bluuebunny/crossref_metadata_2025_split huggingface dataset:
New Criteria for Immunofluorescence Assay for Q Fever Diagnosis in Japan
Authors: Setiyono A. , Ogawa M. , Cai Y. , Shiga S. , Kishimoto T. , Kurane I. 
Published: November 2005

ABSTRACT
A study was made to evaluate the cutoff value of indirect immunofluorescent-antibody (IFA) test for Q fever diagnosis in Japan. We used 346 sera, including 16 from confirmed Q fever cases, 304 from Japanese pneumonia patients, and 26 from negative cases. Thirteen sera from the confirmed Q fever cases with an immunoglobulin M (IgM) titer of ≥1:128 and/or IgG titer of ≥1:256 by the IFA test were positive by both enzyme-linked immunosorbent assay (ELISA) and Western blotting assay (WBA), whereas 298 sera from pneumonia patients and 26 negative sera with an IgM titer of ≤1:16 and an IgG titer of ≤1:32 by the IFA test were negative by both ELISA and WBA. In the proposed “equivocal area,” with an IgM titer of ≥1:32 and ≤1:64 and/or an IgG titer of ≥1:64 and ≤1:128, we found 9 sera, 3 from confirmed Q fever cases and 6 from Japanese pneumonia patients, by the IFA test. Three sera from the confirmed Q fever cases and one of the sera from pneumonia patients were IgM and/or IgG positive by both ELISA and WBA. These results suggest that a single cutoff value for the IFA test may cause false-positive and false-negative results. In conclusion, this study showed that an “equivocal area” should be used for the IFA test rather than a single cutoff value and that sera in the equivocal area should be tested by additional serological assays for confirmation.
Quantitative Nucleic Acid Sequence-Based Assay as a New Molecular Tool for Detection and Quantification of
Leishmania
Parasites in Skin Biopsy Samples
Authors: van der Meide Wendy F. , Schoone Gerard J. , Faber William R. , Zeegelaar Jim E. , de Vries Henry J. C. , Özbel Yusuf , Lai A Fat Rudy F. M. , Coelho Leíla I. A. R. C. , Kassi Masoom , Schallig Henk D. F. H. 
Published: November 2005

ABSTRACT

Currently available methods for the diagnosis of cutaneous leishmaniasis (CL) have low sensitivities or are unable to quantify the number of viable parasites. This constitutes a major obstacle for the diagnosis of the disease and for the study of the effectiveness of treatment schedules and urges the development of improved detection methods. In this study, quantitative nucleic acid sequence-based amplification (QT-NASBA) technology was used to detect and quantify
Leishmania
parasites in skin biopsy samples from CL patients. The assay is based on the detection of a small subunit rRNA (18S rRNA), which may allow for the detection of viable parasites. The QT-NASBA assay was evaluated using in vitro-cultured promastigotes and amastigotes and 2-mm skin biopsy samples from Old and New World CL patients. The study demonstrated that the lower detection limit of the QT-NASBA was two parasites per biopsy sample. Parasites could be quantified in a range of 2 to 11,300,000 parasites per biopsy sample. The QT-NASBA could detect levels of parasites 100-fold lower than those detected by conventional PCR. Test evaluation revealed that the QT-NASBA had a sensitivity of 97.5% and a specificity of 100% in the present study. The QT-NASBA is a highly sensitive and specific method that allows quantification of both Old and New World
Leishmania
parasites in skin biopsy samples and may provide an important tool for diagnosis as well as for monitoring the therapy of CL patients.
TaqMan Real-Time Reverse Transcription-PCR and JDVp26 Antigen Capture Enzyme-Linked Immunosorbent Assay To Quantify Jembrana Disease Virus Load during the Acute Phase of In Vivo Infection
Authors: Stewart Meredith , Desport Moira , Hartaningsih Nining , Wilcox Graham 
Published: November 2005

ABSTRACT

Jembrana disease virus (JDV) is an acutely pathogenic lentivirus that affects Bali cattle in Indonesia. The inability to propagate the virus in vitro has made it difficult to quantitate JDV and determine the kinetics of virus replication during the acute phase of the disease process. We report for the first time two techniques that enable quantification of the virus and the use of these techniques to quantify the virus load during the acute phase of the disease process. A one-step JDV
pol
TaqMan real-time reverse transcription-PCR (RT-PCR) assay was developed for the detection and quantification of JDV RNA in plasma. The limit of detection was 9.8 × 10
2
JDV viral RNA copies over 35 cycles, equivalent to 4.2 × 10
4
JDV genome copies/ml, and a peak virus load of 1.6 × 10
12
during the acute febrile period. An antigen capture enzyme-linked immunosorbent assay (ELISA) was also developed to quantify the levels of JDV capsid (JDVp26) over a linear range of 10 to 200 ng/ml. Viral RNA and JDVp26 levels were correlated in 48 plasma samples obtained from experimentally infected cattle. A significant positive correlation (
R
= 0.860 and
r
2
= 0.740) was observed between the two techniques within the range of their detection limits. The relatively insensitive capture ELISA provides an economical and feasible method for monitoring of virus in the absence of more sensitive techniques.
Resilience of the Dominant Human Fecal Microbiota upon Short-Course Antibiotic Challenge
Authors: De La Cochetière M. F. , Durand T. , Lepage P. , Bourreille A. , Galmiche J. P. , Doré J. 
Published: November 2005

ABSTRACTRecent studies have shown that the human fecal microbiota is composed of a consortium of species specific to the host and resistant to modifications over time. Antibiotics are known to affect the intestinal microflora, and ensuing changes may result in antibiotic-associated diarrhea. It is therefore important to characterize the nature and amplitude of these modifications and the ability of this ecosystem to return to its original profile—i.e., its resilience. Six healthy volunteers received oral amoxicillin (1.5 g/day) for 5 days. Fecal samples were collected at day 0 (D0) before antibiotic treatment and at set intervals until 60 days thereafter. Fecal DNA was isolated, and V6-to-V8 regions of the 16S rRNA genes were amplified by PCR with general primers and analyzed by temporal temperature gradient gel electrophoresis. Dominant species profiles were compared on the basis of similarity (Pearson correlation coefficient). Dominant species profiles at D0 were used as a reference. The fecal microbiota showed a major shift in dominant species upon antibiotic treatment, starting 24 h after treatment initiation and reaching an average similarity of only 74% after 4 days. Within 30 days following antibiotic treatment, the fecal microbiota tended to reach an average similarity of 88% to the D0 value; within 60 days, the average similarity to the D0 value was 89%. However, in one subject, important modifications persisted for at least 2 months, with similarity to the D0 value remaining below 70%. We demonstrated the resilience of the dominant human fecal microbiota upon short-course antibiotic challenge. Yet the persistence of long-term alterations in some subjects may explain susceptibilities to antibiotic-associated diarrhea. Furthermore, these findings suggest that strategies reinforcing the ability of the fecal microbiota to resist modifications would be of clinical relevance.
Multilocus Sequence Typing for Differentiation of Strains of
Candida tropicalis
Authors: Tavanti Arianna , Davidson Amanda D. , Johnson Elizabeth M. , Maiden Martin C. J. , Shaw Duncan J. , Gow Neil A. R. , Odds Frank C. 
Published: November 2005

ABSTRACT

A system is described for typing isolates of the pathogenic fungus
Candida tropicalis
, based on sequence polymorphisms in fragments of six genes:
ICL1
,
MDR1
,
SAPT2
,
SAPT4
,
XYR1
, and
ZWF1a
. The system differentiated 87 diploid sequence types (DSTs) among a total of 106 isolates tested or 80 DSTs among 88 isolates from unique sources. Replicate isolates from the same source clustered together with high statistical similarity, with the exception of one isolate. However, a clade of very closely related isolates included replicate isolates from three different patients, as well as single isolates from eight other patients. This clade, provisionally designated clade 1, was one of three clusters of isolates with high statistical similarity. Five of six isolates in one cluster that may acquire clade status were resistant to flucytosine. This study adds
C. tropicalis
to
Candida albicans
and
Candida glabrata
as
Candida
species for which a multilocus sequence typing (MLST) system has been set up. The
C. tropicalis
MLST database can be accessed at
http://pubmlst.org/ctropicalis/
.
Persistent Detection of Varicella-Zoster Virus DNA in a Previously Healthy Child after Severe Chickenpox
Authors: Vossen Mireille T. M. , Gent Mi-Ran , Peters Karla M. C. , Wertheim-van Dillen Pauline M. E. , Dolman Koert M. , van Breda Alex , van Lier René A. W. , Kuijpers Taco W. 
Published: November 2005

ABSTRACTIn immunocompetent children with primary varicella-zoster virus (VZV) infection, peak viral loads are detected in peripheral blood near the onset of the vesicular rash. VZV DNA concentrations normally diminish and become undetectable within 3 weeks after the appearance of the exanthem. Here, we present a previously healthy, human immunodeficiency virus-negative, 4-year-old boy admitted with severe varicella. High viral loads (>340,000 copies/ml) were found in his blood, and the viral loads remained high for at least 1.5 years. Clinical recovery preceded complete clearance of the virus. General and VZV-specific immune reactivity were intact. NK cells and CD8+T cells were activated during acute infection, and VZV-specific CD4+T cells were detected at high frequencies. VZV DNA was initially detected in B cells, NK cells, and both CD4+and CD8+T cells. In contrast, during the persistent phase of VZV DNA detection, the viral DNA was primarily located in CD8+T cells. For the first time, we describe the persistent detection of VZV DNA in a previously healthy child.
Discriminatory Power and Reproducibility of Novel DNA Typing Methods forMycobacterium tuberculosisComplex Strains
Authors: Kremer Kristin , Arnold Catherine , Cataldi Angel , Gutiérrez M. Cristina , Haas Walter H. , Panaiotov Stefan , Skuce Robin A. , Supply Philip , van der Zanden Adri G. M. , van Soolingen Dick 
Published: November 2005

ABSTRACTIn recent years various novel DNA typing methods have been developed which are faster and easier to perform than the current internationally standardized IS6110restriction fragment length polymorphism typing method. However, there has been no overview of the utility of these novel typing methods, and it is largely unknown how they compare to previously published methods. In this study, the discriminative power and reproducibility of nine recently described PCR-based typing methods forMycobacterium tuberculosiswere investigated using the strain collection of the interlaboratory study of Kremer et al. (J. Clin. Microbiol. 37:2607-2618, 1999). This strain collection contains 90M. tuberculosiscomplex and 10 non-M. tuberculosiscomplex mycobacterial strains, as well as 31 duplicated DNA samples to assess reproducibility. The highest reproducibility was found with variable numbers of tandem repeat typing using mycobacterial interspersed repetitive units (MIRU VNTR) and fast ligation-mediated PCR (FLiP), followed by second-generation spoligotyping, ligation-mediated PCR (LM-PCR), VNTR typing using five repeat loci identified at the Queens University of Belfast (QUB VNTR), and the Amadio speciation PCR. Poor reproducibility was associated with fluorescent amplified fragment length polymorphism typing, which was performed in three different laboratories. The methods were ordered from highest discrimination to lowest by the Hunter-Gaston discriminative index as follows: QUB VNTR typing, MIRU VNTR typing, FLiP, LM-PCR, and spoligotyping. We conclude that both VNTR typing methods and FLiP typing are rapid, highly reliable, and discriminative epidemiological typing methods forM. tuberculosisand that VNTR typing is the epidemiological typing method of choice for the near future.
Evaluation of Techniques for Detection of Carbapenem-Resistant
Klebsiella pneumoniae
in Stool Surveillance Cultures
Authors: Landman D. , Salvani J. K. , Bratu S. , Quale J. 
Published: November 2005

ABSTRACT

Screening for gastrointestinal colonization with multidrug-resistant nosocomial pathogens is an important component of infection control protocols. In the New York City region, carbapenem-resistant
Klebsiella pneumoniae
strains, which harbor the KPC carbapenem-hydrolyzing β-lactamase, have rapidly emerged. The potential utility of screening medium, which involved using 10-μg imipenem disks, was investigated. The method of placing a sample from a fecal surveillance culture into broth containing an imipenem disk appeared to have the greatest sensitivity for detecting KPC-producing
K. pneumoniae
. Gastrointestinal colonization with two other carbapenem-resistant nosocomial pathogens,
Pseudomonas aeruginosa
and
Acinetobacter baumannii
, was also detected using this method. Placing fecal surveillance specimens into broth containing an imipenem disk is an easy method for screening samples for carbapenem-resistant nosocomial pathogens.
Comparison of an Automated Repetitive Sequence-Based PCR Microbial Typing System to Pulsed-Field Gel Electrophoresis for Analysis of Outbreaks of Methicillin-Resistant
Staphylococcus aureus
Authors: Ross T. L. , Merz W. G. , Farkosh M. , Carroll K. C. 
Published: November 2005

ABSTRACT

Rapid and sensitive methods for accurate strain delineation are essential for monitoring and preventing transmission of methicillin-resistant
Staphylococcus aureus
(MRSA). Pulsed-field gel electrophoresis (PFGE) has been the standard technique for strain typing most bacterial species including MRSA. The goal of this study was to compare the performance of the DiversiLab microbial typing system (Bacterial BarCodes, Inc., Houston, TX) (rep-PCR) to that of PFGE for typing MRSA isolates from five well-defined outbreaks. The DiversiLab rep-PCR assay is a rapid, semiautomated method based on PCR amplification of specific regions between noncoding repetitive sequences in the bacterial genome. rep-PCR was performed according to the manufacturer's recommendations, and the results were analyzed and dendrograms were generated using the DiversiLab analysis software (version 2.1.66a). PFGE was performed and interpreted according to published procedures. rep-PCR results using similarity indices (SI) of 80%, 85%, and 90% were compared to PFGE analysis. In addition, intra- and interrun reproducibility was determined for rep-PCR. Overall, correct assignment to outbreak versus nonoutbreak clusters occurred for 91 of 109 isolates (85% agreement) when using a SI of 85%. For each specific outbreak, concordance between rep-PCR and PFGE ranged from 73% to 100%. There were 18 discrepant results (17%). Fourteen isolates were unique by PFGE, but they were placed in clusters by rep-PCR; the other 4 were placed in clusters different from those assigned by PFGE. Intra- and interrun reproducibility was excellent. Times to results were 12 to 24 h for rep-PCR compared to 2 to 4 days for PFGE. Rapid, standardized results and excellent reproducibility make rep-PCR a valuable tool for use in MRSA investigations. However, since rep-PCR was less discriminatory than PFGE, we recommend that it be used to screen isolates, followed by testing isolates which share the same rep-PCR pattern with a more sensitive method, such as PFGE or multilocus sequence typing.
Sensitive EDTA-Based Microbiological Assays for Detection of Metallo-β-Lactamases in Nonfermentative Gram-Negative Bacteria
Authors: Marchiaro Patricia , Mussi María A. , Ballerini Viviana , Pasteran Fernando , Viale Alejandro M. , Vila Alejandro J. , Limansky Adriana S. 
Published: November 2005

ABSTRACTThe worldwide spread of metallo-β-lactamase (MBL)-producing gram-negative bacilli represents a great concern nowadays. Sensitive assays for their specific detection are increasingly demanded to aid infection control and to prevent their dissemination. We have developed a novel microbiological assay employing crude bacterial extracts, designated EDTA-imipenem microbiological assay (EIM), to identify MBLs in nonfermentative gram-negative clinical strains. We also evaluated the ability of EIM to detect MBLs in comparison to those of other currently employed screening methods, such as the EDTA disk synergy test (EDS) with imipenem as a substrate and the Etest method. The sensitivities of EIM and Etest were similar (1 versus 0.92, respectively) and much higher than that of EDS (0.67). Moreover, both EIM and Etest displayed the maximum specificity. Modifications were introduced to EDS, including the simultaneous testing of three different β-lactams (imipenem, meropenem, and ceftazidime) and two different EDTA concentrations. This resulted in a sensitivity improvement (0.92), albeit at a cost to its specificity. A simple strategy to accurately detect MBL producers is proposed; this strategy combines (i) an initial screening of the isolates by the extended EDS assay to select the potential candidates and (ii) confirmation of the true presence of MBL activity by EIM.
Specific andSensitive Detection of
Neisseria gonorrhoeae
in ClinicalSpecimens by Real-Time PCR
Authors: Geraats-Peters C. W. M. , Brouwers M. , Schneeberger P. M. , van der Zanden A. G. M. , Bruisten S. M. , Weers-Pothoff G. , Boel C. H. E. , van den Brule A. J. C. , Harmsen H. G. , Hermans M. H. A. 
Published: November 2005

ABSTRACT

Early
diagnosis of
Neisseria gonorrhoeae
infections is important
with regard to patients' health and infectivity. We report the
development of a specific and sensitive TaqMan assay for the detection
of
N. gonorrhoeae
in clinical samples. The target sequence is
a 76-bp fragment of the 5′ untranslated region of the
opa
genes that encode opacity proteins. A panel of 448
well-defined
N. gonorrhoeae
isolates was used to evaluate and
optimize the assay. The method employs two minor-groove binding probes,
one of them recognizing a newly identified sequence in the
opa
genes. Testing a large panel of related and unrelated microorganisms
revealed that other
Neisseria
strains and other microorganisms
tested negative in the
opa
test. With a lower detection limit
of one genome per reaction, the
opa
test appeared more
sensitive than both the COBAS AMPLICOR (Roche Diagnostics Nederland BV,
Almere, The Netherlands) and a LightCycler 16S rRNA test. Analysis of a
panel of 122 COBAS AMPLICOR-positive samples revealed that 68% were
negative in both the 16S rRNA test and the
opa
assay
(confirming that the COBAS AMPLICOR test produces false positives),
while 30% were positive in both assays. Three samples were
opa
positive and 16S rRNA negative, which may be due to the higher
sensitivity of the
opa
assay. We conclude that the
opa
gene-based real-time amplification assay offers a
sensitive, specific, semiquantitative, and reliable assay suitable for
the detection of
N. gonorrhoeae
in clinical specimens and/or
for confirmation of less specific
tests.
Characteristics of
Haemophilus influenzae
Type b Responsible for Meningitis in Poland from 1997 to 2004
Authors: Skoczyńska Anna , Kadłubowski Marcin , Empel Joanna , Hryniewicz Waleria 
Published: November 2005

ABSTRACT

Two hundred forty-five
H. influenzae
isolates responsible for meningitis in Poland from 1997 to 2004 were studied. Among these, 233 (95.1%) belonged to serotype b (Hib), 2 belonged to serotype f, and 10 were noncapsulated. The relatedness of all isolates was evaluated by pulsed-field gel electrophoresis (PFGE), and selected representatives were evaluated by multilocus sequence typing. Resistance to ampicillin was identified in 34 (14.6%) of the Hib isolates and was associated with the production of β-lactamase only. Except for four isolates nonsusceptible to chloramphenicol, all isolates were susceptible to cefotaxime, ciprofloxacin, and rifampin. The PFGE analysis divided the Hib isolates into five PFGE types; however, all of them were possibly related. The most common PFGE type, with 25 subtypes, was characteristic for 97.4% of the isolates. The most prevalent PFGE subtype found in our study was also the most common among the Hib isolates responsible for invasive disease in Italy and the Czech Republic and was found among isolates causing lower respiratory tract infections in Poland. The most prevalent sequence types (STs) in the studied group were ST6 and ST92. Four new STs were found: ST188, ST189, ST190, and ST268. Results of this study support the evidence that the genetic structure of encapsulated
H. influenzae
is clonal. The continuing high number of meningitis cases due to Hib in Poland underlines the need for mass vaccination against Hib in Poland.
Detection of Anti-Hepatitis C Virus Effects of Interferon and Ribavirin by a Sensitive Replicon System
Authors: Kato Takanobu , Date Tomoko , Miyamoto Michiko , Sugiyama Masaya , Tanaka Yasuhito , Orito Etsuro , Ohno Tomoyoshi , Sugihara Kanji , Hasegawa Izumi , Fujiwara Kei , Ito Kiyoaki , Ozasa Atsushi , Mizokami Masashi , Wakita Takaji 
Published: November 2005

ABSTRACTAlthough combination therapy with interferon and ribavirin has improved the treatment for chronic hepatitis C virus (HCV) infection, the detailed anti-HCV effect of ribavirin in clinical concentrations remains uncertain. To detect the anti-HCV effect of ribavirin in lower concentrations, a sensitive and accurate assay system was developed using the reporter replicon system with an HCV genotype 2a subgenomic replicon (clone JFH-1) that exhibits robust replication in various cell lines. This reporter replicon was generated by introducing the luciferase reporter gene (instead of the neomycin resistance gene) into the subgenomic JFH-1 replicon. To assess the replication of this reporter replicon, luciferase activity was measured serially up to day 3 after transient transfection of Huh7 cells. The luciferase activity increased exponentially over the time course of the experiment. After adjustment for transfection efficiency and transfected cell viability, the impacts of interferon and ribavirin were determined. The administration of interferon and ribavirin resulted in dose-dependent suppression of replicon RNA replications. The 50% inhibitory concentration of interferon and ribavirin was 1.80 IU/ml and 3.70 μg/ml, respectively. In clinical concentrations, replications were reduced to 0.09% and 53.74% by interferon (100 IU/ml) and ribavirin (3 μg/ml), respectively. Combination use of ribavirin and interferon enhanced the anti-HCV effect of interferon by 1.46- to 1.62-fold. In conclusion, we developed an accurate and sensitive replicon system, and the antivirus effect of interferon and ribavirin was easily detected within their clinical concentrations by this replicon system. This system will provide a powerful tool for screening new antiviral compounds against HCV.