bluuebunny/crossref_metadata_2025 huggingface dataset:
Diagnosing Herpesvirus Infections by Real-Time Amplification and Rapid Culture
Authors: van Doornum G. J. J. , Guldemeester J. , Osterhaus A. D. M. E. , Niesters H. G. M.
Published: February 2003

Procedures using real-time technique were developed to demonstrate the presence of herpes simplex virus type 1 (HSV-1) and HSV-2, varicella zoster virus (VZV), and cytomegalovirus (CMV) in miscellaneous clinical specimens. The assays were compared to rapid culture using centrifugation followed by detection with monoclonal antibodies. A total of 711 consecutive samples were collected from different patient groups. Throat swabs were obtained from transplant patients; dermal or oral specimens were collected from patients suspected for VZV or HSV infection. Genital specimens were taken from patients who attended the Clinic for Sexually Transmitted Diseases at the Dijkzigt Hospital Rotterdam presenting with symptoms of a primary genital ulcer. Nucleic acid extraction was carried out using a MagnaPure LC instrument. The amplification steps were performed on the ABI Prism 7700 sequence detection system. To monitor the process of extraction and amplification, a universal control consisting of seal herpesvirus type 1 (PhHV-1) was added to the clinical specimens. By culture 127 of 668 (19%) samples were positive for HSV-1, 72 of 668 (10.8%) specimens were positive for HSV-2, and 17 of 366 (4.6%) were positive for VZV. Using real-time amplification the numbers of positive specimens were 143 of 668 (21.4%), 97 of 668 (14.5%), and 27 of 366 (7.4%), respectively. Eighty-six specimens were tested for CMV, 12 (14.0%) were positive by culture, and 17 (19.8%) were positive by real-time PCR. The clinical data of the patients with discrepant results were reviewed thoroughly. In all cases the patients with only real-time PCR-positive results could be considered as truly infected. We concluded that the real-time amplification technique is suitable for the detection of human herpesvirus infection. It offers a semiquantitative and reliable assay with a quick result that is more sensitive than rapid culture, especially for the diagnosis of HSV-2 and VZV infections.
Clonal Diversity among Streptogramin A-Resistant
Staphylococcus aureus
Isolates Collected in French Hospitals
Authors: Haroche Julien , Morvan Anne , Davi Marilyne , Allignet Jeanine , Bimet François , El Solh Névine
Published: February 2003

We analyzed 62 clinical isolates of streptogramin A-resistant (SGA
r
)
Staphylococcus aureus
collected between 1981 and 2001 in 14 hospitals located in seven French cities. These isolates, including five with decreased susceptibility to glycopeptides, were distributed into 45 antibiotypes and 38
Sma
I genotypes. Each of these genotypes included between 1 and 11 isolates, the
Sma
I patterns of which differed by no more than three bands. Although numerous clones were identified, we observed the spread of monoclonal isolates either within the same hospital or within hospitals in distinct cities and at large time intervals. Hybridization with probes directed against 10 SGA
r
genes (
vatA
,
vatB
,
vatC
,
vatD
,
vatE
,
vgaA
,
vgaB
,
vgaAv
,
vgbA
, and
vgbB
) revealed six patterns:
vgaAv
(21 isolates),
vatA
-
vgbA
(24 isolates),
vgaAv
-
vatB
-
vgaB
(14 isolates),
vgaAv
-
vatA
-
vgbA
(1 isolate),
vgaAv
-
vatA
-
vgbA
-
vatB
-
vgaB
(1 isolate), and
vgaA
(1 isolate). We detected at least one SGA
r
determinant in all of the tested isolates.
vgaAv
, which is part of the recently characterized transposon Tn
5406
, was found in 59.7% of the tested isolates. Of the 16 streptogramin B-susceptible isolates, 14 carried
vgaAv
alone and were susceptible to the mixtures of streptogramins, whereas the 2 isolates carrying
vgaAv
-
vatB
-
vgaB
were resistant to these mixtures.
vatA
-
vgbA
was found on plasmids of the same apparent size in 26 (42%) of the tested clinical isolates from 18 unrelated
Sma
I genotypes. The possible dissemination of some of the multiple clones characterized in the present study with an expected increased selective pressure of streptogramins following the recent licensing of Synercid (quinupristin-dalfopristin) must be carefully monitored.
Comparison of a New Quantitative
ompA
-Based Real-Time PCR TaqMan Assay for Detection of
Chlamydia pneumoniae
DNA in Respiratory Specimens with Four Conventional PCR Assays
Authors: Apfalter Petra , Barousch Wolfgang , Nehr Marion , Makristathis Athanasios , Willinger Birgit , Rotter Manfred , Hirschl Alexander M.
Published: February 2003

Chlamydia pneumoniae
, an important respiratory pathogen, is difficult to culture, and detection rates by conventional PCRs vary considerably. A new quantitative
ompA
-based real-time PCR assay based on TaqMan technology for detection of
C. pneumoniae
in respiratory samples is described, and its performance in terms of sensitivity and reproducibility is compared with those of four published conventional PCRs (one single-step PCR targeting a cloned
Pst
I fragment; two nested PCRs, one targeting the 16S rRNA gene followed by hybridization and the other targeting the
ompA
gene; and a touchdown enzyme time-release [TETR] PCR also targeting the 16S rRNA gene). Both
ompA
-based PCRs showed the best analytical sensitivity. All five assays could detect even lower target levels from spiked sputum, with the 16S rRNA assays performing better than the
ompA
-based nested PCR (10
−6
inclusion-forming units [IFU] were detected in four of four and two of four replicates by the 16S rRNA TETR PCR and the 16S rRNA nested PCR, respectively). In general, the
ompA
-based real-time protocol produced the most consistent positive results for all replicates tested down to 10
−6
IFU. Eight of 45 patient sputum specimens (18%) were
C. pneumoniae
DNA positive in at least one of four replicates tested by at least one assay. Without taking into consideration the analytical sensitivity or the reproducibility of the test results, the numbers of
C. pneumoniae
DNA-positive sputum specimens (
n
= 8) were four, three, two, two, and one for the 16S rRNA TETR assay, the
Pst
I-based single-step PCR, the
ompA
-based real-time PCR, the
ompA
-based nested touchdown PCR, and the 16S rRNA-based nested PCR, respectively. However, the overall rate of concordance of positive results was low. Only one cell culture-positive sputum specimen was positive by four of five assays (14 of 16 replicates; mean cycle threshold value, 25; 10
8
particles/ml of sputum). Thirty-seven specimens were
C. pneumoniae
negative by all five assays for all replicates tested, as were all negative controls (
n
= 65 to 100 per testing panel). No PCR inhibitors were detected by real-time PCR or by the 16S rRNA-based nested assay. We confirm that the analytical sensitivity of an assay for the detection of
C. pneumoniae
does not necessarily predict its ability to detect its target in sputum. A quantitative, fast, and easy-to-handle diagnostic approach such as the
ompA
-based real-time TaqMan PCR described here might improve the detection of
C. pneumoniae
in respiratory samples.
Evaluation of Semiautomated Multiplex PCR Assay for Determination of
Streptococcus pneumoniae
Serotypes and Serogroups
Authors: Lawrence Elliot R. , Griffiths David B. , Martin Siobhán A. , George Robert C. , Hall Lucinda M. C.
Published: February 2003

A semiautomated method for the determination of five serotypes and three serogroups in
Streptococcus pneumoniae
was developed. Primers specific for serotypes 1, 3, 14, 19F, and 23F and serogroups 6, 19, and 23 were combined in three multiplex PCRs. Products were separated by capillary electrophoresis with a 7-min run time, and a serotype or serogroup was assigned on the basis of fragment size. The method was used to test 93 clinical isolates, and all isolates of the serotypes concerned were correctly detected. The strategy would allow the detection of multiple serotypes in a single sample. Detection of additional serotypes could be included as capsule locus sequences become available.
Human Papillomavirus Type 16 Status in Cervical Carcinoma Cell DNA Assayed by Multiplex PCR
Authors: Lukaszuk Krzysztof , Liss Joanna , Wozniak Izabela , Emerich Janusz , Wójcikowski Czesław
Published: February 2003

Integration of human papillomavirus (HPV) DNA into host genome occurs early in cancer development and is probably an important event in malignant transformation of cervical cancer. The HPV genome integration usually disrupts
E2
gene open reading frames. It results in the lack of
E2
gene suppressor of the synthesis of E6 and E7 products which, in turn, leads to the overexpression of
E6
and
E7
genes. The oncogenic HPV types (HPV16, -18, -45, and -58) can be present as episomes or may integrate into human chromosomes. Sixty-six cervical cancer patients positive for HPV16 were tested for the presence of
E6
,
E2
,
E1
, and
L1
genes. Multiplex PCR was carried out in all cases. Using cluster analysis, the calculated ratios of
E1/E6
,
E2/E6
,
L1/E6
,
E1/E2
, and
E2/
(
E1*E6
) gene amplification products were divided into two or three statistically different groups. These were used for statistical analysis of the prevalence of specific gene types in histological types of cancer, different levels of clinical staging, and histologically confirmed nodal metastases. The statistical analysis proved a significant correlation in the ratios of
E2/E6
and
E1/E2
only. The
E2/E6
and
E1/E2
were higher in carcinoma in situ than in advanced squamous cancers. The
E2/E6
ratios were lower in higher clinical stages. The multiplex PCR estimation of the
E2/E6
ratio could be a simple method for selecting patients with a high risk of a poor outcome in a standard stage-dependent treatment procedure.
Analysis of a Viridans Group Strain Reveals a Case of Bacteremia Due to Lancefield Group G Alpha-Hemolytic
Streptococcus dysgalactiae
subsp.
equisimilis
in a Patient with Pyomyositis and Reactive Arthritis
Authors: Woo Patrick C. Y. , Teng Jade L. L. , Lau Susanna K. P. , Lum Peggy N. L. , Leung Kit-Wah , Wong Kee-Lam , Li Kin-Wah , Lam Kui-Chun , Yuen Kwok-Yung
Published: February 2003

Streptococcus dysgalactiae
is classified by a combination of phenotypic and genotypic characteristics into Lancefield group C alpha-hemolytic
Streptococcus dysgalactiae
subsp.
dysgalactiae
and Lancefield group C, group G, and group L beta-hemolytic
Streptococcus dysgalactiae
subsp.
equisimilis
. In this study, we report the isolation of a catalase-negative, alpha-hemolytic, optochin- and bacitracin-resistant viridans group strain, which does not grow in 10 or 40% bile, on MacConkey agar or bile esculin agar, or in 6% NaCl, from the blood culture of a 73-year-old woman with pyomyositis and poststreptococcal reactive arthritis. Lancefield grouping revealed that the strain was a group G streptococcus. The Vitek system (GPI) showed that it was unidentified, and the API system (20 STREP) showed that it was 95.7%
S. dysgalactiae
subsp.
dysgalactiae
. 16S rRNA gene sequencing showed that it was a strain of
S. dysgalactiae
. Based on phylogenetic affiliation with 16S rRNA gene or GroEL amino acid (another bacterial gene, in addition to 16S rRNA gene, that is highly conserved) sequences, the strain is most closely related to Lancefield group C beta-hemolytic
S. dysgalactiae
subsp.
equisimilis
. PCR amplification and sequencing of the streptolysin S structural gene (
sagA
) and M protein gene (
emm
) hypervariable region showed the presence of these suspected primary virulence factors. Further studies would delineate whether the isolate is just a hemolysin-deficient variant of group G beta-hemolytic
S. dysgalactiae
subsp.
equisimilis
or a novel type of
S. dysgalactiae
. The present case showed that group G alpha-hemolytic
S. dysgalactiae
subsp.
equisimilis
can be associated with serious invasive infection and poststreptococcal sequelae.
Evaluation of Three Commercial Assays for Detection of
Giardia
and
Cryptosporidium
Organisms in Fecal Specimens
Authors: Johnston Stephanie P. , Ballard Melissa M. , Beach Michael J. , Causer Louise , Wilkins Patricia P.
Published: February 2003

There is an increasing demand for diagnostic testing for
Giardia intestinalis (G. lamblia)
and
Cryptosporidium parvum
, with a priority being placed on obtaining diagnostic results in an efficient and timely manner. Several commercial companies have developed rapid diagnostic tests that are simple to perform and can be completed in less time than traditional methods for detecting
Giardia
and
Cryptosporidium
. We compared one of these rapid tests, the ImmunoCard STAT! (Meridian Bioscience, Inc.) lateral-flow immunoassay, with the MERIFLUOR direct fluorescent-antibody (DFA) test, the ProSpecT EZ microplate assay for
Giardia
and the ProSpecT microplate assay for
Cryptosporidium
, and modified Kinyoun's acid-fast stained smears for the detection of
Cryptosporidium
using 246 specimens. The MERIFLUOR DFA (Meridian Bioscience, Inc.) test detected the largest number of cases (32
Giardia
and 37
Cryptosporidium
) infections and was used to calculate the sensitivity and specificity of the other tests. For
Giardia
, the sensitivities of the ImmunoCard STAT! and the ProSpecT
Giardia
EZ microplate assay (Alexon-Trend, Inc.) were 81 and 91%, respectively. For detection of
Cryptosporidium
, the sensitivities of the ImmunoCard STAT!, the ProSpecT
Cryptosporidium
microplate assay (Alexon-Trend, Inc.), and modified Kinyoun's acid-fast stained smears were 68, 70, and 78%, respectively. Test specificities were equal to or greater than 99%. Specimens with very small numbers of organisms were not detected by the ImmunoCard STAT!, the ProSpecT microplate assay or modified Kinyoun's acid-fast stained smears.
Characterization of Some
Actinomyces
-Like Isolates from Human Clinical Sources: Description of
Varibaculum cambriensis
gen. nov., sp. nov
Authors: Hall Val , Collins Matthew D. , Lawson Paul A. , Hutson Roger A. , Falsen Enevold , Inganas Elisabeth , Duerden Brian
Published: February 2003

Fifteen strains of an anaerobic, catalase-negative, gram-positive diphtheroid-shaped bacterium recovered from human sources were characterized by phenotypic and molecular chemical and molecular genetic methods. The unidentified bacterium showed some resemblance to
Actinomyces
species and related taxa, but biochemical testing, polyacrylamide gel electrophoresis analysis of whole-cell proteins, and amplified 16S ribosomal DNA restriction analysis indicated the strains were distinct from all currently named
Actinomyces
species and related taxa. Comparative 16S rRNA gene sequencing studies showed that the bacterium represents a hitherto-unknown phylogenetic line that is related to but distinct from
Actinomyces
,
Actinobaculum
,
Arcanobacterium
, and
Mobiluncus
. We propose, on the basis of phenotypic and phylogenetic evidence, that the unknown bacterium from human clinical specimens should be classified as a new genus and species,
Varibaculum cambriensis
gen. nov., sp. nov. The type strain of
Varibaculum cambriensis
sp. nov. is CCUG 44998
T
= CIP 107344
T
.
Use of a Phage-Based Assay for Phenotypic Detection of Mycobacteria Directly from Sputum
Authors: Park D. J. , Drobniewski F. A. , Meyer A. , Wilson S. M.
Published: February 2003

The phage amplified biologically assay is a new method for rapid and low-cost phenotypic determination of the drug sensitivities of
Mycobacterium tuberculosis
isolates and the detection of viable organisms in patient specimens. Infection of slowly growing mycobacteria with phage (phage D29) was followed by chemical virucide destruction of extracellular phage. Infected mycobacteria were mixed in culture with rapidly growing sensor cells, which the phage can also infect; i.e., lytic amplification of phage occurs. The aims of the present study were to optimize the speed and sensitivity of the assay and reduce its cost for developing countries by using an
M. tuberculosis
-spiked sputum model with (i) identification of inhibitory components of sputum and optimization of decontamination methods; (ii) simplification of the washing and development steps; (iii) reduction of the use of high-cost components, e.g., oleate-albumin-dextrose-catalase (OADC) supplement; and (iv) optimization of virucide treatment. The following results were obtained. (i) An inhibitory factor in sputum which could be removed by treatment of the sample with sodium dodecyl sulfate or NaOH decontamination was identified. (ii) A microcentrifuge-based approach with thixotropic silica as a bedding and resuspension agent was developed as an alternative to conventional centrifugation medium exchange. The yield was increased 228-fold, with increased speed and reduced cost. (iii) At present, after extracellular inactivation of phage, the ferrous ammonium sulfate (FAS) virucide is sequestered by dilution with an expensive supplement, OADC. Sodium citrate with calcium chloride was found to be a cost-effective after treatment with the FAS protectant and offered greater protection than OADC. Kinetic-lysis experiments indicated that an infection time of 1 to 3 h prior to FAS addition was optimal. (iv) Amplification of the signal (which corresponded to the burst size) was shown by allowing lysis prior to plating in a spiked medium model (up to 20-fold) and a spiked sputum model (up to 10-fold). A liquid culture detection method capable of detecting approximately 60 viable
M. tuberculosis
organisms in 1 ml of sputum was developed. Taken together, these improvements support the routine application of the assay to sputum specimens.
Molecular Characterization and Drug Resistance Patterns of Strains of
Mycobacterium tuberculosis
Isolated from Patients in an AIDS Counseling Center in Port-au-Prince, Haiti: a 1-Year Study
Authors: Ferdinand Séverine , Sola Christophe , Verdol Béatrice , Legrand Eric , Goh Khye Seng , Berchel Mylène , Aubéry Alexandra , Timothée Maryse , Joseph Patrice , Pape Jean William , Rastogi Nalin
Published: February 2003

Tuberculosis (TB) is one of the most common opportunistic diseases that appear among human immunodeficiency virus (HIV)-positive patients in Haiti. In this context the probable emergence of multidrug-resistant (MDR) strains of
Mycobacterium tuberculosis
is of great epidemiological concern. However, as routine culture of
M. tuberculosis
and drug susceptibility testing are not performed in Haiti, it has not been possible so far to evaluate the rate of drug resistance among
M. tuberculosis
isolates from circulating TB cases. This report describes the first study on the molecular typing and drug resistance of
M. tuberculosis
isolates from patients with culture-positive pulmonary tuberculosis monitored at the GHESKIO Centers in Haiti during the year 2000. Clinical, epidemiological, and drug susceptibility testing results were available for 157 patients with confirmed cases of TB, with a total of 8.9% of patients harboring MDR
M. tuberculosis
. A significant association between the occurrence of resistance and previous TB treatment was observed (
P
< 0.001), suggesting that a previous history of TB treatment was a risk factor associated with MDR TB in Haiti. The DNAs of individual isolates from 106 samples were available and were typed by spoligotyping and determination of the variable number of tandem DNA repeats. Both typing methods provided interpretable results for 96 isolates, and the clusters observed were further confirmed by ligation-mediated PCR to define potential cases of active transmission. Thirty-three (34%) of the isolates were found to be grouped into 11 clusters with two or more identical patterns. However, an assessment of risk factors (sex, HIV positivity, previous treatment, drug resistance) showed that none was significantly associated with the active transmission of TB. These observations suggest that acquired MDR TB is prevalent in Haiti and may be associated with compliance issues during TB treatment since prior TB therapy is the strongest risk factor associated with MDR TB. Prevention of TB transmission in Haiti should target active case investigation, routine detection of drug resistance, and adequate treatment of patients. The use of directly observed short-course therapy should be enforced throughout the country; and relapses, reactivations, or newly acquired infections should be discriminated by genotyping methods.
Clonal Reproduction and Limited Dispersal in an Environmental Population of
Cryptococcus neoformans
var.
gattii
Isolates from Australia
Authors: Halliday C. L. , Carter D. A.
Published: February 2003

Cryptococcus neoformans
var.
gattii
is a causative agent of cryptococcosis and is thought to have a specific ecological association with a number of
Eucalyptus
species in Australia. However, the role that the tree plays in the life cycle of the fungus and the nature of the infectious propagule are not well understood. This study set out to examine whether sexual recombination is occurring in a natural population of
C. neoformans
var.
gattii
and whether the fungus disseminates between colonized trees. Thirty
C. neoformans
var.
gattii
isolates, consisting of both the α and
a
mating types, were collected from 13
Eucalyptus camaldulensis
trees growing along a riverbank in Renmark, South Australia. The genetic diversity within the population was studied by using amplified fragment length polymorphism fingerprinting, and each isolate was assigned a unique multilocus genotype. Population genetic analyses of the multilocus data found no evidence of genetic exchange between members of the population, indicating a clonal population structure. Canonical variate analysis was then used to study the relationship between isolates from different colonized trees. Isolates from individual trees were strongly correlated, and it appeared that dispersal between trees was not occurring to any appreciable extent. These results suggest that the eucalypt may not be the primary niche for
C. neoformans
var.
gattii
but that the decaying wood present in hollows on these trees may provide a favorable substrate for extensive clonal propagation of the yeast cells.
Simple Method for Screening
Candida
Species Isolates for the Presence of Secreted Proteinases: a Tool for the Prediction of Successful Inhibitory Treatment
Authors: Dostál Jií , Hamal Petr , Pavlíčková Libuše , Souček Milan , Ruml Tomáš , Pichová Iva , Hrušková-Heidingsfeldová Olga
Published: February 2003

The yeasts of the genus
Candida
are opportunistic pathogens associated with the rising incidence of life-threatening infections in immunocompromised individuals. Secretion of aspartic proteinases has been determined to be one of the virulence factors of the pathogenic
Candida
species. To analyze the extracellular proteolytic activities of a large number of
Candida
clinical isolates, we developed a screening system based on a solid medium containing hemoglobin as the sole nitrogen source. The cleavage of hemoglobin by the secreted proteinases results in formation of clearance zones. The visibility of such zones was enhanced by addition of an acid-base indicator. Using this system, we assessed 245 clinical isolates of
Candida
from patients in the hospital of the Faculty of Medicine, Palacky University, Olomouc, Czech Republic, for the presence of secreted aspartic proteases (Saps). We also used the test plates for rapid semiquantitative testing of Sap inhibitors. Most of the pepstatin analogs affected the formation of the zones of clearance as well as the growth of
Candida albicans
,
C. tropicalis
, and
C. parapsilosis
colonies. By contrast, the human immunodeficiency virus proteinase inhibitors saquinavir, ritonavir, nelfinavir, and indinavir had no effect on the
Candida
strains tested. These results are in agreement with the inhibition constants obtained for the individual inhibitors with purified Saps. Thus, the plates containing hemoglobin proved to be an appropriate tool for the rapid and reliable assessment of Sap production and inhibition.
Comparison of PCR Assays for Detection of the Agent of Human Granulocytic Ehrlichiosis,
Anaplasma phagocytophilum
Authors: Massung Robert F. , Slater Kimetha G.
Published: February 2003

Human granulocytic ehrlichiosis is an emerging infectious disease in the United States and Europe, and PCR methods have been shown to be effective for the diagnosis of acute infections. Numerous PCR assays and primer sets have been reported in the literature. The analytical sensitivities (limits of detection) of 13 published PCR primer sets were compared using DNA extracted from serial dilutions of
Anaplasma phagocytophilum
-infected HL-60 cells. The specificity of the assays that were able to detect ≤2.5 infected cells was tested by the use of template DNA extracted from
Ehrlichia chaffeensis
,
Rickettsia rickettsii
, and
Bartonella henselae
. The assays with the lowest limits of detection were shown to be a nested assay that amplifies the 16S rRNA gene (primer pairs ge3a-ge10 [primary] and ge9-ge3 [nested]; detects 0.25 infected cell), a direct assay that amplifies the major surface protein gene
msp2
(primer pair msp2-3f-msp2-3r; detects 0.25 infected cell), and a direct assay that amplifies the 16S rRNA gene (primer pair ehr521-ehr790; detects 0.25 infected cell). The specificity and limit of detection of the MSP2 and 16S rRNA direct assays were further tested by use of
A. phagocytophilum
template DNA from both North America and Europe and from human, tick, white-footed mouse, equine, deer, bovine, and wood rat samples and of template DNA from closely related species (
Anaplasma marginale
, the white-tailed deer agent, and additional
E. chaffeensis
-positive samples). Three manufacturers' PCR kits were tested and showed distinct variations in the limit of detection, specificity, and nonspecific background amplification. The importance of these results for the molecular diagnosis of human granulocytic ehrlichiosis is discussed.
Development of a Multilocus Sequence Typing Method for Analysis of
Listeria monocytogenes
Clones
Authors: Salcedo C. , Arreaza L. , Alcalá B. , de la Fuente L. , Vázquez J. A.
Published: February 2003

This study is a first step in the development of multilocus sequence typing (MLST) method for
Listeria monocytogenes.
Nine housekeeping genes were analyzed in a set of 62 strains isolated from different sources and geographic locations in Spain. These strains were previously characterized by pulsed-field gel electrophoresis (PFGE). Because of low diversity, two loci were discarded from the study. The sequence analysis of the seven remaining genes showed 29 different allelic combinations, with 22 of them represented by only one strain. The results of this sequence analysis were generally consistent with those of PFGE. Because MLST allows the easy comparison and exchange of results obtained in different laboratories, the future application of this new molecular method could be a useful tool for the listeriosis surveillance systems that will allow the identification and distribution of analysis of
L. monocytogenes
clones in the environment.
Specimen Processing and Concentration of
Chlamydia trachomatis
Added Can Influence False-Negative Rates in the LCx Assay but Not in the APTIMA Combo 2 Assay When Testing for Inhibitors
Authors: Chong S. , Jang D. , Song X. , Mahony J. , Petrich A. , Barriga P. , Chernesky M.
Published: February 2003

Inhibitors in clinical specimens can be detected by adding the target of nucleic acid amplification to the sample. Introduction of a
Chlamydia trachomatis
L2 434 preparation containing 12 elementary bodies (EBs) into first-void urine (FVU) from 225 nonpregnant women and 190 pregnant women before specimen processing by the assays produced false-negative rates of 0.48% (2 of 415 specimens) and 13% (44 of 338 specimens) by the APTIMA Combo 2 and the Chlamydia LCx tests, respectively. Reducing the amount of
C. trachomatis
added to one EB, a concentration closer to the APTIMA Combo 2 test cutoff, for a subset of 244 FVU specimens increased the number of specimens with false-negative results by the APTIMA Combo 2 assay to 7 (2.9%), suggesting that the strength of the input
C. trachomatis
per specimen has an influence on the number of specimens with false-negative results. Repeat testing after overnight storage and dilution decreased the APTIMA Combo 2 test false-negative rates to 0% (0 of 415 specimens) with the stronger inoculum and 0.8% (2 of 244 specimens) with the weaker inoculum; the false-negative rate of the LCx assay was reduced to 5.4% (18 of 334 specimens). When an additional 70 FVU specimens from women to which 12 EBs were added before specimen processing were tested by the LCx assay, 34 specimens had false-negative results, whereas 21 specimens had false-negative results when the
C. trachomatis
EBs were introduced after processing. Nine of the 21 specimens to which EBs were added after processing and all of the 34 urine specimens to which the target was added before processing remained falsely negative on repeat testing at a 1:2 dilution, suggesting that input
C. trachomatis
DNA was lost during processing by the LCx assay. In contrast, the APTIMA Combo 2 assay appears to have a higher sensitivity and either lost little nucleic acid during processing or demonstrated few problems with inhibitors of transcription-mediated amplification.
Analysis of an IS2404-Based Nested PCR for Diagnosis of Buruli Ulcer Disease in Regions of Ghana Where the Disease Is Endemic
Authors: Stienstra Ymkje , van der Werf Tjip S. , Guarner Jeannette , Raghunathan Pratima L. , Spotts Whitney Ellen A. , van der Graaf Winette T. A. , Asamoa Kwame , Tappero Jordan W. , Ashford David A. , King C. Harold
Published: February 2003

Mycobacterium ulceranscauses Buruli ulcer disease (BUD), an ulcerative skin disease emerging mainly in West Africa. Laboratory confirmation of BUD is complicated as no “gold standard” for diagnosis exists. A nested primer PCR based on IS2404has shown promise as a diagnostic assay. We evaluated the IS2404-based PCR to detectM. ulceransDNA in tissue specimens from 143 BUD patients diagnosed according to the World Health Organization BUD clinical case definition in Ghana. Comparisons were made with culture and histopathology results. Variables influencing detection rate tested in this PCR protocol included the amount of tissue used and the stage of disease. The nested PCR was repeated on DNA extracted from a different part of the same biopsy specimen of 21 culture-positive samples. Of all 143 specimens, 107 (74.8%; 95% confidence interval, 68 to 82%) showed the presence ofM. ulceransDNA by PCR. Of the 78 histology-confirmed BUD patient samples, 64 (83%) were PCR positive. Detection rates were influenced neither by the amount of tissue processed for PCR nor by the stage of disease (preulcerative or ulcerative). Taken together, the two nested PCR tests on the subset of 21 culture-positive samples were able to detectM. ulceransDNA in all 21 culture-confirmed patients. For future studies, small tissue samples, e.g., punch biopsy samples, might be sufficient for case confirmation.
Rapid Detection of Rifampin Resistance in
Mycobacterium tuberculosis
Isolates by Heteroduplex Analysis and Determination of Rifamycin Cross-Resistance in Rifampin-Resistant Isolates
Authors: Sarıbaş Zeynep , Kocagöz Tanıl , Alp Alpaslan , Günalp Ayfer
Published: February 2003

Direct heteroduplex analysis and a universal heteroduplex generator assay were performed to detect rifampin resistance rapidly in Turkish
Mycobacterium tuberculosis
isolates. Cross-resistance to rifapentine, rifabutin, and rifalazil was investigated. A relationship between specific mutations and resistance patterns, which can guide the choice of an appropriate therapeutic regimen for tuberculosis patients, was identified.
Spread of Integron-Associated VIM-Type Metallo-β-Lactamase Genes among Imipenem-Nonsusceptible
Pseudomonas aeruginosa
Strains in Greek Hospitals
Authors: Giakkoupi P. , Petrikkos G. , Tzouvelekis L. S. , Tsonas S. , Legakis N. J. , Vatopoulos A. C.
Published: February 2003

Fifty-eight imipenem-nonsusceptible (MIC ≥ 8 μg/ml)
Pseudomonas aeruginosa
strains isolated during May 2001 in 15 Greek hospitals were studied. Thirty-six isolates derived from nine hospitals carried VIM-type metallo-β-lactamase genes, as found by PCR. In 34 isolates,
bla
VIM
was associated with class 1 integrons of various sizes. DNA sequencing indicated the presence of
bla
VIM-2
gene cassettes in a variety of integron structures. Random amplified polymorphic DNA typing suggested diversity of the
bla
VIM
-positive strains. Synergy between 2-mercaptoacetic acid and imipenem indicated carbapenemase activity in 26
bla
VIM
-positive strains.
Evaluation of the Specificities of Five DNA Amplification Methods for the Detection of
Neisseria gonorrhoeae
Authors: Palmer H. M. , Mallinson H. , Wood R. L. , Herring A. J.
Published: February 2003

The intragenus specificities of five molecular diagnostic methods for
Neisseria gonorrhoeae
were determined. Three assays were considered suboptimal. Molecular detection of
N. gonorrhoeae
from sites where other
Neisseria
spp. commonly occur or from any site in low-prevalence settings should be confirmed by a test targeting a different genetic locus.
Diagnosis of Enterovirus Infection by Genus-Specific PCR and Enzyme-Linked Immunosorbent Assays
Authors: Craig Maria E. , Robertson Peter , Howard Neville J. , Silink Martin , Rawlinson William D.
Published: February 2003

PCR for the diagnosis of enterovirus infections is resource intensive but is increasingly used due to wide availability. Enzyme-linked immunosorbent assays (ELISAs) that detect heterotypical antibodies against enterovirus immunoglobulin M (IgM), IgA, and IgG were compared with reverse transcription-PCR by using primers specific to the 5′ untranslated regions of 60 enterovirus species. The ELISAs were less sensitive than the PCR, and only the ELISA for IgM was highly specific. When retrospective diagnosis is important or when specimens are unsuitable for PCR, the ELISA has a limited role if PCR is not available.
AFLMP1
Encodes an Antigenic Cell Wall Protein in
Aspergillus flavus
Authors: Woo Patrick C. Y. , Chong Ken T. K. , Leung Andy S. P. , Wong Samson S. Y. , Lau Susanna K. P. , Yuen Kwok-Yung
Published: February 2003

We have previously reported the cloning and characterization of the
MP1
gene in
Penicillium marneffei
and the
AFMP1
gene in
Aspergillus fumigatus
and their use for serodiagnosis of penicilliosis and aspergilloma and invasive aspergillosis, respectively. In this study, we describe the cloning of the
AFLMP1
gene, which encodes the homologous antigenic cell wall protein in
Aspergillus flavus
, the most common
Aspergillus
species associated with human disease in our locality and in other Asian countries and the second most common
Aspergillus
species associated with human disease in Western countries.
AFLMP1
codes for a protein, Aflmp1p, of 273 amino acid residues, with a few sequence features that are present in Mp1p and Afmp1p, the homologous antigenic cell wall proteins in
P. marneffei
and
A. fumigatus
, respectively, as well as several other cell wall proteins of
Saccharomyces cerevisiae
and
Candida albicans
. It contains a serine- and threonine-rich region for O glycosylation, a signal peptide, and a putative glycosylphosphatidylinositol attachment signal sequence. Specific anti-Aflmp1p antibody was generated with recombinant Aflmp1p protein purified from
Escherichia coli
to allow further characterization of Aflmp1p. Indirect immunofluorescence analysis indicated that Aflmp1p is present on the surface of the hyphae of
A. flavus
. Finally, it was observed that patients with aspergilloma and invasive aspergillosis due to
A. flavus
develop a specific antibody response against Aflmp1p. This suggested that the recombinant protein and its antibody may be useful for serodiagnosis in patients with aspergilloma or invasive aspergillosis, and the protein may represent a good cell surface target for host humoral immunity.
Catheter-Related Sepsis Due toRhodotorula glutinis
Authors: Hsueh Po-Ren , Teng Lee-Jene , Ho Shen-Wu , Luh Kwen-Tay
Published: February 2003

We describe a central venous catheter-related (Port-A-Cath; Smiths Industries Medical Systems [SIMS] Deltec, Inc., St. Paul, Minn.) infection caused byRhodotorula glutinisin a 51-year-old man with nasopharyngeal carcinoma. He was treated with fluconazole for 8 weeks and had the catheter removed. Two isolates ofR. glutinisrecovered from blood specimens (one obtained via peripheral veins and one via the catheter) before administration of fluconazole and one recovered from the removed catheter 17 days after initiation of fluconazole therapy exhibited high-level resistance to fluconazole (MICs, >256 μg/ml). These three isolates were found to belong to a single clone on the basis of identical antibiotypes determined by the E test (PDM Epsilometer; AB Biodisk, Solna, Sweden) and biotypes determined by API ID32 C (bioMerieux, Marcy I'Etoile, France) and their identical random amplified polymorphic DNA patterns.
Development of a 5′ Fluorogenic Nuclease-Based Real-Time PCR Assay for Quantitative Detection of
Actinobacillus actinomycetemcomitans
and
Porphyromonas gingivalis
Authors: Yoshida Akihiro , Suzuki Nao , Nakano Yoshio , Oho Takahiko , Kawada Miki , Koga Toshihiko
Published: February 2003

A 5′ nuclease TaqMan PCR was developed for the quantitative detection of the periodontopathic bacteria
Actinobacillus actinomycetemcomitans
and
Porphyromonas gingivalis
. The relative numbers of bacteria were measured by the comparative threshold cycle method. This simplified method is a way of obtaining the relative quantities of these organisms from specimens and of monitoring the effect of therapy.
Rapid Identification of the Genus
Fonsecaea
by PCR with Specific Oligonucleotide Primers
Authors: Abliz Paride , Fukushima Kazutaka , Takizawa Kayoko , Nieda Norikazu , Miyaji Makoto , Nishimura Kazuko
Published: February 2003

An oligonucleotide primer set based on internal transcribed spacer regions of ribosomal DNA for PCR which gives the amplicon for only the DNA from
Fonsecaea
species was designed. This set yielded an amplicon with 333 bp for all strains of
Fonsecaea pedrosoi
and
Fonsecaea compacta
examined but no amplicons for related dematiaceous fungi and pathogenic yeasts. PCR using this primer set was considered to be a useful method for the rapid identification of the genus
Fonsecaea
.
Ubiquity of Putative Type III Secretion Genes among Clinical and Environmental
Burkholderia pseudomallei
Isolates in Northern Australia
Authors: Smith-Vaughan H. C. , Gal D. , Lawrie P. M. , Winstanley C. , Sriprakash K. S. , Currie B. J.
Published: February 2003

Horseradish peroxidase-like type III secretion (TTS1) genes were present in all 116 Northern Australian
Burkholderia pseudomallei
isolates tested but were not detected in other common environmental
Burkholderia
species. PCR of TTS1 genes may prove valuable as a diagnostic test.
Direct Identification of
Staphylococcus aureus
from Positive Blood Culture Bottles
Authors: Oliveira Kenneth , Brecher Stephen M. , Durbin Annette , Shapiro Daniel S. , Schwartz Donald R. , De Girolami Paola C. , Dakos Joanna , Procop Gary W. , Wilson Deborah , Hanna Chad S. , Haase Gerhard , Peltroche-Llacsahuanga Heidrun , Chapin Kimberle C. , Musgnug Michael C. , Levi Michael H. , Shoemaker Cynthia , Stender Henrik
Published: February 2003

Fluorescence in situ hybridization (FISH) using peptide nucleic acid (PNA) probes targeting
Staphylococcus aureus
16S rRNA is a novel method for direct identification of
S. aureus
from positive blood culture bottles. The test (S. aureus PNA FISH) is performed on smears made directly from positive blood culture bottles with gram-positive cocci in clusters (GPCC) and provides results within 2.5 h. A blinded comparison of
S. aureus
PNA FISH with standard identification methods was performed in collaboration with eight clinical microbiology laboratories. A total of 564 routine blood culture bottles positive for GPCC recovered from both aerobic and anaerobic media from three different manufacturers (ESP, BACTEC, and BacT/Alert) were included in the study. The sensitivity and specificity of
S. aureus
PNA FISH were 100% (57 of 57) and 99.2% (116 of 117), respectively, with 174 GPCC-positive ESP blood culture bottles, 98.5% (67 of 68) and 98.5% (129 of 131), respectively, with 200 GPCC-positive BACTEC blood culture bottles, and 100% (74 of 74) and 99.1% (115 of 116), respectively, with 190 GPCC-positive BacT/Alert blood culture bottles. It is concluded that
S. aureus
PNA FISH performs well with commonly used continuously monitoring blood culture systems.
PCR Assay To Detect
Bacillus anthracis
Spores in Heat-Treated Specimens
Authors: Fasanella A. , Losito S. , Adone R. , Ciuchini F. , Trotta T. , Altamura S. A. , Chiocco D. , Ippolito G.
Published: February 2003

Recent interest in anthrax is due to its potential use in bioterrorism and as a biowarfare agent against civilian populations. The development of rapid and sensitive techniques to detect anthrax spores in suspicious specimens is the most important aim for public health. With a view to preventing exposure of laboratory workers to viable
Bacillus anthracis
spores, this study evaluated the suitability of PCR assays for detecting anthrax spores previously inactivated at 121°C for 45 min. The results indicate that heat treatment ensures the complete inactivation of
B. anthracis
spores without significantly affecting the efficiency of PCR assays.
Localized Cutaneous Hyalohyphomycosis Caused by a
Fusarium
Species Infection in a Renal Transplant Patient
Authors: Cocuroccia Barbara , Gaido Jeanette , Gubinelli Emanuela , Annessi Giorgio , Girolomoni Giampiero
Published: February 2003

Fusariosis is a hyalohyphomycosis due to
Fusarium
species that mainly occurs in immunocompromised hosts. The clinical spectrum of
Fusarium
infection comprises localized and disseminated forms. A case of localized cutaneous fusariosis caused by
Fusarium solani
in a renal transplant patient is described, and the skin manifestations of the disease are discussed.
Group IIIa Hydrides XH2 and XH2- (X = B, Al, Ga): Electron Affinities and Singlet-Triplet Splittings Revisited
Authors: Černušák Ivan , Zavažanová Alena , Raab Juraj , Neogrády Pavel
Published: February 2003

Geometries, electron affinities (EA) and singlet-triplet (S-T) splittings of XH2/XH2- molecules (X = B, Al, Ga) are calculated by coupled-cluster methods, using the sequence of basis sets. The EA values and S-T splittings for aluminium and gallium dihydrides are an order of magnitude larger (in absolute values) than those for boron. For boron and aluminium dihydrides, two types of extrapolations towards complete basis set limit are applied, leading to EA = 0.24 eV, ST = -0.01 eV (BH2), and EA = 1.10 eV, ST = -0.62 eV. The best calculated values for gallium dihydrides are EA = 1.13 eV and ST = -0.74 eV. All three S-T splittings favour singlet as the ground state, although the S-T splittings of BH2- is exceptionally small. In addition, vertical electron affinities and vertical electron detachments are reported for these molecules.
SOME REMARKS ON TOPOLOGICAL ORBIT EQUIVALENCE OF CANTOR MINIMAL SYSTEMS
Authors: HIROKI MATUI
Published: February 2003

We will study the number of discontinuities of the orbit cocycles associated with orbit equivalence between Cantor minimal systems.
THE ORGANISATION OF EXTERNAL RESOURCES THROUGH GUEST ENGINEERS
Authors: SLACK NIGEL , TWIGG DAVID
Published: February 2003

This paper describes exploratory research which investigates the scope, nature, role and activities of guest engineers. Guest engineers are an inter-firm organisational mechanism which is usually deployed as a compromise between the total outsourcing of design on the one hand, and the total in-house development of products on the other. A classification of guest engineers is presented based on their permanent (or semi-permanent) location and the main focus of their engineering expertise. Four types of guest engineer are identified — the pure form of which is termed the guest design engineer (GDE). Suppliers of GDEs, together with their hosts, are examined to identify their most significant activities and tasks. These are divided into clusters of similar activities which are assessed according to their relative importance and relative performance. Differences are found between suppliers' and customers' perceptions of both the importance and performance of each of these cluster activities. In addition, three sets of significant role design decisions are identified: the scheduling of GDEs; the nature of the required skills; and the direct activities of GDEs during the development process itself.
BIOMEDICAL INNOVATION NETWORKS AND NEW TECHNOLOGIES
Authors: DINNIE NICOLA , McKEE LORNA , BOWER D. JANE
Published: February 2003

This paper analyses a number of factors which are implicated in the success of biomedical innovation processes in the United Kingdom (UK): the observability of the innovation process to the participants; the role and interests of UK public and private sector networks; and the ability of the wider constituency to push or drive the innovation process. This study addresses four ongoing developments (familial breast cancer diagnosis, cystic fibrosis gene therapy, inherited eye disease gene therapy and breast cancer screening). The paper draws directly on research funded by the ESRC. Documentary evidence has been analysed for relevant information together with information derived from semi-structured interviews. This has taken account of the key contributions over time from differing professional groups, public and commercial organisations (including user groups) and sectors.
IMITATION THROUGH TECHNOLOGY LICENSING: STRATEGIC IMPLICATIONS FOR SMALLER FIRMS
Authors: LOWE JULIAN , TAYLOR PETER
Published: February 2003

An important aspect of strategic choice is whether to pioneer or to imitate, that is, whether to be a first-mover or a follower. It has been suggested that this is particularly important for small and medium-size enterprises (SMEs) because of potential disadvantages of scale in design, production and marketing. However, empirical evidence suggests that many SMEs can be successful first-movers and that pioneering strategies enable them to compete against larger firms that have scale advantages and possess important downstream resources. This paper looks at the issue of timing of entry from a different perspective. It is concerned with the role of imitation as a strategy and its success when smaller firms are in competition with larger firms. Acquisition of technology through licensing is a frequently used strategy for firms of all sizes. It is also a classic 'imitation' strategy. The paper reports a study of imitative behaviour in a sample of UK manufacturing firms of varying sizes. A comparison of strategic motives, capabilities and outcomes is examined in the context of technology strategies involving both in-house R&D and that acquired through inward technology licensing. An important distinction is made between licensing that provides a basis for subsequent innovation and licensing for reactive imitative purposes. The study concludes that imitation is a viable policy option for smaller firms, but that complementary assets and appropriate capabilities determine whether such a policy has a major competitive impact or is merely a reaction to unsuccessful in-house innovation. The study calls into question the use of size per se as a competitive indicator, and indicates reasons underpinning imitative behaviour that may influence downstream success.