bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of Serologic and Genetic
porB
-Based Typing of
Neisseria gonorrhoeae
: Consequences for Future Characterization
Authors: Unemo Magnus , Olcén Per , Albert Jan , Fredlund Hans
Published: September 2003

Due to temporal changes in the epidemiology of gonorrhea, a precise characterization of
Neisseria gonorrhoeae
is essential. In the present study genetic heterogeneity in the
porB
genes of
N. gonorrhoeae
was examined, and serovar determination was compared to
porB
gene sequencing. Among 108
N. gonorrhoeae
isolates, phylogenetic analysis of the entire
porB
alleles (924 to 993 bp) identified 87 unique sequences. By analyzing only the four to six most heterogeneous
porB
gene regions (174 to 363 bp), 86 out of these 87 genetic variants were identified. Consequently, analysis of shorter highly variable regions of the
porB
gene generates high-level discriminatory ability as well as fast, objective, reproducible, and portable data for epidemiological characterization of
N. gonorrhoeae
. Regarding putative antigenic epitopes of PorB for Genetic Systems monoclonal antibodies (MAbs), some of the previous findings were confirmed, but new findings were also observed. For several of the MAbs, however, the precise amino acid residues of PorB critical for single-MAb reactivity were difficult to identify. In addition, repeated serovar determination of 108
N. gonorrhoeae
isolates revealed discrepancies for 34 isolates, mostly due to nonreproducible reactivity with single MAbs. Thus, the prospects of a genetic typing system with congruent translation of the serovar determination seem to be limited. In conclusion, analysis of short highly variable regions of the
porB
gene could form the basis for a fast molecular epidemiological tool for the examination of emergence and transmission of
N. gonorrhoeae
strains within the community.
Molecular Typing of, and Distribution of Genetic Markers among,
Burkholderia cepacia
Complex Isolates from Brazil
Authors: Detsika Maria G. , Corkill John E. , Magalhães Marcelo , Glendinning Kerry J. , Hart C. Anthony , Winstanley Craig
Published: September 2003

PCR tests were used to assign genomovar status to 39 non-cystic fibrosis (non-CF) and 11 CF
Burkholderia cepacia
complex isolates from patients in hospitals in Recife, Brazil. Non-CF isolates were assigned to genomovar IIIA (71.8%), genomovar I (15.4%),
B. vietnamiensis
(7.7%), and
B. multivorans
(5.1%). CF isolates were assigned to genomovar IIIA (18.2%),
B. vietnamiensis
(18.2%), and genomovar I (9.1%). Six CF isolates sharing
recA
PCR-restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) patterns could not be assigned to a genomovar. 16S rDNA sequence obtained from these isolates indicated a closest relationship to
B. anthina
, but the
recA
sequence was equally divergent from several genomovars. PCR screening indicated the presence of
cblA
in only two isolates, whereas the
B. cepacia
epidemic strain marker was found in 22 of 28 genomovar IIIA isolates. A type III secretion gene was detected in all but genomovar I isolates. RAPD and PCR-RFLP assays, targeting both
recA
and
fliC
, indicated a large amount of genetic variability among the isolates, with many novel patterns being observed. Nine genomovar IIIA isolates from different non-CF patients and clinical sources had identical genotypes, indicating the presence of a common clone.
Detection of Immune-Complex-Dissociated Nonstructural-1 Antigen in Patients with Acute Dengue Virus Infections
Authors: Koraka Penelopie , Burghoorn-Maas Chantal P. , Falconar Andrew , Setiati Tatty E. , Djamiatun Kiss , Groen Jan , Osterhaus Albert D. M. E.
Published: September 2003

Accurate and timely diagnosis of dengue virus (DEN) infections is essential for the differential diagnosis of patients with febrile illness and hemorrhagic fever. In the present study, the diagnostic value of a newly developed immune-complex dissociated nonstructural-1 (NS-1) antigen dot blot immunoassay (DBI) was compared to a commercially available DEN antigen detection kit (denKEY Blue kit; Globio Co., Beverly, Mass.) and a reverse transcription-PCR (RT-PCR) kit. Serial serum or plasma samples (
n
= 181) obtained from 55 acute DEN-infected patients were used. In samples obtained from 32 of these 55 DEN-infected patients, viral RNA could be detected by RT-PCR. DEN antigen was detected in only 10 of these 55 patient samples by using the denKEY kit. When these samples were treated with acid to release the immune-complex-associated NS-1 antigen for detection by DBI, 43 of these 55 patients were found to be positive for DEN NS-1 antigen. In nondissociated samples, 22 of these patients were found to be positive by the DBI. In the presence of DEN-specific immunoglobulin M antibodies, both viral RNA and DEN (NS-1) antigen could be detected. The number of positive samples identified by RT-PCR and DBI from these patients with primary DEN infections varied between 28 and 78%. In secondary DEN infections, the number of samples that tested positive by the DBI after immune-complex dissociation (DIS-DBI) was 25% higher than the number of samples that tested positive by RT-PCR and was 35% higher than that determined by nondissociated antigen (NDIS-DBI) detection. We conclude that the denKEY kit has limited diagnostic value for acute DEN infections compared to the RT-PCR and the NDIS-DBI and DIS-DBI methods. We clearly demonstrate that in secondary DEN infections the dissociation of NS-1 immune complexes is essential for early diagnosis of DEN infections.
Comparison of Quantitative Reverse Transcription-PCR to Viral Culture for Assessment of Respiratory Syncytial Virus Shedding
Authors: Falsey Ann R. , Formica Maria A. , Treanor John J. , Walsh Edward E.
Published: September 2003

Respiratory syncytial virus (RSV) has recently been recognized as a serious pathogen in elderly and immunocompromised adults. Diagnosis of acute infection in adults is often difficult due to the insensitivity of viral culture, and reverse transcription-PCR (RT-PCR) is a more sensitive alternative. The relationship of quantitative RT-PCR to viable virus has never been studied for RSV. Therefore, we compared a quantitative real-time RT-PCR with viral culture to assess viral load in adult volunteers challenged with the RSV A2 strain. Twelve of 13 volunteers were infected, and there was a high correlation (r= 0.84) between quantitative RT-PCR and viral titer by cell culture. However, RT-PCR was more sensitive, with 73 of 169 (43%) samples positive compared to 58 (34%) samples positive by culture. The correlation between the two tests was highest early in the course of viral shedding (r= 0.91, days 0 to 6), whereas during days 7 to 13, there was more variability (r= 0.70). All subjects were culture negative by day 11, whereas one subject remained RT-PCR positive on day 12. All subjects were RT-PCR negative at day 28 postinfection. Quantitative RT-PCR has an excellent correlation with viral titers, as measured by culture, and should be a useful tool for future studies addressing viral load and disease pathogenesis.
Intraspecies Genotype Variability of the Microsporidian ParasiteEncephalitozoon hellem
Authors: Haro María , del Águila Carmen , Fenoy Soledad , Henriques-Gil Nuno
Published: September 2003

Seven isolates ofEncephalitozoon hellemfrom human immunodeficiency virus-positive patients were genotyped through a series of markers: the internal transcribed spacer (ITS) of ribosomal DNA, the polar tube protein (PTP) gene, and two intergenic spacers (IGS-TH and IGS-HZ) whose polymorphism is newly reported. The genome markers were all analyzed at three levels: PCR amplification followed by polyacrylamide gel electrophoresis, single-strand conformation analysis (SSCA), and DNA sequencing. The polymorphisms detected involve insertions/deletions and point mutations. SSCA can distinguish any pair of sequences, even those differing by a single base pair. The different isolates studied fit into the previously described ITS genotype 1A, except one which seems to be a 2A derivative variant (2D). When PTP and the new markers IGS-TH and IGS-HZ were analyzed, most of the isolates displayed different genotypes, demonstrating thatE. hellemhas a strong intraspecies variability. A set of markers such as those used here may be very useful in genotyping of clinical samples and in the assessment of epidemiological relationships amongE. hellemstrains.
Development and Evaluation of a Seminested PCR for Detection and Differentiation ofBabesia gibsoni(Asian Genotype) andB. canisDNA in Canine Blood Samples
Authors: Birkenheuer Adam J. , Levy Michael G. , Breitschwerdt Edward B.
Published: September 2003

Canine babesiosis has recently been recognized as an emerging infectious disease of dogs in North America. We sought to develop a seminested PCR to detect and differentiateBabesia gibsoni(Asian genotype),B. canissubsp.vogeli,B. canissubsp.canis, andB. canissubsp.rossiDNA in canine blood samples. An outer primer pair was designed to amplify an ∼340-bp fragment of the 18S rRNA genes fromB. gibsoni(Asian genotype),B. canissubsp.vogeli,B. canissubsp.rossi, andB. canissubsp.canisbut not mammalian DNA. Forward primers were designed that would specifically amplify a smaller fragment from each organism in a seminested PCR. The practical limit of detection was 50 organisms/ml of mock-infected EDTA anticoagulated whole blood. The primer pair also amplified an ∼370-bp fragment of theB. gibsoni(USA/California genotype) 18S rRNA gene from the blood of an experimentally infected dog with a high percentage of parasitemia. Amplicons were not detected when DNA extracted from the blood of a dog that was naturally infected withTheileria annaeat a low percentage of parasitemia was amplified. Due to limited sensitivity, this test is not recommended for the routine diagnosis ofB. gibsoni(USA/California genotype) orT. annae. The PCR test did not amplifyToxoplasma gondii,Neospora caninum,Leishmania infantum,Cryptosporidium parvum, or canine DNA under any of the conditions tested. The seminested PCR test was able to detect and discriminateB. gibsoni(Asian genotype),B. canissubsp.vogeli,B. canissubsp.canis, andB. canissubsp.rossiDNA in blood samples from infected dogs.
Antimicrobial Susceptibility and Serotype Distribution of
Streptococcus pneumoniae
and Molecular Characterization of Multidrug-Resistant Serotype 19F, 6B, and 23F Pneumococci in Northern Thailand
Authors: Watanabe Hiroshi , Asoh Norichika , Hoshino Kazuhiko , Watanabe Kiwao , Oishi Kazunori , Kositsakulchai Weerayut , Sanchai Tippaya , Kunsuikmengrai Khemrassamee , Kahintapong Sumpun , Khantawa Banyong , Tharavichitkul Prasit , Sirisanthana Thira , Nagatake Tsuyoshi
Published: September 2003

Penicillin-resistant
Streptococcus pneumoniae
is widely spread worldwide. Our study was undertaken to examine the susceptibility and serotypes of
S. pneumoniae
in northern Thailand. Ninety-three
S. pneumoniae
strains were isolated from 93 patients at Chiang Mai University Hospital, Chiang Mai, Thailand, from September 1999 to June 2000. The strains were isolated from sputum (
n
= 51), blood (
n
= 15), nasopharynges (
n
= 14), and other sources (e.g., pus, ears, ascites, and cerebrospinal fluid) (
n
= 13). Of the 93 isolates, 29 (31.2%) were susceptible, 24 (25.8%) showed intermediate resistance (MIC, 0.12 to 1.0 μg/ml), and 40 (43.0%) were fully resistant (MIC, ≥2.0 μg/ml) to penicillin G. Seven (46.7%) from blood, 5 (35.7%) from nasopharynges, 15 (29.4%) from sputum, and 2 (15.4%) from other sources were susceptible isolates. Serotyping with the use of antiserum revealed differences in the predominant types that were susceptible (6A, 11A, and 19A), intermediately resistant (6B and 23F), and fully resistant (6B, 19F, and 23F). Molecular typing by pulsed-field gel electrophoresis of multidrug-resistant pneumococci showed four patterns (A, B, C, and D) for 16 isolates of serotype 19F, with pattern B being predominant (12 isolates). This finding was different from that with the Taiwan multidrug-resistant serotype 19F clone. Eleven isolates of serotype 6B all showed pattern E, and nine isolates of serotype 23F showed two patterns (F and G), with pattern F being predominant (seven isolates). This finding was similar to that with the Spanish multidrug-resistant serotype 23F clone. Our results indicated that the resistance of pneumococci to antibiotics in northern Thailand is progressing rapidly and that effort should be intensified to prevent any spread of pandemic multidrug-resistant serotypes 19F, 6B, and 23F.
Recent Emergence of an Epidemic Clindamycin-Resistant Clone of
Clostridium difficile
among Polish Patients with
C. difficile
-Associated Diarrhea
Authors: Pituch Hanna , van Belkum Alex , van den Braak Nicole , Obuch-Woszczatynski Piotr , Verbrugh Henri , Meisel-Mikołajczyk Felicja , Łuczak Mirosław
Published: September 2003

Analysis of both the antibiotic resistance and the virulence characteristics of anaerobic human microbial pathogens is important in order to improve our understanding of a number of clinically significant infectious diseases, including
Clostridium difficile
-associated diarrhea (CDAD). We determined the presence of the clindamycin resistance-associated gene
ermB
and the ribotype of 33
C. difficile
strains isolated from Polish patients suffering from CDAD. While all strains produced cytotoxin B (TcdB), enterotoxin A (TcdA) was produced by a subset of 15 strains only. The results showed that a single
ermB
-positive, TcdA
−
B
+
C. difficile
strain with ribotype A has disseminated widely in the two Warsaw hospitals under investigation. Although different strains with the same phenotype were detected, the genotype A strain appeared to be the only one with a clear epidemic character. Apparently, enhanced local spread of CDAD-causing
C. difficile
may be restricted to a limited number of bacterial genotypes only.
Detection and Analysis of Iron Uptake Components Expressed by
Acinetobacter baumannii
Clinical Isolates
Authors: Dorsey Caleb W. , Beglin Melanie S. , Actis Luis A.
Published: September 2003

The
Acinetobacter baumannii
19606 prototype strain produces a 78-kDa iron-regulated outer membrane protein immunologically related to FatA, which is required for iron acquisition by the fish pathogen
Vibrio anguillarum
via the anguibactin-mediated system. This
A. baumannii
strain also secretes histamine, a biosynthetic precursor of the siderophore anguibactin. In contrast, the
A. baumannii
8399 clinical strain isolated in Oregon produces a siderophore and a putative 73-kDa iron-regulated outer membrane (OM73) receptor that are different from those produced by
V. anguillarum
and
A. baumannii
19606. These observations suggest that different
A. baumannii
clinical isolates express unrelated iron uptake systems. This hypothesis is supported by differences in outer membrane protein profiles among
A. baumannii
isolates obtained from Oregon and Europe. The 19606 isolate and some European isolates expressed a FatA-like protein, while neither 19606 nor any of the European isolates expressed proteins related to OM73. Some European isolates failed to express FatA- and OM73-like proteins. All but one of the Oregon isolates expressed OM73-like proteins, while none of them contained a FatA-like protein. The presence of these proteins always correlated with the presence of the
om73
- and
fatA
-like genes in the cognate strains. While 19606 and a few European isolates produced histamine, none of the Oregon isolates had this capability. Interestingly, one strain each from the Oregon and European isolates did not express any of these products involved in iron acquisition, indicating that they could acquire iron through siderophore-mediated transport systems different from those expressed by the 19606 and 8399 clinical isolates.
Clonal Distribution of Invasive Pneumococcal Isolatesfrom Children and Selected Adults in the United States Prior to7-Valent Conjugate VaccineIntroduction
Authors: Gertz Robert E. , McEllistrem M. Catherine , Boxrud David J. , Li Zhongya , Sakota Varja , Thompson Terry A. , Facklam Richard R. , Besser John M. , Harrison Lee H. , Whitney Cynthia G. , Beall Bernard
Published: September 2003

Theseven-valent pneumococcal conjugated polysaccharide vaccine PC7V was
licensed for use among children in 2000. Since 90 serotypes of
pneumococci exist, an increase in nonvaccine serotypes could occur
through immune selection for capsular type switching. Eleven hundred
sixty-eight invasive isolates (24 serotypes), recovered primarily from
pediatric patients (855 isolates = 73%) and 22 reference
strains of known multilocus sequence types (STs) were subjected to
macrorestriction profiling (pulsed-field gel electrophoresis
[PFGE]). The correlation of 187 ST results (including 49
newly discovered STs) with the PFGE data assigned 1,042 (89.2%)
study isolates to 46 defined clonal complexes or genetic lineages based
on related multilocus STs (BURST). Seventeen clonal complexes were
represented by 2 to 10 related allelic profiles (STs), while 33
lineages (including reference strains) consisted of single STs with 4
or fewer allelic identities to other STs found in the study. Expansion
of the BURST analysis to a global analysis of all known pneumococcal
STs (as of 27 November 2002) reduced the number of single ST lineages
from 33 to 8, and the number of multi-ST clonal complexes was reduced
from 17 to 13. In this work, we established the basic genetic structure
within individual serotypes prior to PC7V use. The resultant database
will be useful for detecting potential selective effects of this
vaccine in postvaccine
surveillance.
Immunoassay Targeting Nonstructural Protein 5 To Differentiate West Nile Virus Infection from Dengue and St. Louis Encephalitis Virus Infections and from Flavivirus Vaccination
Authors: Wong Susan J. , Boyle Rebekah H. , Demarest Valerie L. , Woodmansee Anh N. , Kramer Laura D. , Li Hongmin , Drebot Michael , Koski Raymond A. , Fikrig Erol , Martin Denise A. , Shi Pei-Yong
Published: September 2003

West Nile virus (WNV) is an emerging flavivirus that has caused frequent epidemics since 1996. Besides natural transmission by mosquitoes, WNV can also be transmitted through blood transfusion and organ transplantation, thus heightening the urgency of development of a specific and rapid serologic assay of WNV infection. The current immunoassays lack specificity because they are based on detection of antibodies against WNV structural proteins and immune responses to structural proteins among flaviviruses cross-react to each other. Here, we describe microsphere immunoassays that detect antibodies to nonstructural proteins 3 and 5 (NS3 and NS5). In contrast to immunoassays based on viral envelope and NS3 proteins, the NS5-based assay (i) reliably discriminates between WNV infections and dengue virus or St. Louis encephalitis virus infections, (ii) differentiates between flavivirus vaccination and natural WNV infection, and (iii) indicates recent infections. These unique features of the NS5-based immunoassay will be very useful for both clinical and veterinary diagnosis of WNV infection.
Molecular Typing ofMycobacterium tuberculosisby Using Nine Novel Variable-Number Tandem Repeats across the Beijing Family and Low-Copy-Number IS6110Isolates
Authors: Spurgiesz R. Scott , Quitugua Teresa N. , Smith Kimothy L. , Schupp James , Palmer Eldon G. , Cox Rebecca A. , Keim Paul
Published: September 2003

Molecular epidemiological tools for genotyping clinical isolates ofMycobacterium tuberculosishave been developed and used to help track and contain transmission of tuberculosis. We identified 87 short sequence repeat loci within the genome of theM. tuberculosisH37Rv strain. Nine tandem repeats were found to be variable (variable-number tandem repeats [VNTRs]) in a set of 91 isolates. Fifty-seven of the isolates had only four IS6110bands. The other 34 isolates were members of the Beijing strain family. The number of alleles of each these nine VNTRs was determined by examining each isolate. Six of the loci (Mtb-v1, -v4, -v10, -v15, -v18, and -v20) were able to differentiate the Beijing spoligotype identical isolates into seven distinct genotypes. Five of the loci (Mtb-v3, -v5, -v6, -v10, and -v15) were informative in discriminating the four-band IS6110restriction fragment length polymorphism isolates from each other. The Nei's diversity values of each marker ranged from 0.02 to 0.59, with the number of alleles ranging from two to eight across the entire strain set. These nine loci provide a useful, discriminatory extension of VNTR typing methods for application to molecular epidemiologic studies ofM. tuberculosis.
Development and Application of Real-Time PCR Assay for Quantification of
Mycobacterium ulcerans
DNA
Authors: Rondini S. , Mensah-Quainoo E. , Troll H. , Bodmer T. , Pluschke G.
Published: September 2003

Buruli
ulcer, an infection caused by
Mycobacterium ulcerans
, is,
after tuberculosis and leprosy, the third most common mycobacterial
disease. The mode of transmission of
M. ulcerans
is not
exactly known, but since Buruli ulcer often occurs in focalized swampy
areas, it is assumed that there is a reservoir of the pathogen in
stagnant water. Buruli ulcer usually starts as a painless nodule and
can lead to massive destruction of skin, subcutaneous tissue, and
eventually muscle and bone. Currently the only recommended treatment is
wide surgical excision. In this report we describe the development of a
real-time PCR method for the quantification of
M. ulcerans
DNA
(IS2404 TaqMan). The highly specific assay is based on the detection of
the
M. ulcerans
specific insertion sequence IS
2404
.
The IS2404 TaqMan assay turned out to be about 10 times more sensitive
than the available conventional PCR-based diagnostic test. It is
demonstrated that the IS2404 TaqMan assay is suitable for the
quantitative assessment of the dissemination of the mycobacteria in
Buruli ulcer lesions. Prototype results obtained with excised tissue
from a patient with a late preulcerative Buruli ulcer lesion
reconfirmed earlier histopathological findings indicating that tissue
damage occurs far beyond the regions in which large numbers of
mycobacteria are detectable. The IS2404 TaqMan assay should be a useful
tool for both diagnosis and research into the pathology and mode of
transmission of this still inadequately investigated mycobacterial
disease.
Cohort Study of Guinean Children: Incidence, Pathogenicity, Conferred Protection, and Attributable Risk for Enteropathogens during the First 2 Years of Life
Authors: Valentiner-Branth Palle , Steinsland Hans , Fischer Thea K. , Perch Michael , Scheutz Flemming , Dias Francisco , Aaby Peter , Mølbak Kåre , Sommerfelt Halvor
Published: September 2003

We recruited 200 children shortly after birth and collected stool specimens weekly, irrespective of whether the children had diarrhea, until up to 2 years of age. All children were recruited during the first year of the study and were monitored for a median of 18.4 months. To measure pathogenicity, the odds ratio for diarrhea, adjusted for age, sex, and coinfections with other enteropathogens, was determined by logistic regression. Standard estimation of the population attributable risk indicated that rotavirus, enterotoxigenicEscherichia colithat produced only the heat-stable toxin ST,Isosporaspp.,Cryptosporidium parvum, Shiga toxin (Stx)-producingE. coli(STEC), andShigellaspp. or enteroinvasiveE. coliwere the most important contributors to diarrhea in this population. Stx2- but not Stx1-producing STEC strains were pathogenic. EnteroaggregativeE. coli, diffusely adherentE. coli, and attaching-and-effacingE. colistrains, which were the most commonly isolated microorganisms, were not associated with diarrhea. For most of the microorganisms, primary infections did not confer protection against reinfection with the same organism, but some conferred protection against diarrhea from reinfection.
Presence of
Pseudomonas putida
Strains Harboring Plasmids Bearing the Metallo-β-Lactamase Gene
bla
IMP
in a Hospital in Japan
Authors: Yomoda Sachie , Okubo Toyoji , Takahashi Ayako , Murakami Masami , Iyobe Shizuko
Published: September 2003

To determine the persistence and spread of antibiotic-resistant strains in Gunma University Hospital, 83
Pseudomonas putida
strains (each from a different patient) were isolated from January 1997 through December 2001. Of the 83 strains isolated, 27 were resistant to carbapenems. All 27 produced metallo-β-lactamase and were found to be PCR positive for the
bla
IMP
gene. Most (22 strains) were primarily isolated from the wards (W7 [9 strains] and W4 [8 strains]). Another five
bla
IMP
-positive
P. putida
strains from wards W7 and W4 were obtained by swabbing around the water pipes. A total of 32
bla
IMP
-positive
P. putida
strains were assessed by pulsed-field gel electrophoresis (PFGE) and testing of drug susceptibility to 10 chemotherapeutic agents. Both PFGE and MIC patterns revealed that there were long-term resident strains among inpatients and hospital environments. The
bla
IMP
genes of 22 of 32 strains were all transferable to a recipient strain of
Pseudomonas aeruginosa
by conjugation or transformation and conferred resistance to carbapenems and cephems. The
bla
IMP
plasmids were conjugally transmissible among
P. aeruginosa
strains and mediated resistance to amikacin as well as β-lactams. Ten of the 22 plasmids mediated additional resistance to gentamicin and tobramycin. Plasmids with identical DNA and drug resistance patterns were found in
P. putida
strains with identical PFGE patterns and with different PFGE patterns. We presumed that
P. putida
was one of the resident species in inpatients and especially in hospital environments, spreading drug resistance genes via plasmids among
P. putida
strains and supplying them to more pathogenically important species, such as
P. aeruginosa
.
Modification of Rapid Susceptibility Assay for Antifungal Susceptibility Testing of
Aspergillus fumigatus
Authors: Wetter Tracy J. , Hazen Kevin C. , Cutler Jim E.
Published: September 2003

To improve objectivity and speed of current antifungal mold susceptibility testing, the yeast Rapid Susceptibility Assay (RSA) was adapted for
Aspergillus
species. The RSA is based on glucose utilization in the presence of an antifungal drug.
Aspergillus fumigatus
conidia were incubated in 0.2% glucose RPMI 1640 containing 0.03 to 16 μg of amphotericin B or itraconazole/ml. Drug-related inhibition of glucose utilization correlated with suppression of conidial germination. Following incubation of conidia with various concentrations of antifungal drug, the percentage of residual glucose in the growth medium was determined colorimetrically and plotted against drug concentration to determine the MIC (MIC
RSA
). National Committee for Clinical Laboratory Standards (NCCLS) M38-P testing was also performed to obtain NCCLS MICs (MIC
NCCLS
) for direct comparison with MIC
RSA
s. Conidial inocula of an optical density at 530 nm (OD
530
) of 0.11 facilitated determination of amphotericin B and itraconazole MIC
RSA
s at 16 h equal to or within a single twofold dilution of MIC
NCCLS
s obtained at 48 h. Preliminary testing with a 0.11-OD
530
conidial inoculum of the slower-growing
Aspergillus terreus
resulted in itraconazole and amphotericin B MIC
RSA
s at 16 h equal to or within a single twofold dilution of MIC
NCCLS
s obtained at 48 h. These data indicate that the mold RSA provides a more objective and rapid method for
Aspergillus
spp. susceptibility testing than the NCCLS M38-P assay.
Use of the VITEK 2 System for Rapid Identification of Clinical Isolates of Staphylococci from Bloodstream Infections
Authors: Spanu Teresa , Sanguinetti Maurizio , Ciccaglione Daniela , D'Inzeo Tiziana , Romano Lucio , Leone Fiammetta , Fadda Giovanni
Published: September 2003

Staphylococci are an increasing cause of bloodstream infections. Rapid reliable identification of these organisms is essential for accurate diagnosis and prompt effective treatment. We evaluated the ability of the VITEK 2 system (bioMérieux, Inc, Hazelwood, Mo.) to identify these organisms rapidly and accurately. A total of 405 clinically relevant nonduplicate staphylococcal isolates (
Staphylococcus aureus
,
n
= 130; coagulase-negative staphylococci,
n
= 275) collected from blood cultures were tested. VITEK 2 results were considered correct when they were identical to those furnished by the comparison method based on the ID 32 STAPH system (bioMérieux, Marcy l'Etoile, France) plus supplementary manual testing. When discrepancies occurred, isolate identity was verified by molecular typing. The VITEK 2 correctly identified 387 (95.6%) isolates at the species level: 379 (including all but one [99.2%] of 130
S. aureus
isolates and 249 of 275 [90.5%] coagulase-negative isolates) were identified by the automated reading; for the other eight, supplemental tests suggested by the manufacturer had to be used. Only one strain (0.2%) was misidentified (
Staphylococcus hominis
as
Staphylococcus epidermidis
), and four (1%), all
S. epidermidis
, were not identified. For the remaining 13 strains (including 10
S. hominis
), the VITEK 2 system was unable to discriminate among two species, and no supplemental tests were suggested for conclusive identification. Over 90% of results were obtained within 4 h. These results suggest that the VITEK 2 system can provide rapid, accurate, and reliable species-level identification of staphylococci responsible for bloodstream infections, although there is room for improvement in the identification of certain coagulase-negative species, especially
S. hominis
.
Multiple CTX-M-Type Extended-Spectrum β-Lactamases in Nosocomial Isolates of
Enterobacteriaceae
from a Hospital in Northern Italy
Authors: Pagani Laura , Dell'Amico Emanuela , Migliavacca Roberta , D'Andrea Marco Maria , Giacobone Ernesto , Amicosante Gianfranco , Romero Egidio , Rossolini Gian Maria
Published: September 2003

Twelve isolates of
Enterobacteriaceae
(1 of
Klebsiella pneumoniae
, 8 of
Escherichia coli
, 1 of
Proteus mirabilis
, and 2 of
Proteus vulgaris
) classified as extended-spectrum β-lactamase (ESBL) producers according to the ESBL screen flow application of the BD-Phoenix automatic system and for which the cefotaxime MICs were higher than those of ceftazidime were collected between January 2001 and July 2002 at the Laboratory of Clinical Microbiology of the San Matteo University Hospital of Pavia (northern Italy). By PCR and sequencing, a CTX-M-type determinant was detected in six isolates, including three of
E. coli
(carrying
bla
CTX-M-1
), two of
P. vulgaris
(carrying
bla
CTX-M-2
), and one of
K. pneumoniae
(carrying
bla
CTX-M-15
). The three CTX-M-1-producing
E. coli
isolates were from different wards, and genotyping by pulsed-field gel electrophoresis (PFGE) revealed that they were clonally unrelated to each other. The two CTX-M-2-producing
P. vulgaris
isolates were from the same ward (although isolated several months apart), and PFGE analysis revealed probable clonal relatedness. The
bla
CTX-M-1
and
bla
CTX-M-2
determinants were transferable to
E. coli
by conjugation, while conjugative transfer of the
bla
CTX-M-15
determinant from
K. pneumoniae
was not detectable. Present findings indicate that CTX-M enzymes of various types are present also in Italy and underscore that different CTX-M determinants can be found in a single hospital and can show different dissemination patterns. This is also the first report of CTX-M-2 in
P. vulgaris
.
Molecular Properties of
Salmonella enterica
Serotype Paratyphi B Distinguish between Its Systemic and Its Enteric Pathovars
Authors: Prager Rita , Rabsch Wolfgang , Streckel Wiebke , Voigt Wolfgang , Tietze Erhardt , Tschäpe Helmut
Published: September 2003

Salmonella enterica
serotype O1,4,5,12:Hb:1,2, designated according to the current Kauffmann-White scheme as
S. enterica
serotype Paratyphi B, is a very diverse serotype with respect to its clinical and microbiological properties. PCR and blot techniques, which identify the presence, polymorphism, and expression of various effector protein genes, help to distinguish between strains with systemic and enteric outcomes of disease. All serotype Paratyphi B strains from systemic infections have been found to be somewhat genetically related with respect to the pattern of their virulence genes
sopB
,
sopD
,
sopE1
,
avrA
, and
sptP
as well as other molecular properties (multilocus enzyme electrophoresis type, pulsed-field gel electrophoresis [PFGE] type, ribotype, and IS
200
type). They have been classified as members of the systemic pathovar (SPV). All these SPV strains possess a new
sopE1
-carrying bacteriophage (designated ΦSopE309) with high SopE1 protein expression but lack the commonly occurring
avrA
determinant. They exhibit normal SopB protein expression but lack SopD protein production. In contrast, strains from enteric infections classified as belonging to the enteric pathovar possess various combinations of the respective virulence genes, PFGE pattern, and ribotypes. We propose that the PCR technique for testing for the presence of the virulence genes
sopE1
and
avrA
be used as a diagnostic tool for identifying both pathovars of
S. enterica
serotype Paratyphi B. This will be of great public health importance, since strains of serotype Paratyphi B have recently reemerged worldwide.
Phage-Based Typing Scheme for
Salmonella enterica
Serovar Heidelberg, a Causative Agent of Food Poisonings in Canada
Authors: Demczuk Walter , Soule Geoff , Clark Clifford , Ackermann Hans-W. , Easy Russell , Khakhria Rasik , Rodgers Frank , Ahmed Rafiq
Published: September 2003

Salmonella enterica
serovar Heidelberg is perhaps the second most frequent
Salmonella
serovar isolated from humans and the most common isolated from animals in Canada. This pathogen has shown increasing resistance to antimicrobial agents and mimics the multidrug resistance observed in
S. enterica
serovar Typhimurium strain DT 104. However, unlike for serovar Typhimurium, a rapid and inexpensive subtyping method has not been available for large-scale surveillance efforts. We developed a phage typing scheme and subtyped 2,523 strains of serovar Heidelberg from outbreaks, sporadic infections, and environmental sources in Canada between January 1991 and December 2000. All strains were sensitive to one or more phages and could be subdivided into 49 phage types. A total of 196 isolates from 13 major outbreaks could be subtyped into six phage types, while 86 strains from family outbreaks were assigned to seven phage types. All strains were typeable, and epidemiologically related strains isolated from patients and implicated foods had identical phage types, antibiograms, and pulsed-field gel electrophoresis (PFGE) patterns. Combining PFGE with phage typing increased the discriminatory power of the analysis beyond that of either method alone. We concluded that this phage typing scheme, in conjunction with PFGE, enhances subtyping of serovar Heidelberg strains. Furthermore, this phage typing scheme is a rapid, economical, stable, and reliable epidemiologic tool for tracing the origin of food-borne disease and for the surveillance of sporadic infections.
Production of Cytolethal Distending Toxins by Pathogenic
Escherichia coli
Strains Isolated from Human and Animal Sources: Establishment of the Existence of a New
cdt
Variant (Type IV)
Authors: Tóth István , Hérault Fréderique , Beutin Lothar , Oswald Eric
Published: September 2003

Three types of cytolethal distending toxin (CDT), namely, CDT-I, CDT-II, and CDT-III, have been described in
Escherichia coli
. Using primers designed for the detection of sequences common to the
cdtB
genes, we analyzed by PCR a set of 21 CDT-producing
E. coli
strains of intestinal and extraintestinal origins isolated from human and different animal species in several European countries and in the United States. On the basis of the existing differences in the
cdtB
genes,
cdt
-I-,
cdt
-II-, and
cdt
-III-specific primer pairs were designed and used for
cdt
typing. These new primers successfully differentiated all of the previously described
cdt
genes. Six strains proved to be
cdt-
I; eight strains proved to be
cdt
-III. However, none of the type I-, II-, and III-specific primers generated amplicons from six CDT
+
strains, suggesting the existence of a new
cdt
variant. Sequence analysis of the amplicons from two untypeable genes confirmed the existence of a new
cdt
variant that we called
cdt
-IV. Using the new specific primers,
cdt
-IV was detected in human, porcine, and poultry strains of intestinal and extraintestinal origins. To validate all sets of
cdt
specific primers, a group of 353 human
E. coli
strains isolated in Hungary was then investigated for the presence of
cdt
genes. This included 190 strains isolated from patients with urinary tract infections (UTI), 51 strains isolated from other (nonurinary) extraintestinal infections, and 112 intestinal strains isolated from healthy individuals. Of 190 UTI strains, 15 (7.9%) had
cdt
genes. Of 51 non-UTI extraintestinal strains 3 (5.9%) contained the
cdt
gene, and 1 (0.9%) of 112 healthy intestinal strains was PCR positive. Five strains proved to be
cdt
-I, and fourteen strains proved to be
cdt
-IV. The CDT-producing extraintestinal strains belonged to a wide variety of serogroups, including O2, O6, O75, and O170. In conclusion, we have developed a new PCR typing system for CDT able to detect a new CDT variant present in pathogenic
E. coli
strains obtained from animals and humans.
Discrimination of
d
-Tartrate-Fermenting and -Nonfermenting
Salmonella enterica
subsp.
enterica
Isolates by Genotypic and Phenotypic Methods
Authors: Malorny Burkhard , Bunge Cornelia , Helmuth Reiner
Published: September 2003

A multiplex PCR and an improved lead acetate test were developed to discriminate
d
-tartrate-fermenting and -nonfermenting
Salmonella enterica
subsp.
enterica
strains. Both methods showed an accuracy of 100% when 125
Salmonella
strains belonging to 15 serovars were tested. Special emphasis was given to
S. enterica
subsp.
enterica
serovar Paratyphi B isolates because of the clinical importance of its
d
-tartrate-nonfermenting variant and the recently increasing numbers of cases of human outbreaks caused by its fermenting variant (formerly
Salmonella
serovar Java). The lead acetate test described previously (G. A. Alfredsson, R. M. Barker, D. C. Old, and J. P. Duguid, J. Hyg. 70:651-666, 1972) was modified in the inoculation and incubation procedure. The PCR assay was based on the genotypic difference of the presence (
d
-tartrate-fermenting strains) or absence (
d
-tartrate-nonfermenting strains) of the ATG start codon for the gene STM 3356, which encodes a putative cation transporter. Sequence data revealed a nucleotide exchange from G to A within the ATG start codon of gene STM 3356 in the
d
-tartrate-nonfermenting strains. In order to increase the reliability of the PCR assay, a positive control based on a
Salmonella
genus-specific primer set for the detection of
Salmonella
DNA was included. The PCR-based discrimination needs only several hours compared to 6 days needed by the improved lead acetate test to obtain reliable results. Consequently, the PCR
d
-tartrate assay should be the method of choice for the discrimination of
d
-tartrate-fermenting and -nonfermenting
Salmonella
strains in the future.
GeneScan Reverse Transcription-PCR Assay for Detection of Six Common Respiratory Viruses in Young Children Hospitalized with Acute Respiratory Illness
Authors: Erdman Dean D. , Weinberg Geoffrey A. , Edwards Kathryn M. , Walker Frances J. , Anderson Barbara C. , Winter Jörn , González Monica , Anderson Larry J.
Published: September 2003

A reverse transcription-PCR (RT-PCR) assay based on automated fluorescent capillary electrophoresis and GeneScan software analysis was developed to detect six common respiratory viruses in clinical specimens from young children. Assays for human respiratory syncytial virus (HRSV); human parainfluenza viruses 1, 2, and 3 (HPIV1, -2, and -3, respectively); and influenza A and B viruses were incorporated into a single standard assay format. The optimized assay panel was used to test 470 respiratory specimens obtained from 462 children hospitalized with acute respiratory illness that had been previously tested by viral culture (405 specimens) or direct immunofluorescence staining (DIF) (65 specimens). Of 93 specimens positive for respiratory viruses by culture or DIF, 86 (92%) were positive by RT-PCR, including 66 HRSV, 2 HPIV2, 5 HPIV3, 3 influenza A virus, and 10 influenza B virus specimens. An additional 119 respiratory viruses were identified by RT-PCR in 116 patients for whom results were negative by viral isolation or DIF. We conclude that the GeneScan RT-PCR panel can markedly improve detection of acute respiratory virus infections in young children.
Semiquantitative Detection by Real-Time PCR ofAspergillus fumigatusin Bronchoalveolar Lavage Fluids and Tissue Biopsy Specimens from Patients with Invasive Aspergillosis
Authors: Rantakokko-Jalava Kaisu , Laaksonen Sanna , Issakainen Jouni , Vauras Jukka , Nikoskelainen Jukka , Viljanen Matti K. , Salonen Juha
Published: September 2003

A real-time PCR method was developed and used to detectAspergillus fumigatusmitochondrial DNA (mtDNA) in bronchoalveolar lavage (BAL) fluids and tissue biopsy specimens. The analytical sensitivity of the assay was oneA. fumigatusconidium per reaction, and the assay was linear at least over 4 orders of magnitude above the detection limit. BAL fluids from 66 immunocompromised patients at risk of invasive pulmonary aspergillosis (IPA) and 33 immunocompetent controls and tissue biopsy specimens from 10 immunocompromised patients were analyzed. The results were related to the clinical diagnosis established according to recently published consensus criteria.A. fumigatusmtDNA positivity was encountered in 16 of 81 (20%) BAL fluid specimens from patients at risk and 1 of 33 (3%) specimens from immunocompetent controls. PCRs were positive in six of seven, two of four, and four of five of the patients with proven, probable, and possible IPA, respectively, as well as in four patients at risk but without any other evidence of IPA. With qualitative detection, the diagnostic sensitivity of PCR was 73%, specificity was 93%, and predictive values of positive (PPV) and negative (NPV) results were 73 and 95%, respectively. Using a threshold cycle of <35 as a limit for positive PCR, the specificity and PPV of PCR in the diagnosis of invasive aspergillosis were 100%, but its sensitivity was only 45% and NPV was 92%. PCR was positive in tissue biopsy specimens from all patients with invasive aspergillosis caused byA. fumigatus. Semiquantitative detection ofA. fumigatusmtDNA in BAL fluid may be helpful in the diagnosis of IPA. PCR is well suited for the verification of the presence ofA. fumigatusin tissue biopsy specimens.
Use of Real-Time PCR with Multiple Targets To Identify
Pseudomonas aeruginosa
and Other Nonfermenting Gram-Negative Bacilli from Patients with Cystic Fibrosis
Authors: Qin Xuan , Emerson Julia , Stapp Jenny , Stapp Lynn , Abe Patrick , Burns Jane L.
Published: September 2003

Pseudomonas aeruginosa
and other gram-negative isolates from patients with cystic fibrosis (CF) may be difficult to identify because of their marked phenotypic diversity. We examined 200 gram-negative clinical isolates from CF respiratory tract specimens and compared identification by biochemical testing and real-time PCR with multiple different target sequences using a standardized combination of biochemical testing and molecular identification, including 16S rRNA partial sequencing and
gyrB
PCR and sequencing as a “gold standard.” Of 50 isolates easily identified phenotypically as
P. aeruginosa
, all were positive with PCR primers for
gyrB
or
oprI
, 98% were positive with exotoxin A primers, and 90% were positive with
algD
primers. Of 50
P. aeruginosa
isolates that could be identified by basic biochemical testing, 100% were positive by real-time PCR with
gyrB
or
oprI
primers, 96% were positive with exotoxin A primers, and 92% were positive with
algD
primers. For isolates requiring more-extensive biochemical evaluation, 13 isolates were identified as
P. aeruginosa
; all 13 were positive with
gyrB
primers, 12 of 13 were positive with
oprI
primers, 11 of 13 were positive with exotoxin A primers, and 10 of 13 were positive with
algD
primers. A single false-positive
P. aeruginosa
result was seen with
oprI
primers. The best-performing commercial biochemical testing was in exact agreement with molecular identification only 60% of the time for this most difficult group. Real-time PCR had costs similar to those of commercial biochemical testing but a much shorter turnaround time. Given the diversity of these CF isolates, real-time PCR with a combination of two target sequences appears to be the optimum choice for identification of atypical
P. aeruginosa
and for non-
P. aeruginosa
gram-negative isolates.
Antimicrobial Susceptibility Testing of Aquatic Bacteria: Quality Control Disk Diffusion Ranges for
Escherichia coli
ATCC 25922 and
Aeromonas salmonicida
subsp.
salmonicida
ATCC 33658 at 22 and 28°C
Authors: Miller R. A. , Walker R. D. , Baya A. , Clemens K. , Coles M. , Hawke J. P. , Henricson B. E. , Hsu H. M. , Mathers J. J. , Oaks J. L. , Papapetropoulou M. , Reimschuessel R.
Published: September 2003

Quality control (QC) ranges for disk diffusion susceptibility testing of aquatic bacterial isolates were proposed as a result of a multilaboratory study conducted according to procedures established by the National Committee for Clinical Laboratory Standards (NCCLS). Ranges were proposed for
Escherichia coli
ATCC 25922 and
Aeromonas salmonicida
subsp.
salmonicida
ATCC 33658 at 22 and 28°C for nine different antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, gentamicin, oxolinic acid, oxytetracycline, ormetoprim-sulfadimethoxine, and trimethoprim-sulfamethoxazole). All tests were conducted on standard Mueller-Hinton agar. With ≥95% of all data points fitting within the proposed QC ranges, the results from this study comply with NCCLS guidelines and have been accepted by the NCCLS Subcommittee for Veterinary Antimicrobial Susceptibility Testing. These QC guidelines will permit greater accuracy in interpreting results and, for the first time, the ability to reliably compare susceptibility test data between aquatic animal disease diagnostic laboratories.
Evaluation of Three Rapid Methods for the Direct Identification of
Staphylococcus aureus
from Positive Blood Cultures
Authors: Chapin Kimberle , Musgnug Michael
Published: September 2003

Staphylococci represent the most commonly encountered blood culture isolates. Differentiating
Staphylococcus aureus
from coagulase-negative staphylococci (CoNS) is important in guiding empirical therapy, especially since the majority of CoNS are contaminants. This study evaluated three rapid methods for the direct identification of
S. aureus
from blood cultures. A total of 157 patient blood cultures with gram stains showing gram-positive cocci in clusters were included. The following assays were evaluated: API RAPIDEC staph (API) (bioMerieux, Durham, N.C.), the tube coagulase test (TCT) read at 4 h, and peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) (AdvanDx, Woburn, Mass.). All assays yielded results of
S. aureus
or non-
S. aureus.
The direct rapid results were compared to results obtained with isolated colonies using the AccuProbe
Staphylococcus aureus
Culture Identification Test (Gen-Probe, San Diego, Calif.). API, TCT, and PNA FISH exhibited sensitivities of 96, 84, and 99% and specificities of 99, 100, and 100%, respectively. Direct identification testing by any of these three assays yielded acceptable performance and timely results. This ability to accurately detect
S. aureus
in blood cultures gives the physician information with which to initiate or tailor antimicrobial therapy. Coupled with direct susceptibility testing of positive blood culture broths, the patient and institution may experience improved outcomes.