage at −70
o
C, passaged three times before testing, and inoculated into the panels according to the manufacturer's directions before being inserted into a Walk/Away 96 instrument loaded with version 22.28 software. At the end of the initial 2.5-h incubation period, 89 isolates (66.4%) were correctly identified at a probability level of ≥85%. After additional testing recommended by the manufacturer was completed, another 11 isolates (8.2%) were correctly identified at probability levels of ≥85%. Twenty-five (18.7%) isolates were correctly identified after additional testing, but the probability levels were less than 85%. Two isolates were unidentified, and seven (5.2%) were incorrectly identified. The seven misidentified strains were not concentrated in any one genus. With an accuracy approaching 75%, this product may be used for the identification of the commonly isolated non-
Enterobacteriaceae
bacteria but may present problems in identification of other non-glucose-fermenting gram-negative bacilli.
Multicenter Evaluation of a Nonweekend Reading Schedule for Radiometric Pyrazinamide Susceptibility Testing of
Mycobacterium tuberculosis
Authors: Madison B. , Gross W. , George I. , Sloutsky A. , Washabaugh G. , Robinson-Dunn B. , Lipman H. , Metchock B. , Mazurek G. , Ridderhof J.
Published: September 2002

Pyrazinamide (PZA) is an integral component of the short-course chemotherapy regimen for tuberculosis. The BACTEC 460TB PZA susceptibility test for
Mycobacterium tuberculosis
with a daily (D) reading schedule has been available for more than 10 years, but weekend laboratory staffing is necessary. A nonweekend (NW) reading schedule has not been validated in a multicenter study. This prospective multicenter study compares the interlaboratory reproducibility of PZA susceptibility results by following both the D and NW schedules. A total of 181 cultures were shared among four laboratories. Isolates were selected based on resistance or borderline resistance to at least one streptomycin-isoniazid-rifampin-ethambutol drug or PZA. One laboratory used a D reading schedule, and three laboratories used a NW schedule. Both reading schedules are based on the standard BACTEC 460TB PZA protocol. With the NW schedule, the growth index (GI) is not available for test interpretation on Saturday, Sunday, and Monday. Of the 181 shared cultures, 154 were found to be susceptible by all laboratories, 19 were found to be resistant, and 8 had discordant results. The overall pairwise interlaboratory agreement was 97.7%. The discrepancies were not associated with the type of reading schedule used. However, the median control GI was significantly higher for the NW schedule (321) than for the D schedule (259) (
P
< 0.0001) although results were available on average in about 7 days from setup for both schedules. These results show that the NW schedule is a suitable alternative for laboratories that do not read and interpret PZA susceptibility tests on weekends.
Head-to-Head Multicenter Comparison of DNA Probe and Nucleic Acid Amplification Tests for
Chlamydia trachomatis
Infection in Women Performed with an Improved Reference Standard
Authors: Black Carolyn M. , Marrazzo Jeanne , Johnson Robert E. , Hook Edward W. , Jones Robert B. , Green Timothy A. , Schachter Julius , Stamm Walter E. , Bolan Gail , St. Louis Michael E. , Martin David H.
Published: September 2002

Few evaluations of tests for
Chlamydia trachomatis
have compared nucleic acid amplification tests (NAATs) with diagnostic tests other than those by culture. In a five-city study of 3,551 women, we compared the results of commercial ligase chain reaction (LCR) and PCR tests performed on cervical swabs and urine with the results of PACE 2 tests performed on cervical swabs, using independent reference standards that included both cervical swabs and urethral swab-urine specimens. Using cervical culture as a standard, the sensitivities of PACE 2, LCR, and PCR tests with cervical specimens were 78.1, 96.9, and 89.9%, respectively, and the specificities were 99.3, 97.5, and 98.2%, respectively. Using either cervical swab or urine LCR-positive tests as the standard decreased sensitivities to 60.8% for PACE 2 and to 75.8 and 74.9% for PCR with cervical swabs and urine, respectively. Specificities increased to 99.7% for PACE 2 and to 99.7 and 99.4% for PCR with cervical swabs and urine, respectively. Sensitivities with a cervical swab-urine PCR standard were 61.9% for PACE 2 and 85.5 and 80.8% for LCR with cervical swabs and urine, respectively. Specificities were 99.6% for PACE 2 and 99.0 and 98.9% for LCR with cervical swabs and urine, respectively. Cervical swab versus urine differences were significant only for PCR specificities (
P
= 0.034). Overall, LCR sensitivity exceeded that of PCR, and sensitivities obtained with cervical swabs exceeded those obtained with urine specimens by small amounts. These data have substantiated, using a large multicenter sample and a patient standard, that LCR and PCR tests performed on endocervical swabs and urine are superior to PACE 2 tests for screening
C. trachomatis
infections in women. In our study, NAATs improved the detection of infected women by 17 to 38% compared to PACE 2.
Comparison of Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis as Tools for Typing
Staphylococcus aureus
Isolates in a Microepidemiological Setting
Authors: Peacock S. J. , de Silva G. D. I. , Justice A. , Cowland A. , Moore C. E. , Winearls C. G. , Day N. P. J.
Published: September 2002

Multilocus sequence typing (MLST) of
Staphylococcus aureus
is well suited to the study of global or long-term epidemiology, but its role in local epidemiology has not been defined. The present study has compared MLST with pulsed-field gel electrophoresis (PFGE) by using
S. aureus
isolates associated with carriage and disease in a busy regional renal unit. One hundred forty-four patients were prospectively recruited, of whom 103 were receiving hemodialysis and 41 were on continuous ambulatory peritoneal dialysis. Three nasal swab specimens were obtained 1 month apart on entering the study. A nasal swab was positive for
S. aureus
on at least one occasion in 50 patients (35%). Typing of the 104 carriage isolates demonstrated 21 PFGE types and 21 sequence types (STs). Thirty-one carriers had two or more positive nasal swabs; of these, the isolates in all swabs from a given carrier had identical PFGE types for 29 carriers; the isolates in all of the same 29 swabs had identical STs. The carriage strain in two patients changed both PFGE type and STs during the period of swabbing. Eight patients (6%) had an episode of
S. aureus
bacteremia during the 12-month study period, and two of these were nasal carriers. One of these invasive isolates had the same PFGE type and ST as the carriage isolate. There were no differences between Simpson's index of diversity for PFGE and Simpson's index of diversity for MLST for both invasive and carriage isolates, suggesting that the two methods have very similar discriminatory abilities. We conclude that PFGE and MLST performed equally in this study.
Evaluation of an Enzyme-Linked Immunosorbent Assay with Recombinant Rhoptry-Associated Protein 1 Antigen against
Babesia bovis
for the Detection of Specific Antibodies in Cattle
Authors: Boonchit Suthisak , Xuan Xuenan , Yokoyama Naoaki , Goff Will L. , Wagner Gale , Igarashi Ikuo
Published: September 2002

The gene encoding
Babesia bovis
rhoptry-associated protein 1 (RAP-1) was used to develop an enzyme-linked immunosorbent assay (ELISA) to measure specific antibodies against
B. bovis.
The
B. bovis
RAP-1 gene was subcloned into a baculovirus transfer vector, and the RAP-1 protein was expressed in insect cells infected with a recombinant baculovirus. The recombinant
B. bovis
RAP-1 of 65 kDa was detected with anti-RAP-1 mouse serum by Western blotting, and this recombinant RAP-1 was used as an antigen in the ELISA. The ELISA was able to differentiate between
B. bovis
-infected sera and
B. bigemina
-infected sera or noninfected normal bovine sera. The results demonstrate that the recombinant RAP-1 expressed in insect cells might be a useful antigen for the detection of antibodies to
B. bovis.
Optimal Testing Conditions for Determining MICs and Minimum Fungicidal Concentrations of New and Established Antifungal Agents for Uncommon Molds: NCCLS Collaborative Study
Authors: Espinel-Ingroff A. , Chaturvedi V. , Fothergill A. , Rinaldi M. G.
Published: September 2002

This collaborative three-center study evaluated NCCLS M38-A document testing conditions and other testing conditions for the antifungal susceptibility testing of 35 isolates of
Aspergillus nidulans
,
A. terreus
,
Bipolaris hawaiiensis
,
B. spicifera
,
Cladophialophora bantiana
,
Dactylaria constricta
,
Fusarium solani
,
Paecilomyces lilacinus
,
Scedosporium prolificans
,
Trichoderma longibrachiatum
, and
Wangiella dermatitidis
for itraconazole, three new triazoles (voriconazole, posaconazole, and ravuconazole), and amphotericin B. MICs and minimum fungicidal concentrations (MFCs) were determined in each center by using four media (standard RPMI-1640 [RPMI], RPMI with 2% dextrose [RPMI-2%], antibiotic medium 3 [M3], and M3 with 2% dextrose [M3-2%]) and two criteria of MIC determination (complete growth inhibition [MICs-0] and prominent growth inhibition [MICs-2]) at 24, 48 and 72 h. MFCs were defined as the lowest drug concentrations that yielded <3 colonies (approximately 99 to 99.5% killing activity). The reproducibility (within three wells) was higher among MICs-0 (93 to 99%) with either RPMI or M3 media than among all MICs-2 (86 to 95%) for the five agents at 48 to 72 h. The agreement for MFCs was lower (86 to 94%). Based on interlaboratory agreement, the optimal testing conditions were RPMI broth, 48 to 72 h of incubation and 100% growth inhibition (MIC-0); MFCs can be obtained after MIC determination with the above optimal testing parameters. These results warrant consideration for inclusion in the future version of the NCCLS M38 document. However, the role of these in vitro values as predictors of clinical outcome remains to be established in clinical trials.
Serodiagnosis of Tuberculosis: Comparison of Immunoglobulin A (IgA) Response to Sulfolipid I with IgG and IgM Responses to 2,3-Diacyltrehalose, 2,3,6-Triacyltrehalose, and Cord Factor Antigens
Authors: Julián Esther , Matas Lurdes , Pérez Andrés , Alcaide José , Lanéelle Marie-Antoinette , Luquin Marina
Published: September 2002

Nonpeptidic antigens from the
Mycobacterium tuberculosis
cell wall are the focus of extensive studies to determine their potential role as protective antigens or serological markers of tuberculous disease. Regarding this latter role and using an enzyme-linked immunosorbent assay, we have made a comparative study of the immunoglobulin G (IgG), IgM, and IgA antibody responses to four trehalose-containing glycolipids purified from
M. tuberculosis
: diacyltrehaloses, triacyltrehaloses, cord factor, and sulfolipid I (SL-I). Sera from 92 tuberculosis patients (taken before starting antituberculosis treatment) and a wide group of control individuals (84 sera from healthy donors, including purified protein derivative-negative, -positive, healed, and vaccinated individuals, and 52 sera from nontuberculous pneumonia patients), all from Spain, were studied. The results indicated a significantly elevated IgG and IgA antibody response in tuberculosis patients, compared with controls, with all the antigens used. SL-I was the best antigen studied, showing test sensitivities and specificities for IgG of 81 and 77.6%, respectively, and of 66 and 87.5% for IgA. Using this antigen and combining IgA and IgG antibody detection, high test specificity was achieved (93.7%) with a sensitivity of 67.5%. Currently, it is widely accepted that it is not possible to achieve sensitivities above 80% in tuberculosis serodiagnosis when using one antigen alone. Thus, we conclude that SL-I, in combination with other antigenic molecules, could be a useful antigen for tuberculosis serodiagnosis.
Mutations in the
rpoB
and
katG
Genes Leading to Drug Resistance in
Mycobacterium tuberculosis
in Latvia
Authors: Tracevska T. , Jansone I. , Broka L. , Marga O. , Baumanis V.
Published: September 2002

To characterize the genetic basis of drug resistance in
Mycobacterium tuberculosis
in Latvia, mutations involved in rifampin (
rpoB
gene) and isoniazid (
katG
gene) resistance in DNA from 19 drug-susceptible and 51 multidrug-resistant
M. tuberculosis
complex isolates were analyzed. The most frequent
rpoB
gene mutations found by the Line Probe assay were the S531L (14 of 34 isolates), D516V (7 of 34), H526D (4 of 34), and D516Y plus P535S (4 of 34) mutations. Direct sequencing of seven isolates with unclear results from Line Probe assay showed the presence of the L533P mutation and the Q510H plus H526Y (1 of 34) and D516V plus P535S (4 of 34) double mutations, neither of which has been described previously. Single-strand conformation polymorphism analysis showed strand mobility differences between the rifampin-susceptible and -resistant samples for the D516V, H526D, and D516Y plus P535S mutations but not for the S531L mutation. Nucleotide substitution at codon 315 (AGC→ACC) of the
katG
gene was found in 48 of 51 multidrug-resistant samples by sequencing. Furthermore,
katG
gene restriction fragment length polymorphism analysis with endonuclease
Aci
I confirmed the nucleotide change in codon 315.
Use of 16S rRNA Gene Sequencing for Identification of Nonfermenting Gram-Negative Bacilli Recovered from Patients Attending a Single Cystic Fibrosis Center
Authors: Ferroni Agnes , Sermet-Gaudelus Isabelle , Abachin Eric , Quesne Gilles , Lenoir Gerard , Berche Patrick , Gaillard Jean-Louis
Published: September 2002

During 1999, we used partial 16S rRNA gene sequencing for the prospective identification of atypical nonfermenting gram-negative bacilli isolated from patients attending our cystic fibrosis center. Of 1,093 isolates of nonfermenting gram-negative bacilli recovered from 148 patients, 46 (4.2%) gave problematic results with conventional phenotypic tests. These 46 isolates were genotypically identified as
Pseudomonas aeruginosa
(19 isolates, 12 patients),
Achromobacter xylosoxidans
(10 isolates, 8 patients),
Stenotrophomonas maltophilia
(9 isolates, 9 patients),
Burkholderia cepacia
genomovar I/III (3 isolates, 3 patients),
Burkholderia vietnamiensis
(1 isolate),
Burkholderia gladioli
(1 isolate), and
Ralstonia mannitolilytica
(3 isolates, 2 patients), a recently recognized species.
Imipenem-EDTA Disk Method for Differentiation of Metallo-β-Lactamase-Producing Clinical Isolates of
Pseudomonas
spp. and
Acinetobacter
spp
Authors: Yong Dongeun , Lee Kyungwon , Yum Jong Hwa , Shin Hee Bong , Rossolini Gian Maria , Chong Yunsop
Published: September 2002

Rapid detection of metallo-β-lactamase (MBL)-producing gram-negative bacilli is necessary to prevent their dissemination. The method using a disk with imipenem plus 750 μg of EDTA differentiated all MBL-producing pseudomonads, and the sensitivity and specificity for acinetobacters were 95.7 and 91.0%, respectively. The imipenem-EDTA disks were stable for 12 and 16 weeks at 4 and −20°C, respectively.
PCR Detection of
Borrelia burgdorferi
Sensu Lato, Tick-Borne Encephalitis Virus, and the Human Granulocytic Ehrlichiosis Agent in
Ixodes persulcatus
Ticks from Western Siberia, Russia
Authors: Morozova Olga V. , Dobrotvorsky Andrey K. , Livanova Natalya N. , Tkachev Sergey E. , Bakhvalova Valentina N. , Beklemishev Anatoly B. , Cabello Felipe C.
Published: September 2002

PCR assays were used to test adult
Ixodes persulcatus
ticks from Western Siberia, Russia, for
Borrelia burgdorferi
sensu lato, tick-borne encephalitis virus (TBEV), and the human granulocytic ehrlichiosis (HGE) agent. Of the 150 ticks that were studied, 38% were infected with
B. burgdorferi
, 46% were infected with TBEV, and 8% were infected with the HGE agent. These three pathogens were distributed in the ticks independently of one another.
Rapid Detection of Human Immunodeficiency Virus Type 1 Subtype E Infection by PCR
Authors: Chen Mao-Yuan , Wang Wei-Kung , Lee Ming-Cheng , Twu Shing-Jer , Wu Shiow-Ing , Lee Chun-Nan
Published: September 2002

The CRF01_AE (subtype E) strain of human immunodeficiency virus type 1 (HIV-1), originally reported in Thailand, spread rapidly to and showed prevalence in several countries in Southeast Asia, including Taiwan. This strain was also found in other regions of the world. Based on sequence analysis of the
vpu
gene, a nested PCR assay including an outer primer pair and a subtype E-specific inner primer pair was developed in this study for rapid detection of subtype E viruses. It was tested with 397 HIV-1-positive samples of known subtypes. For these samples, the sensitivity of detection of subtype E viruses was 100% (127 of 127), and the specificity was 97.8% (264 of 270). Although six samples of either subtype A or G showed a positive PCR, most of the cross-reactivity could be reduced by raising the annealing temperature from 54°C to 63°C. When tested with 195 HIV-positive samples of unknown subtypes, the assay had a sensitivity of 98.0% and a specificity of 98.6%. This is a simple, convenient, and sensitive method for rapid detection of subtype E viruses, especially in regions in which viruses of subtypes B and E are predominant.
Detection of
embB306
Mutations in Ethambutol-Susceptible Clinical Isolates of
Mycobacterium tuberculosis
from Northwestern Russia: Implications for Genotypic Resistance Testing
Authors: Mokrousov Igor , Otten Tatiana , Vyshnevskiy Boris , Narvskaya Olga
Published: September 2002

A total of 183 epidemiologically unlinked
Mycobacterium tuberculosis
isolates collected in the St. Petersburg area of Russia from 1996 to 2001 were screened for alterations in codon 306 of the
embB
gene; mutations in this codon are reported to confer resistance to ethambutol (EMB). The
embB306
mutations were detected in 14 (48.3%) of 29 EMB-resistant strains and, quite surprisingly, in 48 (31.2%) of 154 EMB-susceptible strains. A discrepancy between the results of phenotypic and genotypic EMB resistance tests was restricted to the strains already resistant to other antitubercular (anti-TB) drugs. In particular, 40 (60%) of the 69 EMB-susceptible strains resistant to rifampin, isoniazid, and streptomycin but none of the 43 pansusceptible strains harbored an
embB306
mutation. We hypothesize that the phenomenon observed could reflect the presence of a target other than EmbB for the drug in tubercle bacilli; this unknown target could be sensitized and affected, sensu lato, by EMB during treatment with other first-line anti-TB drugs. Comparison with DNA fingerprinting data showed that, irrespectively of the phenotypic susceptibility profiles, 46 (50.6%) of 91 Beijing family strains and 16 (17.4%) of 92 strains of other genotypes had a mutation in
embB306
.
Direct Detection and Differentiation of
Legionella
spp. and
Legionella pneumophila
in Clinical Specimens by Dual-Color Real-Time PCR and Melting Curve Analysis
Authors: Reischl Udo , Linde Hans-Jörg , Lehn Norbert , Landt Olfert , Barratt Kevin , Wellinghausen Nele
Published: September 2002

A dual-color LightCycler PCR assay targeting the 16S rDNA gene of
Legionella
spp. was established. By using two pairs of hybridization probes,
Legionella
spp. and
Legionella pneumophila
could be detected and differentiated simultaneously. With 26 culture-positive and 42 culture-negative respiratory specimens from patients with atypical pneumonia, 100% sensitivity and specificity was observed for
L. pneumophila
.
DNA Differential Diagnosis of Human Taeniid Cestodes by Base Excision Sequence Scanning Thymine-Base Reader Analysis with Mitochondrial Genes
Authors: Yamasaki Hiroshi , Nakao Minoru , Sako Yasuhito , Nakaya Kazuhiro , Sato Marcello Otake , Mamuti Wulamu , Okamoto Munehiro , Ito Akira
Published: September 2002

For DNA differential diagnosis of human
Taenia
cestodes, a base excision sequence scanning thymine-base method using the cytochrome
c
oxidase subunit I and cytochrome
b
genes as targets was used. The characteristic thymine-base peak profiles provide four distinct types, unique for
T. saginata
,
T. asiatica
, and two genotypes of
T. solium
. This approach provides a useful tool for the identification and diagnosis of human taeniid cestodes without DNA sequencing if nucleotide sequence databases are available.
Assessment of a Combined Testing Strategy for Detection of Antibodies to Human Herpesvirus 8 (HHV-8) in Persons with Kaposi's Sarcoma, Persons with Asymptomatic HHV-8 Infection, and Persons at Low Risk for HHV-8 Infection
Authors: Casper Corey , Krantz Elizabeth , Taylor Heather , Dalessio Julie , Carrell David , Wald Anna , Corey Lawrence , Ashley Rhoda
Published: September 2002

The performance of a human herpesvirus 8 (HHV-8) enzyme immunoassay (EIA) and selective subsequent use of an HHV-8 immunofluorescence assay (IFA) was tested in persons unlikely to be infected with HHV-8 and those who had HHV-8 detected in their saliva. The IFA was performed on samples within a range of EIA optical densities (0.15 to 0.35) where there was considerable overlap between HHV-8-infected and uninfected individuals. The sensitivity of the testing strategy was 88%, with a specificity of 97%.
Phylogenetic Analysis of Newcastle Disease Virus Genotypes Isolated in Japan
Authors: Mase Masaji , Imai Kunitoshi , Sanada Yasuyuki , Sanada Naoko , Yuasa Noboru , Imada Tadao , Tsukamoto Kenji , Yamaguchi Shigeo
Published: September 2002

We genetically analyzed field isolates of the Newcastle disease (ND) virus isolated in Japan from 1930 to 2001. The coding region of the fusion protein was amplified by reverse transcriptase PCR and directly sequenced. Phylogenetic analysis revealed the presence of viruses belonging to six of the eight known genotypes. It can be concluded from this study that ND outbreaks in Japan have been of multiple etiologies.
Erythromycin-Resistant Group A Streptococcal Isolates Recovered in Sofia, Bulgaria, from 1995 to 2001
Authors: Detcheva Antoaneta , Facklam Richard R. , Beall Bernard
Published: September 2002

The frequency of erythromycin resistance within group A streptococci in Sofia, Bulgaria, from 1995 to 2001 was 2.1% (26 isolates). Of this, 57.7% was macrolide-lincosamide-streptogramin (MLS) inducible, 7.7% was MLS constitutive, and 34.6% had the M phenotype. Eleven different
emm
sequence types were found among 25 erythromycin-resistant isolates tested. Nineteen of 26 erythromycin-resistant isolates were additionally resistant to tetracycline and/or chloramphenicol.
Association of the
prtF1
Gene (Encoding Fibronectin-Binding Protein F1) and the
sic
Gene (Encoding the Streptococcal Inhibitor of Complement) with
emm
Types of Group A Streptococci Isolated from Japanese Children with Pharyngitis
Authors: Ma Xiaoming , Kikuta Hideaki , Ishiguro Nobuhisa , Yoshioka Mikio , Ebihara Takashi , Murai Teiko , Kobayashi Intetu , Kobayashi Kunihiko
Published: September 2002

A total of 66 clinical isolates of group A streptococci (GAS) were obtained from 66 Japanese children with pharyngitis. The
prtF1
gene (encoding fibronectin-binding protein F1) and the
sic
gene (encoding the streptococcal inhibitor of complement) were present in 51 (77.3%) and 48 (72.7%) of the 66 isolates, respectively. These results indicated that a high prevalence of two virulence genes,
prtF1
and
sic
, is characteristic of GAS in Japan.
Impact of Nasopharyngeal Swab Types on Detection of
Bordetella pertussis
by PCR and Culture
Authors: Cloud Joann L. , Hymas Weston , Carroll Karen C.
Published: September 2002

A study was designed to assess the performance of various swabs and transport media routinely used to collect specimens submitted for
Bordetella
culture and PCR. Calcium-alginate swabs inhibited the PCR. No inhibition was detected in any PCRs with dacron or rayon swabs. All swab materials performed similarly for recovery of
Bordetella pertussis
in culture. The Amies with charcoal transport system performed poorly for culture. Calcium-alginate swabs are not recommended for PCR-based detection of
B. pertussis
. Dacron and rayon swabs are an excellent choice for both PCR and culture.