bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotypic and Genotypic Characterization of Two Penicillin-Susceptible Serotype 6B
Streptococcus pneumoniae
Clones Circulating in Italy
Authors: Gherardi Giovanni , Del Grosso Maria , Scotto d'Abusco Anna , D'Ambrosio Fabio , Dicuonzo Giordano , Pantosti Annalisa
Published: July 2003

Twenty-nine penicillin-susceptible serotype 6B strains isolated from patients with invasive diseases and from healthy carriers were examined by different genotyping methods. Ten groups were identified on the basis of the pulsed-field gel electrophoresis (PFGE) profiles, and two of these contained multiple isolates and were analyzed further. PFGE group 1 comprised 12 isolates, the majority of which had a multiresistant phenotype (resistance to erythromycin, clindamycin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole), corresponding to that of a clone previously described in the Mediterranean area and related to penicillin-resistant clone Spain
6B
-2. The
pbp2b
,
pbp2x
,
dhf
, and
pspA
genes of the isolates had identical restriction profiles; and the partial sequence of
pspA
was identical to that of clone Spain
6B
-2. In all isolates the resistance determinants
erm
(B) and
tet
(M) were inserted in a Tn
1545
-like element; 11 isolates carried
cat
as part of the integrated plasmid pC194. Multilocus sequence typing (MLST) performed with two isolates confirmed that their profiles corresponded to that of the Mediterranean clone. PFGE group 2 comprised nine strains, of which the majority showed no antibiotic resistance. Their
pspA
profiles were different, and the partial sequences obtained for two representative isolates indicated the presence of PspA proteins of different clades. The MLST profile of one strain was identical to that of a serotype 6B strain from the United Kingdom, while two other isolates were novel one-allele variants. This clone appears to be related (five of seven identical alleles) to two other internationally disseminated clones, Hungary
19A
-6 and Poland
23F
-16, both of which are penicillin resistant. The presence of antibiotic-susceptible isolates of this clone suggests that traits other than antibiotic resistance can make a clone successful.
Genotyping by Amplified Fragment Length Polymorphism Analysis Reveals Persistence and Recurrence of Infection with
Streptococcus anginosus
Group Organisms
Authors: Jacobs Jan A. , Tjhie Jeroen H. T. , Smeets Monique G. J. , Schot Corrie S. , Schouls Leo M.
Published: July 2003

Streptococcus anginosus
,
Streptococcus constellatus
, and
Streptococcus intermedius
, commonly referred to as the
Streptococcus anginosus
group (SAG), are commensal organisms known for their propensity to cause purulent infections which are difficult to eradicate. In this study, we determined the genetic similarities between SAG isolates consecutively recovered from single patients to assess the duration of infection or colonization. A total of 97 SAG isolates recovered from 30 patients were included; 65 (67.0%) of the isolates were abscess related. The isolates were identified by the 16S rRNA reverse line blot hybridization assay as
S. anginosus
(
n
= 34),
S. constellatus
(
n
= 55), and
S. intermedius
(
n
= 8). Amplified fragment length polymorphism (AFLP) analysis of the SAG isolates produced discriminatory and reproducible patterns. Consecutive SAG isolates with identical AFLP types were found in 27 of 30 (90.0%) patients, and consecutive isolates with only a single AFLP type were demonstrated in 21 (70.0%) patients. The median delay between the times of recovery of the first and last isolates of identical AFLP types from each patient was 36 days, and this delay extended for more than 1 year in patients with both colonizing and abscess-related SAG isolates. In six bacteremic patients, paired blood and nonblood SAG isolates showed identical AFLP types.
Usefulness of a New Mycobacteriophage-Based Technique for Rapid Diagnosis of Pulmonary Tuberculosis
Authors: Alcaide Fernando , Galí Nuria , Domínguez José , Berlanga Pilar , Blanco Silvia , Orús Pilar , Martín Rogelio
Published: July 2003

A new mycobacteriophage-based technique (PhageTek MB) was compared with standard culture and staining techniques for diagnosis of pulmonary tuberculosis. A total of 2,048 respiratory specimens from 1,466 patients collected from February 2000 to March 2001 were studied by both (i) conventional methods (direct microscopic examination [auramine-rhodamine fluorochrome], and culture in BacT/ALERT 3D and solid media) and (ii) the PhageTek MB assay. This phenotypic test utilizes specific mycobacteriophages to detect the presence of live
Mycobacterium tuberculosis
complex organisms within a decontaminated clinical sample. Overall, 205 (10%) specimens were positive for mycobacteria (134 patients): 144 (70.2%)
M. tuberculosis
isolates and 61 (29.8%) nontuberculous mycobacterium isolates (30
Mycobacterium kansasii
, 12
Mycobacterium xenopi
, 9
Mycobacterium gordonae
, 7
Mycobacterium avium
complex, 2
Mycobacterium chelonae
, and 1
Mycobacterium fortuitum
isolate). PhageTek MB was more likely to give a positive result with specimens in which high numbers of acid-fast bacilli were observed on the smear. The sensitivity, specificity, and positive and negative predictive values of this mycobacteriophage-based technique versus culture for
M. tuberculosis
were 58.3, 99.1, 83.2, and 96.9%, respectively. PhageTek MB is a rapid (48-h), specific, safe, and easy-to-perform test. According to the prevalence of the disease in the population studied, the test would require improved sensitivity in order to be used as a screening test for routine diagnosis of respiratory tuberculosis in our setting.
Different Cytomegalovirus Glycoprotein B Genotype Distribution in Serum and Cerebrospinal Fluid Specimens Determined by a Novel Multiplex Nested PCR
Authors: Tarragó David , Quereda Carmen , Tenorio Antonio
Published: July 2003

A novel and highly sensitive multiplex nested PCR assay has been developed for the simultaneous glycoprotein B (gB) typing of four gB genotypes of human cytomegalovirus (CMV) directly from clinical specimens. Specifically, a pair of primers to conserved regions of all gB genotypes within the gB gene (
gB
and
gpUL55
) was used for primary amplification. A mixture of nested primers to specific and conserved regions of each gB genotype was used for a secondary PCR amplification, yielding amplicons of different sizes for each gB genotype that could easily be differentiated by agarose gel electrophoresis. A total of 40 of 44 serum specimens and 26 of 26 cerebrospinal fluid (CSF) samples, which had previously tested positive for CMV in 66 of 70 AIDS patients with different CMV disease conditions, were gB genotyped by this novel assay. Significant differences in glycoprotein B genotype distribution between serum and CSF specimens were found (
P
= 0.001). gB genotype 3 (gB3) and gB2 were the most prevalent genotypes in sera (42.5%) and CSF (38.5%), respectively. A different distribution was also observed when only patients with CMV retinitis were studied (
P
= 0.005), suggesting a gB2 neuron tropism. Neither CMV disease nor any particular clinical manifestation of CMV disease was associated with gB genotypes (
P
> 0.05). A high incidence of mixed infection with the gB1 and gB3 genotypes (27.5%) was detected in serum specimens, indicating that reinfection and reactivation are common traits in advanced AIDS patients.
Widespread Pyrazinamide-Resistant
Mycobacterium tuberculosis
Family in a Low-Incidence Setting
Authors: Nguyen Dao , Brassard Paul , Westley Jennifer , Thibert Louise , Proulx Melanie , Henry Kevin , Schwartzman Kevin , Menzies Dick , Behr Marcel A.
Published: July 2003

An unusually high prevalence of pyrazinamide (PZA) monoresistance in
Mycobacterium tuberculosis
has been observed in Quebec. In the absence of a recognized outbreak, we hypothesized that these isolates most likely represented reactivation of an old endemic strain in this low-incidence area. A case-control study of 77 PZA-resistant isolates with a specific Quebec mutation and 253 PZA-susceptible control
M. tuberculosis
isolates was undertaken. By molecular analysis, all 77 case isolates shared a unique mutation profile in the
pncA
gene which was not present in control isolates. While control isolates manifested diverse IS
6110
restriction fragment length polymorphism (RFLP) patterns, spoligotypes, and major genetic groups, case isolates had similar but nonidentical IS
6110
RFLP patterns, had common spoligotypes, and were confined to one major genetic group, suggesting a common clonal ancestor. By epidemiologic and geographic analyses, however, there were no significant differences between the cases and the controls. We conclude that a clonally related family of PZA-monoresistant
M. tuberculosis
isolates in Quebec represents historic rather than recent transmission.
Detection and Characterization of Verocytotoxin-Producing
Escherichia coli
by Automated 5′ Nuclease PCR Assay
Authors: Nielsen Eva Møller , Andersen Marianne Thorup
Published: July 2003

In recent years increased attention has been focused on infections caused by isolates of verocytotoxin-producing
Escherichia
coli (VTEC) serotypes other than O157. These non-O157 VTEC isolates are commonly present in food and food production animals. Easy detection, isolation, and characterization of non-O157 VTEC isolates are essential for improving our knowledge of these organisms. In the present study, we detected VTEC isolates in bovine fecal samples by a duplex 5′ nuclease PCR assay (real-time PCR) that targets
vtx1
and
vtx2
. VTEC isolates were obtained by colony replication by use of hydrophobic-grid membrane filters and DNA probe hybridization. Furthermore, we have developed 5′ nuclease PCR assays for the detection of virulence factors typically present in VTEC isolates, including subtypes of three genes of the locus of enterocyte effacement (LEE) pathogenicity island. The 22 assays included assays for the detection of verocytotoxin genes (
vtx1
,
vtx2
), pO157-associated genes (
ehxA
,
katP
,
espP
, and
etpD
), a recently identified adhesin (
saa
), intimin (
eae
, all variants), seven subtypes of
eae
, four subtypes of
tir
, and three subtypes of
espD
. A number of reference strains (VTEC and enteropathogenic
E. coli
strains) and VTEC strains isolated from calves were tested to validate the PCR assays. The expected virulence profiles were detected for all reference strains. In addition, new information on the subtypes of LEE genes was obtained. For reference strains as well as bovine isolates, a consistent relationship between subtypes of the LEE genes was found, so that a total of seven different combinations of these were recognized (corresponding to the seven subtypes of
eae
). Isolates with 15 different serogroup-virulence profiles were isolated from 16 calves. Among these, 53% harbored LEE and 73% harbored factors carried by the large virulence plasmid. One LEE-negative isolate had the gene for the adhesin Saa. The most common virulence profile among the bovine isolates was
vtx1
,
eae
-ζ,
tir
-α,
ehxA
, and
espP
. This panel of assays offers an easy method for the extensive characterization of VTEC isolates.
Rapid Screening and Identification of Methicillin-Resistant
Staphylococcus aureus
from Clinical Samples by Selective-Broth and Real-Time PCR Assay
Authors: Fang Hong , Hedin Göran
Published: July 2003

A screening method for methicillin-resistant
Staphylococcus aureus
(MRSA) by using selective broth and real-time PCR (broth-PCR) was developed and evaluated. The samples (
n
= 304) were cultured in the broth overnight, followed by
nuc
gene detection by real-time PCR.
nuc
-negative samples were further checked for the presence of
nuc
amplification inhibitors by a PCR internal inhibitor assay.
nuc
-positive samples and
nuc
-negative samples with PCR inhibitors were cultured onto plates and processed further. The diagnostic values for this MRSA screening method were 93.3% sensitivity, 89.6% specificity, 31.8% positive predictive value, and 99.6% negative predictive value. The application of the broth-PCR method will be able to report most of the negative samples (258 of 289 [89.3%]) on the next morning and can save as much as 84.9% (258 of 304) of the labor and cost spent on processing the
nuc
-negative specimens on plates. In the study, all the samples were processed in parallel by the broth enrichment method and the plating method for comparison. To identify MRSA, the isolated oxacillin-resistant
S. aureus
strains were tested by a duplex real-time PCR targeting the
mecA
gene and the
nuc
gene. A collection of MRSA, methicillin-susceptible
Staphylococcus aureus
, methicillin-resistant
Staphylococcus epidermidis
, and methicillin-susceptible
Staphylococcus epidermidis
strains and a panel of standard strains of 11 bacterial species other than
S. aureus
were also tested by this method, which was proved to be a valuable tool for MRSA identification in a routine microbiological laboratory.
Sources and Magnitude of Intralaboratory Variability in a Sequence-Based Genotypic Assay for Human Immunodeficiency Virus Type 1 Drug Resistance
Authors: Galli R. A. , Sattha B. , Wynhoven B. , O'Shaughnessy M. V. , Harrigan P. R.
Published: July 2003

We assessed the intralaboratory reproducibility of a system for sequencing human immunodeficiency virus type 1 (HIV-1) protease (PR) and reverse transcriptase (RT) by using replicate subanalyses of 46 plasma samples collected from HIV-1-infected, antiretroviral-experienced patients in order to determine the relative contributions of the different procedural steps to final sequence variability. Complete sequence concordance between duplicates of each sample was 99.4%. Complete and partial mismatches occurred scattered throughout the PR-RT genome segment at >300 positions. Approximately 75% of the discordances involved mixtures, some of which appeared at key resistance sites. Most differences were the result of the first-round RT-PCR procedure. Inter-rater concordance for sequence analysis and assembly was >99.9%. There was no observed correlation between the number or frequency of mismatches and plasma viral loads. A separate longitudinal analysis of a single routine control sample sequenced 103 times over 9 months consistently gave highly reproducible sequences (median percentage of nucleotide discordances, 0.04%; range, 0 to 0.2%). Finally, sequence data from 168 sequential samples collected from 22 patients with long-term, predominantly wild type HIV showed that intrapatient nucleotide concordance with individual index sequences ranged from 96.5 to 100%. Together, these results confirm that sequence-based genotyping can be a precise and reliable tool for monitoring HIV drug resistance, and they suggest that efforts to reduce variability should focus on the first RT-PCR step. Consequently, the data suggest that the composition of external quality assessment panels should be based on clinical HIV isolates rather than DNA clones.
Identification of
Bacillus anthracis
by
rpoB
Sequence Analysis and Multiplex PCR
Authors: Ko Kwan Soo , Kim Jong-Man , Kim Jong-Wan , Jung Byeong Yeal , Kim Wonyong , Kim Ik Jung , Kook Yoon-Hoh
Published: July 2003

Comparative sequence analysis was performed upon
Bacillus anthracis
and its closest relatives,
B. cereus
and
B. thuringiensis
. Portions of
rpoB
DNA from 10 strains of
B. anthracis
, 16 of
B. cereus
, 10 of
B. thuringiensis
, 1 of
B. mycoides
, and 1 of
B. megaterium
were amplified and sequenced. The determined
rpoB
sequences (318 bp) of the 10
B. anthracis
strains, including five Korean isolates, were identical to those of Ames, Florida, Kruger B, and Western NA strains. Strains of the “
B. cereus
group” were separated into two subgroups, in which the
B. anthracis
strains formed a separate clade in the phylogenetic tree. However,
B. cereus
and
B. thuringiensis
could not be differentiated. Sequence analysis confirmed the five Korean isolates as
B. anthracis
. Based on the
rpoB
sequences determined in the present study, multiplex PCR generating either
B. anthracis
-specific amplicons (359 and 208 bp) or
cap
DNA (291 bp) in a virulence plasmid could be used for the rapid differential detection and identification of virulent
B. anthracis
.
Detection and Verification of
Mycobacterium avium
subsp.
paratuberculosis
in Fresh Ileocolonic Mucosal Biopsy Specimens from Individuals with and without Crohn's Disease
Authors: Bull Tim J. , McMinn Elizabeth J. , Sidi-Boumedine Karim , Skull Angela , Durkin Damien , Neild Penny , Rhodes Glenn , Pickup Roger , Hermon-Taylor John
Published: July 2003

Mycobacterium avium
subsp.
paratuberculosis
is a robust and phenotypically versatile pathogen which causes chronic inflammation of the intestine in many species, including primates.
M. avium
subsp.
paratuberculosis
infection is widespread in domestic livestock and is present in retail pasteurized cows' milk in the United Kingdom and, potentially, elsewhere. Water supplies are also at risk. The involvement of
M. avium
subsp.
paratuberculosis
in Crohn's disease (CD) in humans has been uncertain because of the substantial difficulties in detecting this pathogen. In its Ziehl-Neelsen staining-negative form,
M. avium
subsp.
paratuberculosis
is highly resistant to chemical and enzymatic lysis. The present study describes the development of optimized sample processing and DNA extraction procedures with fresh human intestinal mucosal biopsy specimens which ensure access to
M. avium
subsp.
paratuberculosis
DNA and maximize detection of these low-abundance pathogens. Also described are two nested PCR methodologies targeted at IS
900
, designated IS
900
[L/AV] and IS
900
[TJ1-4], which are uniquely specific for IS
900
. Detection of
M. avium
subsp.
paratuberculosis
in mucosal biopsy specimens was also evaluated by using mycobacterial growth indicator tube (MGIT) cultures (Becton Dickinson). IS
900
[L/AV] PCR detected
M. avium
subsp.
paratuberculosis
in 34 of 37 (92%) patients with CD and in 9 of 34 (26%) controls without CD (noninflammatory bowel disease [nIBD] controls) (
P
= 0.0002; odds ratio = 3.47).
M. avium
subsp.
paratuberculosis
was detected by IS
900
[L/AV] PCR in MGIT cultures after 14 to 88 weeks of incubation in 14 of 33 (42%) CD patients and 3 of 33 (9%) nIBD controls (
P
= 0.0019; odds ratio = 4.66). Nine of 15 (60%) MGIT cultures of specimens from CD patients incubated for more than 38 weeks were positive for
M. avium
subsp.
paratuberculosis
. In each case the identity of IS
900
from
M. avium
subsp.
paratuberculosis
was verified by amplicon sequencing. The rate of detection of
M. avium
subsp.
paratuberculosis
in individuals with CD is highly significant and implicates this chronic enteric pathogen in disease causation.
Genome-Wide DNA Microarray Analysis of
Francisella tularensis
Strains Demonstrates Extensive Genetic Conservation within the Species but Identifies Regions That Are Unique to the Highly Virulent
F. tularensis
subsp.
tularensis
Authors: Broekhuijsen Martien , Larsson Pär , Johansson Anders , Byström Mona , Eriksson Ulla , Larsson Eva , Prior Richard G. , Sjöstedt Anders , Titball Richard W. , Forsman Mats
Published: July 2003

Francisella tularensis
is a potent pathogen and a possible bioterrorism agent. Little is known, however, to explain the molecular basis for its virulence and the distinct differences in virulence found between the four recognized subspecies,
F. tularensis
subsp.
tularensis
,
F. tularensis
subsp.
mediasiatica
,
F. tularensis
subsp.
holarctica
, and
F. tularensis
subsp.
novicida
. We developed a DNA microarray based on 1,832 clones from a shotgun library used for sequencing of the highly virulent strain
F. tularensis
subsp.
tularensis
Schu S4. This allowed a genome-wide analysis of 27 strains representing all four subspecies. Overall, the microarray analysis confirmed a limited genetic variation within the species
F. tularensis
, and when the strains were compared, at most 3.7% of the probes showed differential hybridization. Cluster analysis of the hybridization data revealed that the causative agents of type A and type B tularemia, i.e.,
F. tularensis
subsp.
tularensis
and
F. tularensis
subsp.
holarctica
, respectively, formed distinct clusters. Despite marked differences in their virulence and geographical origin, a high degree of genomic similarity between strains of
F. tularensis
subsp.
tularensis
and
F. tularensis
subsp.
mediasiatica
was apparent. Strains from Japan clustered separately, as did strains of
F. tularensis
subsp.
novicida
. Eight regions of difference (RD) 0.6 to 11.5 kb in size, altogether comprising 21 open reading frames, were identified that distinguished strains of the moderately virulent subspecies
F. tularensis
subsp.
holarctica
and the highly virulent subspecies
F. tularensis
subsp.
tularensis
. One of these regions, RD1, allowed for the first time the development of an
F. tularensis
-specific PCR assay that discriminates each of the four subspecies.
Sequence-Based Typing of
Legionella pneumophila
Serogroup 1 Offers the Potential for True Portability in Legionellosis Outbreak Investigation
Authors: Gaia Valeria , Fry Norman K. , Harrison Timothy G. , Peduzzi Raffaele
Published: July 2003

Seven gene loci of
Legionella pneumophila
serogroup 1 were analyzed as potential epidemiological typing markers to aid in the investigation of legionella outbreaks. The genes chosen included four likely to be selectively neutral (
acn
,
groES
,
groEL
, and
recA
) and three likely to be under selective pressure (
flaA
,
mompS
, and
proA
). Oligonucleotide primers were designed to amplify 279- to 763-bp fragments from each gene. Initial sequence analysis of the seven loci from 10 well-characterized isolates of
L. pneumophila
serogroup 1 gave excellent reproducibility (
R
) and epidemiological concordance (
E
) values (
R
= 1.00;
E
= 1.00). The three loci showing greatest discrimination and nucleotide variation,
flaA
,
mompS
, and
proA
, were chosen for further study. Indices of discrimination (
D
) were calculated using a panel of 79 unrelated isolates. Single loci gave
D
values ranging from 0.767 to 0.857, and a combination of all three loci resulted in a
D
value of 0.924. When all three loci were combined with monoclonal antibody subgrouping, the
D
value was 0.971. Sequence-based typing of
L. pneumophila
serogroup 1 using only three loci is epidemiologically concordant and highly discriminatory and has the potential to become the new “gold standard” for the epidemiological typing of
L. pneumophila
.
Molecular Epidemiology and Characterization of Plasmid-Encoded β-Lactamases Produced by Tunisian Clinical Isolates of
Salmonella enterica
Serotype Mbandaka Resistant to Broad-Spectrum Cephalosporins
Authors: Makanera Abdoulaye , Arlet Guillaume , Gautier Valérie , Manai Mohamed
Published: July 2003

We studied 31 clinical isolates of
Salmonella enterica
serotype Mbandaka resistant to broad-spectrum cephalosporins and recovered in Tunisia over a 5-year period. The transferability of this resistance was demonstrated by conjugation experiments. Thirty of the 31 isolates were positive in the double-disk synergy test. By isoelectric focusing analysis, all of the isolates were found to produce a band of β-lactamase activity with a pI of 5.9. Three of these isolates produced an additional band with a pI of 7.6. PCR and DNA sequencing identified these β-lactamases as TEM-4 and SHV-2a, respectively. The remaining isolate, highly resistant to ceftazidime but susceptible to cefepime, produced a β-lactamase that focused at pI 7.8. No synergy was detected by the double-disk synergy test. Sequence analysis of the
bla
gene amplified by PCR showed that the plasmid-mediated AmpC-type enzyme was ACC-1a. Fingerprinting analysis by repetitive-element PCR and enterobacterial repeat intergenic consensus-PCR suggested that 29 of the 31
Salmonella
serotype Mbandaka isolates belonged to the same clonal population.
Genetic Relationship between Methicillin-Sensitive and Methicillin-Resistant
Staphylococcus aureus
Strains from France and from International Sources: Delineation of Genomic Groups
Authors: Branger Catherine , Gardye Carole , Galdbart Jacques-Olivier , Deschamps Catherine , Lambert Nicole
Published: July 2003

Cluster analysis of the
Sma
I patterns, generated by pulsed-field gel electrophoresis, of 44 methicillin-resistant (MRSA) and 118 methicillin-sensitive (MSSA)
Staphylococcus aureus
strains isolated in various French hospitals and 61 MRSA and 48 MSSA strains from 20 other countries revealed 20 genomic groups distributed into four distantly related phylogenic branches. Eighty-three of the 105 MRSA strains (79%) were clustered in the six genomic groups of phylogenic branch I; and 154 of the 166 MSSA strains (92.8%) were clustered in the 14 genomic groups of phylogenic branches II, III, and IV. Agreement between genomic group and two other markers, esterase type and phage group, was obtained, emphasizing the clonal structure of the population. The genomic groups were delineated by esterase type. The distribution of the strains within the genomic groups was independent of their geographical origin; French strains were clustered with strains from other countries. The three types of the staphylococcal cassette chromosome
mec
(SCC
mec
) complex were distributed according to genomic groups. Most of the time, type I and type II SCC
mec
complexes were found in the MRSA strains belonging to the same genomic groups. In contrast, the type III SCC
mec
complex was specific to the MRSA strains belonging to the three genomic groups characterized by a common esterase type.
Differentiation of Rickettsiae by
groEL
Gene Analysis
Authors: Lee Jung-Hee , Park Hyo-Soon , Jang Won-Jong , Koh Seong-Eun , Kim Jong-Moon , Shim Soo-Kyoung , Park Mi-Yeoun , Kim Yoon-Won , Kim Bum-Joon , Kook Yoon-Hoh , Park Kyung-Hee , Lee Seung-Hyun
Published: July 2003

The nucleotide sequences (534 to 546 bp) of the
groEL
gene, which encodes the 60-kDa heat shock protein GroEL, from 15 rickettsial strains were determined and compared. In the phylogenetic tree created by the unweighted pair group method with arithmetic averages and the neighbor-joining method, rickettsial strains could be distinguished from
Ehrlichia
strains. Five spotted fever group strains, four typhus group strains, and six scrub typhus group (STG) strains were differentiated as distinct entities. Unlike
gltA
and
ompA
gene analyses, differentiation between members of the genus
Rickettsia
and the STG rickettsiae by
groEL
gene analysis was possible. In comparison with 16S rRNA gene analysis, the
groEL
gene has a higher degree of divergence among the rickettsiae. We therefore successfully developed rapid differentiation methods, PCR-restriction fragment length polymorphism analysis and a species-specific PCR, based on the
groEL
gene sequences. Four Korean isolates were identified by these methods and
groEL
gene analysis. The results suggest that the
groEL
gene is useful for the identification and characterization of rickettsiae
.
Biofilm-Forming Ability ofCandida albicansIs Unlikely To Contribute to High Levels of Oral Yeast Carriage in Cases of Human Immunodeficiency Virus Infection
Authors: Jin Y. , Yip H. K. , Samaranayake Y. H. , Yau J. Y. , Samaranayake L. P.
Published: July 2003

An increased prevalence of candidal carriage and oral candidiasis is common in cases of human immunodeficiency virus (HIV) infection, and the reasons for this may include the enhanced ability of colonizing yeasts to produce biofilms on mucosal surfaces. The aim of the present study was therefore to examine the differences, if any, in the biofilm-forming abilities of 26Candida albicansyeast isolates from HIV-infected individuals and 20 isolates from HIV-free individuals, as this attribute of yeast isolates from patients with HIV disease has not been examined before. Biofilm formation in microtiter plate wells was quantitatively determined by both the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction method and the crystal violet method. Although candidal biofilm formation could be quantitatively evaluated by either technique, the better reproducibility (P< 0.05) of the XTT reduction assay compared with that of the crystal violet method led us to conclude that the former is more reliable. There were no significant quantitative differences in biofilm formation betweenC. albicansisolates from HIV-infected patients and isolates from HIV-free individuals during in vitro incubation in a multiwell culture system over a period of 66 h. Three of eight host factors in the HIV-infected group were found to be associated with candidal biofilm formation. Thus, yeasts isolated from older individuals and those with higher CD4-cell counts exhibited decreased biofilm formation, while the findings for yeasts from individuals receiving zidovudine showed the reverse (P< 0.05 for all comparison). Our data indicate that attributes other than biofilm formation may contribute to the increased oral yeast carriage rates in cases of HIV infection.
Continued Low Rates of Transmission of
Mycobacterium tuberculosis
in Norway
Authors: Dahle Ulf R. , Sandven Per , Heldal Einar , Caugant Dominique A.
Published: July 2003

In this study, we determined the genetic diversity of
Mycobacterium tuberculosis
isolated in Norway from 1999 to 2001. The results were compared to those for strains isolated from 1994 to 1998. A total of 818 patients were diagnosed with tuberculosis (TB) during the last 3-year period. Of these cases, 576 (70%) were verified by culturing, and strains from 551 patients (96%) were analyzed by the IS
6110
restriction fragment length polymorphism (RFLP) method. We excluded 13 strains (2.4%) from the analyses, since they were found to represent false-positive samples. A total of 67 strains (12%) that carried fewer than five copies of IS
6110
were analyzed by spoligotyping. The strains were from 157 patients (29%) of Norwegian origin and 381 patients (71%) of foreign origin. The rate of diversity among all of the strains was 90%, while in 1994 to 1998 it was 87%. Clusters were assumed to have arisen from recent transmission; the degree of such transmission was 10% in 1999 to 2001, while for the whole 8-year period (1994 to 2001), it was 11%. Of the 109 patients diagnosed as being part of a cluster in 1999 to 2001, 89 were infected with a strain that carried more than four copies of IS
6110
. Among these 89 patients, 52 (58%) were infected with a strain that had already been identified in 1994 to 1998. The results indicated that most cases of TB in Norway were due to the import of new strains rather than to transmission within the country. This finding demonstrates that screening of immigrants for TB upon arrival in Norway needs to be improved. Outbreaks, however, were caused mainly by strains that have been circulating in Norway for many years.
Characterization of a Lipoprotein Common to
Legionella
Species as a Urinary Broad-Spectrum Antigen for Diagnosis of Legionnaires' Disease
Authors: Kim Min Ja , Sohn Jang Wook , Park Dae Won , Park Seung Chul , Chun Byung Chul
Published: July 2003

We have previously identified the
Legionella
19-kDa peptidoglycan-associated lipoprotein (PAL) as a species-common immunodominant antigen. We describe here for the first time the excretion and detection of the PAL antigen in infected urine specimens, which is useful for the diagnosis of Legionnaires' disease. Rabbit anti-PAL immunoglobulin G (IgG) antibody was produced by immunization with the purified, recombinant PAL of
Legionella pneumophila
serogroup 1 and used in the PAL antigen capture enzyme-linked immunosorbent assay (ELISA) to detect urinary PAL antigen. A soluble-antigen capture ELISA using rabbit IgG antibodies against
Legionella
soluble antigens was prepared independently and used as a broad-spectrum standard test to detect soluble antigens of several
Legionella
species. Urine samples were obtained from guinea pigs experimentally infected with each of
L. pneumophila
serogroups 1, 3, and 6, and other
Legionella
species. The absorbance values of the PAL antigen ELISA highly correlated with those of the soluble-antigen ELISA in infected urine samples, with a correlation coefficient of 0.84 (
P
< 0.01). When applied to 17 infected urine samples and 67 negative controls from guinea pigs, the sensitivity and specificity of the PAL antigen capture ELISA were 88.2 and 95.5%, respectively. Compared to the commercial Biotest enzyme immunoassay, the PAL antigen ELISA was more efficient for detecting pneumophila non-serogroup 1 and nonpneumophila species. None of the 161 control human urine specimens obtained from healthy adults and patients with either non-
Legionella
pneumonia or urinary tract infections tested positive in the PAL antigen ELISA. The present study shows that the
Legionella
PAL is a very useful broad-spectrum antigen for urinary diagnostic testing. Moreover, since recombinant PAL antigen can be produced more efficiently than the soluble antigens, the development of a broad-spectrum diagnostic immunoassay based on the detection of the PAL antigen appears to be warranted.
Evaluation of Culture Methods and a DNA Probe-Based PCR Assay for Detection of
Campylobacter
Species in Clinical Specimens of Feces
Authors: Maher Majella , Finnegan Cathriona , Collins Evelyn , Ward Brid , Carroll Cyril , Cormican Martin
Published: July 2003

Campylobacter
species are the leading agents of bacterial gastroenteritis in developed countries. In this study 320 specimens of feces from patients with symptoms of acute gastroenteritis were cultured for
Campylobacter
species by direct plating on modified charcoal cefoperazone deoxycholate agar and by enrichment in modified Preston broth, with or without blood added, for 48 h at 37°C prior to plating. A 16S/23S PCR/DNA probe membrane-based colorimetric detection assay was evaluated on a subset of the feces (
n
= 127), including 18 culture-positive and 109 culture-negative specimens. DNA was extracted directly from the fecal specimens by using the QIAamp DNA stool Minikit for the DNA probe-based PCR assay (PCR/DNA probe assay). A second PCR/DNA probe assay based on the 16S rRNA gene in C
ampylobacter
spp. was applied to all specimens that were culture negative, PCR/DNA positive on initial analysis.
Campylobacter
species were cultured in 20 of the 320 specimens. The 16S/23S PCR/DNA probe assay detected campylobacter DNA in 17 of 18 (94% sensitivity) culture-positive specimens and in 41 (38%) culture-negative specimens. The presence of campylobacter DNA in 35 of 41 culture-negative specimens was confirmed by the 16S PCR/DNA probe assay. DNA sequence analysis of seven 16S/23S PCR products and five 16S PCR products amplified from a selection of these specimens confirmed the presence of campylobacter DNA and more specifically
Campylobacter jejuni
,
C. concisus
,
C. curvus
, and
C. gracilis
DNA in these specimens. The molecular assays described in this study are rapid methods for the detection and identification of
Campylobacter
species in fecal specimens. The finding of
Campylobacter
spp. DNA in a large number of specimens of feces from patients with no other identified cause of diarrhea may suggest that
Campylobacter
spp. other than
C. jejuni
and
C. coli
may account for a proportion of cases of acute gastroenteritis in which no etiological agent is currently identified.
Human Metapneumovirus Associated with Respiratory Tract Infections in a 3-Year Study of Nasal Swabs from Infants in Italy
Authors: Maggi Fabrizio , Pifferi Massimo , Vatteroni Marialinda , Fornai Claudia , Tempestini Elena , Anzilotti Silvia , Lanini Letizia , Andreoli Elisabetta , Ragazzo Vincenzo , Pistello Mauro , Specter Steven , Bendinelli Mauro
Published: July 2003

The newly described human metapneumovirus (hMPV) is reported here to be more commonly associated with lower respiratory tract disease. The present study examined nasal swab specimens from 90 infants with acute respiratory tract infections in Pisa, Italy, over a period of three respiratory virus seasons. The incidence of infection varied in each of the 3 years, with the rates of positivity for hMPV being 7% in 2001 but 37 and 43% in 2000 and 2002, respectively. hMPV was noted to occur seasonally in a pattern typical of the frequency of occurrence of respiratory syncytial virus. More than one-half (14 of 23) of the infants infected with hMPV had bronchopneumonia. One-third (9 of 23) of the hMPV-infected patients were also infected with another respiratory virus, a relationship that has not previously been reported. Mixed infections did not account for a higher percentage of cases of bronchopneumonia than hMPV infection alone did. Furthermore, 7 of 17 infants whose plasma was also tested for hMPV RNA were demonstrated to have virus in both nasal swab and blood specimens. The study indicates that hMPV is seen as commonly as other respiratory viruses, may be associated with severe respiratory disease in infants, can establish mixed infections with other respiratory viruses, and has a seasonal occurrence.
Determination of Amoebicidal Activities of Multipurpose Contact Lens Solutions by Using a Most Probable Number Enumeration Technique
Authors: Beattie Tara K. , Seal David V. , Tomlinson Alan , McFadyen Angus K. , Grimason Anthony M.
Published: July 2003

Six multipurpose contact lens solutions [All-in-One, All-in-One (Light), ReNu MultiPlus, Optifree Express, Complete, and Solo-care soft] were tested for their efficacies against
Acanthamoeba castellanii
trophozoites and cysts by using a most probable number (MPN) technique for amoebic enumeration. Against trophozoites, All-in-One, ReNu Multiplus, and Optifree Express achieved total kill (log reduction of >3) after the manufacturer's minimum recommended disinfection time (MMRDT), with the remaining solutions failing to reach a log reduction of 1. After 24 h of exposure, all solutions proved trophozoiticidal, achieving, with the exception of Complete (log reduction of 3.13), total kill. Against cysts, All-in-One gave a log reduction of >3 within the MMRDT, with all other solutions failing to achieve a log reduction of 1. After 24 h of exposure, All-in-One achieved total kill of cysts (log reduction of 3.74), ReNu MultiPlus gave a log reduction of 3.15, and the remaining solutions reached log reductions of between 1.09 and 2.27. The MPN technique provides a simple, reliable, and reproducible method of amoebic enumeration that depends on simply establishing the presence or absence of growth on culture plates inoculated with a series of dilutions and determining the MPN of amoebae present from statistical tables. By use of this technique, two of the multipurpose solutions tested, ReNu MultiPlus and Optifree Express, demonstrated effective trophozoiticidal activities within the recommended disinfection times; however, only All-in-One proved effective against both trophozoites and cysts over the same time period. This MPN technique, which uses axenically produced trophozoites and mature, double-walled cysts, has the potential to form the basis of a national standard for amoebicidal efficacy testing of multipurpose contact lens disinfecting solutions.
Molecular Epidemiology of Methicillin-ResistantStaphylococcus aureusStrains Causing Neonatal Toxic Shock Syndrome-Like Exanthematous Disease in Neonatal and Perinatal Wards
Authors: Kikuchi Ken , Takahashi Naoto , Piao Chuncheng , Totsuka Kyoichi , Nishida Hiroshi , Uchiyama Takehiko
Published: July 2003

Neonatal toxic shock syndrome-like exanthematous disease (NTED) is a new neonatal disease caused by toxic shock syndrome toxin 1 (TSST-1). We conducted a prospective surveillance study and characterized the methicillin-resistantStaphylococcus aureus(MRSA) strains isolated from patients with NTED and compared them with the strains from patients with other MRSA infections and asymptomatic carriers. The study was performed in the neonatal intensive care unit and a general neonatal and maternal ward in the Tokyo Women's Medical University Hospital (TWMUH) from September to December 1998. Among 103 patients eligible for the study, MRSA was detected in 62 (60.2%) newborns; of these 62 newborns, 8 (12.9%) developed NTED, 1 (1.6%) had another MRSA infection, and 53 (85.5%) were asymptomatic MRSA carriers. Sixty-nine MRSA strains were obtained from the 62 newborns. DNA fingerprinting by pulsed-field gel electrophoresis showed two clusters: clone A with 8 subtypes and clone B. Sixty-seven of the 69 MRSA strains (97.1%) belonged to clone A, and type A1 was the most predominant (42 of 69 strains; 60.9%) in every neonatal and perinatal ward. All but one of the clone A strains had the TSST-1 and staphylococcal enterotoxin C genes. We also analyzed eight MRSA strains from eight NTED patients in five hospitals in Japan other than TWMUH. All the MRSA strains from NTED patients also belonged to clone A. These results suggest that a single clone that predominated in the neonatal wards of six hospitals might have caused NTED. However, the occurrence of NTED might not be dependent on the presence of an NTED-specific strain.
High Prevalence of M184 Mutation among Patients with Viroimmunologic Discordant Responses to Highly Active Antiretroviral Therapy and Outcomes after Change of Therapy Guided by Genotypic Analysis
Authors: Nicastri Emanuele , Sarmati Loredana , d'Ettorre Gabriella , Parisi Saverio G. , Palmisano Lucia , Galluzzo Clementina , Montano Marco , Uccella Ilaria , Amici Roberta , Gatti Francesca , Vullo Vincenzo , Concia Ercole , Vella Stefano , Andreoni Massimo
Published: July 2003

Whether highly active antiretroviral therapy (HAART) should be modified in patients with persistent increases in CD4
+
T cells despite detectable viral loads is an unresolved question. Forty-three heavily pretreated human immunodeficiency virus (HIV)-infected patients with virologic failure during HAART were studied before a change of therapy guided by genotypic analysis and during follow-up. Patients with an increase in CD4
+
cell count (>100 cells/ml) over pre-HAART values were considered to be discordant patients (20 individuals), whereas patients with a lower increase or no increase in CD4
+
cell count were considered failing patients (23 individuals). Based on univariate analysis, a high CD4
+
cell count before antiretroviral treatment, homosexual behavior as a risk factor for HIV infection, reduced drug exposure to nonnucleoside reverse transcriptase inhibitors, low replicative capacity of HIV isolates, and more frequent detection of HIV isolates with a non-B subtype, an R5 biological phenotype, and M184V and T215Y/F mutations were factors associated with a discordant response to HAART. Based on multivariate analysis, only the M184V mutation remained significantly associated with a viroimmunologic discordant response (odds ratio, 25.48; 95% confidence interval, 1.43 to 453.93). No difference in lamivudine exposure was found between discordant (95%) and failing (91%) patients. Twelve months after the genotypic analysis-guided change of therapy, 3 discordant (15%) and 6 failing patients (26%) achieved undetectable viral loads (<50 copies/ml), whereas in patients with HIV RNA loads of >500 copies/ml, discordant responses were observed in 5 out of 15 discordant patients and in 4 out of 16 failing patients. A relationship between the M184V mutation and a viroimmunologic discordant response to HAART was found. After the genotypic analysis-driven change of therapy, similar rates of virologic suppression were detected in the two groups.
Reliability of Nucleic Acid Amplification Methods for Detection of
Chlamydia trachomatis
in Urine: Results of the First International Collaborative Quality Control Study among 96 Laboratories
Authors: Verkooyen Roel P. , Noordhoek Gerda T. , Klapper Paul E. , Reid Jim , Schirm Jurjen , Cleator Graham M. , Ieven Margareta , Hoddevik Gunnar
Published: July 2003

The first European Quality Control Concerted Action study was organized to assess the ability of laboratories to detect
Chlamydia trachomatis
in a panel of urine samples by nucleic acid amplification tests (NATs). The panel consisted of lyophilized urine samples, including three negative, two strongly positive, and five weakly positive samples. Ninety-six laboratories in 22 countries participated with a total of 102 data sets. Of 204 strongly positive samples 199 (97.5%) were correctly reported, and of 506 weakly positive samples 466 (92.1%) were correctly reported. In 74 (72.5%) data sets correct results were reported on all samples, and 17 data sets (16.7%) showed either one false-negative or one false-positive result. In another 11 data sets, two or more incorrect results were reported, and two data sets reported a false-positive result on one negative sample. The Roche COBAS Amplicor test was performed in 44 (43%) data sets, the Abbott LCx assay was performed in 31 (30%) data sets, the Roche Amplicor manual assay was performed in 9 (9%) data sets, an in-house PCR was performed in 9 (9%) data sets, the Becton Dickinson ProbeTec ET assay was performed in 5 (4.9%) data sets, and the GenProbe TMA assay was performed in 4 (3.9%) data sets. The results of the Roche Amplicor manual (95.6% correct), COBAS Amplicor (97.0%), and Abbott LCx (94.8%) tests were comparable (
P
= 0.48). The results with the in-house PCR, BD ProbeTec ET, and GenProbe TMA tests were reported correctly in 88.6, 98, and 92.5% of the tests, respectively. Freeze-drying of clinical urine specimens proved to be a successful method for generating standardized, stable, and easy-to-transport samples for the detection of
C. trachomatis
by using NATs. Although the results, especially the specificity, for this proficiency panel were better than most quality control studies, sensitivity problems occurred frequently, underlining the need for good laboratory practice and reference reagents to monitor the performance of these assays.
Severity of Nonbullous
Staphylococcus aureus
Impetigo in Children Is Associated with Strains Harboring Genetic Markers for Exfoliative Toxin B, Panton-Valentine Leukocidin, and the Multidrug Resistance Plasmid pSK41
Authors: Koning Sander , van Belkum Alex , Snijders Susan , van Leeuwen Willem , Verbrugh Henri , Nouwen Jan , Op ′t Veld Mariet , van Suijlekom-Smit Lisette W. A. , van der Wouden Johannes C. , Verduin Cees
Published: July 2003

Nonbullous impetigo is a common skin infection in children and is frequently caused by
Staphylococcus aureus
. Staphylococcal toxins and especially exfoliative toxin A are known mediators of bullous impetigo in children. It is not known whether this is also true for nonbullous impetigo. We set out to analyze clonality among clinical isolates of
S. aureus
from children with nonbullous impetigo living in a restricted geographical area in The Netherlands. We investigated whether staphylococcal nasal carriage and the nature of the staphylococcal strains were associated with the severity and course of impetigo. Bacterial isolates were obtained from the noses and wounds of children suffering from impetigo. Strains were genetically characterized by pulsed-field gel electrophoresis-mediated typing and binary typing, which was also used to assess toxin gene content. In addition, a detailed clinical questionnaire was filled in by each of the participating patients. Staphylococcal nasal carriage seems to predispose the patients to the development of impetigo, and 34% of infections diagnosed in the Rotterdam area are caused by one clonal type of
S. aureus
. The
S. aureus
strains harbor the exfoliative toxin B (ETB) gene as a specific virulence factor. In particular, the numbers (
P
= 0.002) and sizes (
P
< 0.001) of the lesions were increased in patients infected with an ETB-positive strain. Additional predictors of disease severity and development could be identified. The presence of a staphylococcal plasmid encoding multiple antibiotic resistance traits, as detected by binary typing, was associated with a reduction in the cure rate. Our results recognize that a combination of staphylococcal virulence and resistance genes rather than a single gene determines the development and course of nonbullous impetigo. The identification of these microbial genetic markers, which are predictive of the severity and the course of the disease, will facilitate guided individualized antimicrobial therapy in the future.