bluuebunny/crossref_metadata_2025 huggingface dataset:
Epidemiological Survey of Human Metapneumovirus Infection in a Large Pediatric Tertiary Care Center
Authors: Chano Frédéric , Rousseau Céline , Laferrière Céline , Couillard Michel , Charest Hugues
Published: November 2005

The human metapneumovirus (hMPV) was recently identified and linked to acute respiratory tract infections (ARTI). To assess the clinical importance of this virus in infants and children, we developed a rapid and efficient reverse transcription-PCR-based screening method for a large volume of samples and tested retrospectively a collection of 1,132 respiratory specimens submitted over a full year period to the virology laboratory of a large tertiary care pediatric center in Montreal, Canada. A total of 41 samples from 37 patients were positive by this method. During the winter months of 2001, up to 8% of specimens submitted for respiratory virus testing were hMPV positive. Sequencing data of the hMPV M gene revealed that two genogroups of the virus, each of which can be divided into two subgroups, cocirculated during this time period. A case-controlled study was conducted to compare the symptoms associated with hMPV infection with those involving other etiologic agents causing ARTI. Symptoms most frequently observed in hMPV-positive patients were cough, wheezing, and dyspnea, although the symptomatology could differ substantially from patient to patient. No distinct symptom profile could be associated with hMPV. Three nosocomial cases of hMPV infection were identified. Together, our data suggest that hMPV is a significant cause of symptomatic respiratory tract infections in infants and children. The incidence of the disease and the morbidity associated with the infection justify adding hMPV to the list of common respiratory viruses routinely screened for by clinical laboratories.
Multicenter Evaluation of BBL CHROMagar MRSA Medium for Direct Detection of Methicillin-Resistant
Staphylococcus aureus
from Surveillance Cultures of the Anterior Nares
Authors: Flayhart Diane , Hindler Janet F. , Bruckner David A. , Hall Geraldine , Shrestha Rabin K. , Vogel Sherilynn A. , Richter Sandra S. , Howard Wanita , Walther Rhonda , Carroll Karen C.
Published: November 2005

Active surveillance for methicillin-resistant
Staphylococcus aureus
(MRSA) is among the strategies recommended by the Society for Healthcare Epidemiology of America for control of nosocomial MRSA infections. Infection control and laboratory personnel desire rapid, sensitive, and inexpensive methods to enhance surveillance activities. A multicenter study was performed to evaluate a new selective and differential chromogenic medium, BBL CHROMagar MRSA (C-MRSA) medium (BD Diagnostics, Sparks, MD), which enables recovery and concomitant identification of MRSA strains directly from nasal swab specimens taken from the anterior nares. Specimens were inoculated to C-MRSA and Trypticase soy agar with 5% sheep blood agar (TSA II, BD Diagnostics). Mauve colonies on C-MRSA at 24 h and 48 h and suspicious colonies on TSA II were confirmed as
Staphylococcus aureus
by Gram stain morphology and a coagulase test. In addition, the results of C-MRSA were compared to results of susceptibility testing (five different methods) of
S. aureus
strains isolated on TSA II. A total of 2,015 specimens were inoculated to C-MRSA and TSA II. Three hundred fifty-four
S. aureus
isolates were recovered; 208 (59%) were oxacillin (methicillin) susceptible and 146 (41%) were oxacillin resistant (MRSA). On C-MRSA, 139/146 or 95.2% of MRSA isolates were recovered, whereas recovery on TSA II was 86.9% (127/146) (
P
= 0.0027). The overall specificity of C-MRSA was 99.7%. When C-MRSA was compared to each susceptibility testing method, the sensitivity and specificity, respectively, were as follows: oxacillin MIC by broth microdilution, 94.4% and 96.7%; oxacillin screen agar, 94.3% and 96.7%; PBP2′ latex agglutination, 93.7% and 98.5%; cefoxitin disk diffusion, 95.0% and 98.1%; and
mecA
PCR, 95.1% and 98.1%. In this study, C-MRSA was superior to TSA II for recovery of MRSA from surveillance specimens obtained from the anterior nares and was comparable to conventional, rapid, and molecular susceptibility methods for the identification of MRSA isolates.
Relationship between MIC and Minimum Sterol 14α-Demethylation-Inhibitory Concentration as a Factor in Evaluating Activities of Azoles against Various Fungal Species
Authors: Shimokawa Osamu , Niimi Masakazu , Kikuchi Ken , Saito Mitsumasa , Kajiwara Hideko , Yoshida Shin-ichi
Published: November 2005

The minimum growth-inhibitory concentrations (MICs) of azole antifungals were compared to their minimum sterol 14α-demethylation-inhibitory concentrations (MDICs) for clinical fungal isolates. The ascomycetous
Candida
yeasts tested were clearly divided into two groups: group I, consisting of
C. albicans
,
C. tropicalis
, and
C. lusitaniae
, had MICs that were much higher than the MDICs, whereas group II, comprising
C. glabrata
,
C. parapsilosis
,
C. guilliermondii
, and
C. krusei
, had MICs that were approximately equal to the MDICs. In the ascomycetous fungi
Aspergillus fumigatus
and
Sporothrix schenckii
, the MICs were indistinguishable from the MDICs. In the basidiomycetous fungi
Cryptococcus
(
Filobasidiella
)
neoformans
,
C. curvatus
, and
Trichosporon asahii
, the MICs and the MDICs were practically identical. These results support the notion that there are two distinct classes of fungi differing in their degree of tolerance to sterol 14α-demethylation deficiency. These findings have significant implications for both fungal physiology and antifungal chemotherapy.
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

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

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

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

Recent 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

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

In 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

In 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

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

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

The 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

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

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

Although 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.
Use of Resequencing Oligonucleotide Microarrays for Identification of
Streptococcus pyogenes
and Associated Antibiotic Resistance Determinants
Authors: Davignon Louis , Walter Elizabeth A. , Mueller Kate M. , Barrozo Christopher P. , Stenger David A. , Lin Baochuan
Published: November 2005

Group A streptococci (GAS) are responsible for a wide variety of human infections associated with considerable morbidity and mortality. Ever since the first systematic effort by Lancefield to group
Streptococcus
species by M protein variants, the detection and characterization of
Streptococcus
by different methods have been an evolving process. The ideal assay for GAS identification not only would provide quick and accurate diagnostic results but also would reveal antibiotic resistance patterns and genotype information, aiding not only in treatment but in epidemiologic assessment as well. The oligonucleotide microarray is a promising new technology which could potentially address this need. In this study, we evaluated the usefulness of oligonucleotide resequencing microarrays for identifying GAS and its associated antibiotic resistance markers. We demonstrated an assay platform that combines the use of resequencing DNA microarrays with either random nucleic acid amplification or multiplex PCR for GAS detection. When detecting
Streptococcus pyogenes
from coded clinical samples, this approach demonstrated an excellent concordance with a more established culture method. To this end, we showed the potential of resequencing microarrays for efficient and accurate detection of GAS and its associated antibiotic resistance markers with the benefit of sequencing information from microarray analysis.
Distribution of
tccP
in Clinical Enterohemorrhagic and Enteropathogenic
Escherichia coli
Isolates
Authors: Garmendia Junkal , Ren Zhihong , Tennant Sharon , Midolli Viera Monica Aparecida , Chong Yuwen , Whale Andrew , Azzopardi Kristy , Dahan Sivan , Sircili Marcelo Palma , Franzolin Marcia Regina , Trabulsi Luiz R. , Phillips Alan , Gomes Tânia A. T. , Xu Jianguo , Robins-Browne Roy , Frankel Gad
Published: November 2005

Enterohemorrhagic
Escherichia coli
(EHEC) and enteropathogenic
E. coli
(EPEC) are diarrheagenic pathogens that colonize the gut through the formation of attaching and effacing lesions, which depend on the translocation of effector proteins via a locus of enterocyte effacement-encoded type III secretion system. Recently, two effector proteins, EspJ and TccP, which are encoded by adjacent genes on prophage CP-933U in EHEC O157:H7, have been identified. TccP consists of a unique N-terminus region and several proline-rich domains. In this project we determined the distribution of
tccP
in O157:H7, in non-O157 EHEC, and in typical and atypical EPEC isolates. All the EHEC O157:H7 strains tested were
tccP
+
. Unexpectedly,
tccP
was also found in non-O157 EHEC, and in typical and atypical EPEC isolates, particularly in strains belonging to serogroups O26 (EHEC), O119 (typical EPEC), and O55 (atypical EPEC). We recorded some variation in the length of
tccP
, which reflects diversity in the number of the proline-rich repeats. These results show the existence of a class of “attaching and effacing” pathogens which express a combination of EPEC and EHEC virulence determinants.
Molecular Epidemiology of Clinical
Cryptococcus neoformans
Strains from India
Authors: Jain N. , Wickes B. L. , Keller S. M. , Fu J. , Casadevall A. , Jain P. , Ragan M. A. , Banerjee U. , Fries B. C.
Published: November 2005

Little is known about the molecular epidemiology of the human pathogenic fungus
Cryptococcus neoformans
in India, a country now in the midst of an epidemic of AIDS-related cryptococcosis. We studied 57 clinical isolates from several regions in India, of which 51 were
C. neoformans
var.
grubii
, 1 was
C. neoformans
var.
neoformans
, and 5 were
C. neoformans
var.
gattii
. This strain set included 18 additional sequential isolates from 14 patients. Strains were characterized phenotypically by measuring the polysaccharide capsule and by determining the MICs of standard antifungals. Molecular typing was performed by a PCR-based method using the minisatellite-specific core sequence (M13), by electrophoretic karyotyping, by restriction fragment length polymorphisms with the
C. neoformans
transposon 1 (TCN-1), and by
URA5
DNA sequence analysis. Overall, Indian isolates were less heterogeneous than isolates from other regions and included a subset that clustered into one group based on
URA5
DNA sequence analysis. In summary, our results demonstrate (i) differences in genetic diversity of
C. neoformans
isolates from India compared to isolates from other regions in the world; (ii) that DNA typing with the TCN-1 probe can adequately distinguish
C. neoformans
var.
grubii
strains; (iii) that TCN-1 sequences are absent in many
C. neoformans
var.
gattii
strains, supporting previous studies indicating that these strains have a limited geographical dispersal; and (iv) that human cryptococcal infection can be associated with microevolution of the infecting strain and by simultaneous coinfection with two distinct
C. neoformans
strains.
PCR Analysis of Egyptian Respiratory Adenovirus Isolates, Including Identification of Species, Serotypes, and Coinfections
Authors: Metzgar David , Osuna Miguel , Yingst Samuel , Rakha Magda , Earhart Kenneth , Elyan Diaa , Esmat Hala , Saad Magdi D. , Kajon Adriana , Wu Jianguo , Gray Gregory C. , Ryan Margaret A. K. , Russell Kevin L.
Published: November 2005

Eighty-eight adenovirus (Ad) isolates and associated clinical data were collected from walk-in patients with influenza-like illness in Egypt during routine influenza surveillance from 1999 through 2002. Respiratory Ad distributions are geographically variable, and serotype prevalence has not been previously characterized in this region. Serotype identity is clinically relevant because it predicts vaccine efficacy and correlates strongly with both clinical presentation and epidemiological pattern. Species and serotype identities were determined using several well-validated multiplex PCR protocols culled from the literature and supplemented with a few novel primer sets designed to identify rare types. The isolates included common species B1 serotypes (Ad3 and Ad7), common species C serotypes (Ad1, Ad2, and Ad5), the less common species B2 serotype Ad11, and three isolates of the rare species B1 serotype Ad16. Two isolates that appear to be variant Ad16 were also identified. Fifteen coinfections of multiple adenoviral types, primarily AdB/AdC and Ad3/Ad7 dual infections, were detected. The majority of these were verified using redundant PCR tests targeted at multiple genes. PCR is able to resolve coinfections, in contrast to traditional serum neutralization tests. PCR is also comparatively rapid and requires very little equipment. Application of the method allowed an inclusive determination of the serotypes found in the Egyptian respiratory sample set and demonstrated that coinfections are common and may play a previously unrecognized role in adenovirus pathogenesis, evolution, and epidemiology. In particular, coinfections may influence adenoviral evolution, as interserotypic recombination has been identified as a source of emerging strains.
Microbial Risk Indicators of Early Childhood Caries
Authors: Corby P. M. , Lyons-Weiler J. , Bretz W. A. , Hart T. C. , Aas J. A. , Boumenna T. , Goss J. , Corby A. L. , Junior H. M. , Weyant R. J. , Paster B. J.
Published: November 2005

The aim of this study was to use molecular identification methods, such as 16S RNA gene sequence and reverse-capture checkerboard hybridization, for identification of the bacteria associated with dental caries and with dental health in a subset of 204 twins aged 1.5 to 7 years old. A total of 448 plaque samples (118 collected from caries-free subjects and 330 from caries-active subjects) were used for analysis. We compared the bacteria found in biofilms of children exhibiting severe dental caries, with different degrees of lesion severity, with those found in biofilms of caries-free children. A panel of 82 bacterial species was selected, and a PCR-based reverse-capture checkerboard method was used for detection. A simple univariate test was used to determine the overabundance and underabundance of bacterial species in the diseased and in the healthy groups. Features identified with this univariate test were used to construct a probabilistic disease prediction model. Furthermore, a method for the analysis of global patterns of gene expression was performed to permit simultaneous analysis of the abundance of significant species by allowing cross-bacterial comparisons of abundance profiles between caries-active and caries-free subjects. Our results suggested that global patterns of microbial abundance in this population are very distinctive. The top bacterial species found to be overabundant in the caries-active group were
Actinomyces
sp. strain B19SC,
Streptococcus mutans
, and
Lactobacillus
spp., which exhibited an inverse relationship to beneficial bacterial species, such as
Streptococcus parasanguinis
,
Abiotrophia defectiva
,
Streptococcus mitis
,
Streptococcus oralis
, and
Streptococcus sanguinis
.
Supplementation of CHROMagar Candida Medium with Pal's Medium for Rapid Identification of
Candida dubliniensis
Authors: Sahand Ismail H. , Moragues María D. , Eraso Elena , Villar-Vidal María , Quindós Guillermo , Pontón José
Published: November 2005

CHROMagar Candida medium is used for the isolation and identification of
Candida
species, but it does not differentiate
Candida albicans
from
Candida dubliniensis
. This differentiation can be achieved by using Pal's agar, which cannot be used in primary isolation. We have combined both media to obtain a new medium that can be used for the isolation and identification of
C. dubliniensis
in primary cultures.
Comparison of the RSV Respi-Strip with Direct Fluorescent-Antigen Detection for Diagnosis of Respiratory Syncytial Virus Infection in Pediatric Patients
Authors: Gregson D. , Lloyd T. , Buchan S. , Church D.
Published: November 2005

The RSV Respi-Strip was compared to the SimulFluor respiratory screen for detecting respiratory syncytial virus in nasopharyngeal aspirates from pediatric patients. Of samples tested, 115/239 (49%) were positive by direct fluorescent-antigen detection. The sensitivity and specificity of the RSV Respi-Strip were 92% (95% confidence interval [CI], 86% to 96%) and 98% (95% CI, 94% to 100%), respectively, with a diagnostic efficiency of 95%.
Use of Four Dengue Virus Antigens for Determination of Dengue Immune Status by Enzyme-Linked Immunosorbent Assay of Immunoglobulin G Avidity
Authors: Matheus Séverine , Deparis Xavier , Labeau Bhety , Lelarge Josiane , Morvan Jacques , Dussart Philippe
Published: November 2005

We used an enzyme-linked immunosorbent assay (ELISA) of immunoglobulin G avidity to determine the dengue immune status of 105 pairs of serum samples from patients infected with dengue virus. This study shows that a simple avidity test, for which only one acute-phase serum sample is required, is potentially more useful than the hemagglutination inhibition test for the discrimination of primary from secondary dengue virus infection, whatever the type of dengue antigen used.
Epidemiology and Molecular Analysis of Intestinal Colonization by Vancomycin-Resistant Enterococci in Greek Hospitals
Authors: Gikas Achilleas , Christidou Athanasia , Scoulica Efstathia , Nikolaidis Paulos , Skoutelis Athanasios , Levidiotou Stamatina , Kartali Sofia , Maltezos Efstratios , Metalidis Simeon , Kioumis John , Haliotis George , Dima Sofia , Roumbelaki Maria , Papageorgiou Nikoleta , Kritsotakis Evangelos I. , Tselentis Yannis
Published: November 2005

From 1,246 specimens collected from 13 Greek hospitals, 266 vancomycin-resistant enterococci strains were isolated from 255 patients (20.5%). The VanA phenotype was present in 82 (30.8%) strains, the VanB phenotype in 17 (6.4%) strains, the VanC1 phenotype in 152 (57.1%) strains, and the VanC2/C3 phenotypes in 15 (5.6%) strains. When only VanA and VanB phenotypes were considered, the overall prevalence was 7.5%. Eighty-six isolates exhibiting the VanA or VanB phenotype were analyzed by pulsed-field gel electrophoresis (PFGE), and 46 PFGE groups were found.
Broth Microdilution Susceptibility Testing of
Brucella
Species: Quality Control Limits for Ten Antimicrobial Agents against Three Standard Quality Control Strains
Authors: Brown Steven D. , Traczewski Maria M.
Published: November 2005

Brucella broth without supplementation is the recommended medium for broth microdilution susceptibility tests of
Brucella abortus
,
B. melitensis
, and
B. suis
. Based on an eight-laboratory collaborative study using a pH-adjusted modification of this medium, we propose MIC quality control ranges for three control strains against 10 antimicrobials that are potentially efficacious for treating infections caused by these agents of bioterrorism.
Laryngeal Scleroma Associated with
Klebsiella pneumoniae
subsp.
ozaenae
Authors: De Champs C. , Vellin J. F. , Diancourt L. , Brisse S. , Kemeny J. L. , Gilain L. , Mom T.
Published: November 2005

Klebsiella pneumoniae
subsp.
ozaenae
was isolated from the pharynx of a woman with laryngeal scleroma.
K. pneumoniae
subsp.
ozaenae
is rarely isolated from clinical infections and has never been reported in laryngeal scleroma, which is usually caused by
K. pneumoniae
subsp.
rhinoscleromatis
.
Three Nonfatal Cases of Neonatal Adenovirus Infection
Authors: Elnifro Elfateh M. , Cooper Robert J. , Dady Ian , Hany Sa'id , Mughal Zolf M. , Klapper Paul E.
Published: November 2005

Neonatal adenovirus infection is considered a rare and fatal disease. Three nonfatal neonatal adenovirus infections manifesting as conjunctivitis or conjunctivitis with other nonspecific symptoms are described. Adenovirus DNA was detected by PCR in eye swabs from two patients and in both cerebrospinal fluid and eye swabs in the third patient.
Group A Streptococcal Meningitis in a Pediatric Patient following Cochlear Implantation: Report of the First Case and Review of the Literature
Authors: Pettersen Géraldine , Ovetchkine Philippe , Tapiero Bruce
Published: November 2005

The Food and Drug Administration published a public health warning on the association of bacterial meningitis and cochlear implants in June 2002. This article reports the first case of group A streptococcal (GAS) meningitis in a cochlear-implanted patient, followed by a review on cochlear implantation and GAS meningitis.
Molecular Identification of
Rhizomucor pusillus
as a Cause of Sinus-Orbital Zygomycosis in a Patient with Acute Myelogenous Leukemia
Authors: Iwen Peter C. , Freifeld Alison G. , Sigler Lynne , Tarantolo Stefano R.
Published: November 2005

Sinus-orbital zygomycosis caused by
Rhizomucor pusillus
in a patient with acute myelogenous leukemia is described. Identification was achieved by sequencing of the internal transcribed spacer (ITS) regions of the rRNA gene and by expression of zygospores in mating. This report highlights the value of ITS sequencing as a diagnostic tool for the identification of
R. pusillus
and expands the understanding of infection types caused by this zygomycete.
Non-Culture-Based Analysis of Bacterial Populations from Patients with Chronic Rhinosinusitis
Authors: Power Daniel A. , Burton Jeremy P. , Chilcott Chris N. , Tagg John R. , Dawes Patrick J.
Published: November 2005

Middle meatus aspirates from patients with chronic rhinosinusitis were analyzed by bacterial culture, denaturing gradient gel electrophoresis (DGGE), and antibiotic sensitivity techniques. DGGE detected a greater bacterial diversity than culture methods. Although resistance to antibiotics was low, there was evidence of changes in the composition of the bacterial microbiota over time, and the presence of noncultured bacteria was demonstrated.
Intraabdominal Zygomycosis Caused bySyncephalastrum racemosumInfection Successfully Treated with Partial Surgical Debridement and High-Dose Amphotericin B Lipid Complex
Authors: Schlebusch Sanmarié , Looke David F. M.
Published: November 2005

Invasive zygomycosis rarely complicates trauma. We describe the first recorded case of invasive infection of the anterior abdominal wall and omentum with the zygomyceteSyncephalastrum racemosum, which was successfully treated with partial surgical debridement and amphotericin B lipid complex.
THE EOSINOPHIL
Authors: Rothenberg Marc E. , Hogan Simon P.
Published: November 2005

Eosinophils have been considered end-stage cells involved in host protection against parasites. However, numerous lines of evidence have now changed this perspective by showing that eosinophils are pleiotropic multifunctional leukocytes involved in initiation and propagation of diverse inflammatory responses, as well as modulators of innate and adaptive immunity. In this review, we summarize the biology of eosinophils, focusing on the growing properties of eosinophil-derived products, including the constituents of their granules as well as the mechanisms by which they release their pleiotropic mediators. We examine new views on the role of eosinophils in homeostatic function, including developmental biology and innate and adaptive immunity (as well as interaction with mast cells and T cells). The molecular steps involved in eosinophil development and trafficking are described, with special attention to the important role of the transcription factor GATA-1, the eosinophil-selective cytokine IL-5, and the eotaxin subfamily of chemokines. We also review the role of eosinophils in disease processes, including infections, asthma, and gastrointestinal disorders, and new data concerning genetically engineered eosinophil-deficient mice. Finally, strategies for targeted therapeutic intervention in eosinophil-mediated mucosal diseases are conceptualized.
Multiple transcripts of Ca2+ channel α1-subunits and a novel spliced variant of the α1C-subunit in rat ductus arteriosus
Authors: Yokoyama Utako , Minamisawa Susumu , Adachi-Akahane Satomi , Akaike Toru , Naguro Isao , Funakoshi Kengo , Iwamoto Mari , Nakagome Masamichi , Uemura Nobuyuki , Hori Hideaki , Yokota Shumpei , Ishikawa Yoshihiro
Published: November 2005

Voltage-dependent Ca2+ channels (VDCCs), which consist of multiple subtypes, regulate vascular tone in developing arterial smooth muscle, including the ductus arteriosus (DA). First, we examined the expression of VDCC subunits in the Wistar rat DA during development. Among α1-subunits, α1C and α1G were the most predominant isoforms. Maternal administration of vitamin A significantly increased α1C- and α1G-transcripts. Second, we examined the effect of VDCC subunits on proliferation of DA smooth muscle cells. We found that 1 μM nitrendipine (an L-type Ca2+ channel blocker) and kurtoxin (a T-type Ca2+ channel blocker) significantly decreased [3H]thymidine incorporation and that 3 μM efonidipine (an L- and T-type Ca2+ channel blocker) further decreased [3H]thymidine incorporation, suggesting that L- and T-type Ca2+ channels are involved in smooth muscle cell proliferation in the DA. Third, we found that a novel alternatively spliced variant of the α1C-isoform was highly expressed in the neointimal cushion of the DA, where proliferating and migrating smooth muscle cells are abundant. The basic channel properties of the spliced variant did not differ from those of the conventional α1C-subunit. We conclude that multiple VDCC subunits were identified in the DA, and, in particular, α1C- and α1G-subunits were predominant in the DA. A novel spliced variant of the α1C-subunit gene may play a distinct role in neointimal cushion formation in the DA.