bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of C
18
-Carboxypropylbetaine and Standard
N
-Acetyl-
l
-Cysteine-NaOH Processing of Respiratory Specimens for Increasing Tuberculosis Smear Sensitivity in Brazil
Authors: Scott Cherise P. , dos Anjos Filho Luciano , de Queiroz Mello Fernanda Carvalho , Thornton Charles G. , Bishai William R. , Fonseca Leila S. , Kritski AfrÂnio L. , Chaisson Richard E. , Manabe Yukari C. 
Published: August 2002

ABSTRACT

Techniques to improve the sensitivity of smear microscopy would facilitate early tuberculosis (TB) diagnosis and disease control, especially in low-income countries where the positive predictive value is high. C
18
-carboxypropylbetaine (CB-18) is a zwitterionic detergent that helps to compensate for the innate buoyancy of mycobacteria, potentially enhancing recovery by centrifugation. Previous data suggest that CB-18 may increase the sensitivity of smear, culture, and molecular amplification diagnostic testing. The goal of the present study was to evaluate if the sensitivity of the smear technique using light microscopy could be improved by treating respiratory samples with CB-18. In the first phase, respiratory specimens were collected consecutively from patients with suspected pulmonary tuberculosis in a tertiary-care hospital in Rio de Janeiro, Brazil (236 specimens were analyzed). After protocol modifications, another 120 respiratory specimens were evaluated. The standard technique was
N
-acetyl-
l
-cysteine with sodium hydroxide (NALC-NaOH) treatment, smear concentration with centrifugation, and Ziehl-Neelsen staining. Culture on Löwenstein-Jensen slants was performed on all specimens for use as the “gold standard.” No specimens from patients undergoing active TB treatment were included. The initial protocol for CB-18 processing resulted in a sensitivity of 59.6% and specificity of 96.8% compared to standard processing with a sensitivity of 66.0% and specificity of 96.8%. Using the modified protocol, the sensitivity of CB-18 increased to 71.4% with a specificity of 97.0% versus standard processing with a sensitivity of 61.9% and a specificity of 99.0%. The diagnostic yield of acid-fast bacillus smear with CB-18 in the absence of fluorescence microscopy and PCR compared to standard processing with NALC-NaOH was not significantly different, although the power to detect a difference by the modified assay was low.
PCR-Based Identification of Bacteria Associated with Endodontic Infections
Authors: Fouad Ashraf F. , Barry Jody , Caimano Melissa , Clawson Michael , Zhu Qiang , Carver Rachaele , Hazlett Karsten , Radolf Justin D. 
Published: August 2002

ABSTRACT

PCR primers that target the bacterial 16S rRNA genes (or the
tuf
gene for the genus
Enterococcus
) were used to identify 10 putative bacterial pathogens in root canals with necrotic pulp. In addition, the associations of these microorganisms with symptoms and a history of diabetes mellitus were investigated. Microbial samples from the root canals of 24 teeth with necrotic pulp were included in the study. PCR with universal bacterial primers identified bacterial DNA in 22 specimens; the remaining 2 specimens were from intact teeth that had been traumatized 6 months prior to treatment. PCR with specific primers showed that preoperative symptoms were significantly associated with the presence of
Streptococcus
spp. (
P
< 0.001 by chi-square analysis). There was also a nonsignificant trend for symptoms to be associated with
Fusobacterium nucleatum
and
Porphyromonas gingivalis
(odds ratio, >2) and for diabetes mellitus to be associated with
P. gingivalis
and
Porphyromonas endodontalis
(odds ratio, >2). Cloning and sequencing of the universal PCR product in one specimen revealed the presence of an organism related to the genus
Olsenella
, which has not previously been described in endodontic infections.
Sensitivity of Three Urinary Antigen Tests Associated with Clinical Severity in a Large Outbreak of Legionnaires' Disease in The Netherlands
Authors: Yzerman Ed P. F. , Boer Jeroen W. den , Lettinga Kamilla D. , Schellekens Joop , Dankert Jacob , Peeters Marcel 
Published: August 2002

ABSTRACT
In 1999 an outbreak involving 188 patients with Legionnaires' disease (LD) occurred among visitors to a flower show in the Netherlands. Two enzyme immunoassays (Binax and Biotest) and one immunochromatographic assay (Binax NOW) were tested, using urine samples from LD patients from the 1999 outbreak. Sensitivity was calculated using positive culture and/or seroconversion as the “gold standard” in outbreak-related patients with radiographically confirmed pneumonia who fulfilled the epidemiological critera. The Binax EIA, Biotest EIA, and Binax NOW assay showed overall sensitivities of 69, 71, and 72%, respectively. When the tests were performed with concentrated urine samples, the overall sensitivities increased to 79, 74, and 81%, respectively. Using multiple logistic regression analysis with backward elimination, a statistically significant association was found between clinical severity and test sensitivity for all tests. For patients with mild LD, the test sensitivities ranged from 40 to 53%, whereas for patients with severe LD who needed immediate special medical care, the sensitivities reached 88 to 100%. These findings have major implications for the diagnostic process in patients with mild pneumonia and suggest that patients with mild pneumonia may go underdiagnosed if urine antigen tests alone are used.
Detection and Genotyping of Oocysts of
Cryptosporidium parvum
by Real-Time PCR and Melting Curve Analysis
Authors: Tanrıverdi Sultan , Tanyeli Atila , Başlamışlı Fikri , Köksal Fatih , Kılınç Yurdanur , Feng Xiaochuan , Batzer Glenda , Tzipori Saul , Widmer Giovanni 
Published: August 2002

ABSTRACT

Several real-time PCR procedures for the detection and genotyping of oocysts of
Cryptosporidium parvum
were evaluated. A 40-cycle amplification of a 157-bp fragment from the
C. parvum
β-tubulin gene detected individual oocysts which were introduced into the reaction mixture by micromanipulation. SYBR Green I melting curve analysis was used to confirm the specificity of the method when DNA extracted from fecal samples spiked with oocysts was analyzed. Because
C. parvum
isolates infecting humans comprise two distinct genotypes, designated type 1 and type 2, real-time PCR methods for discriminating
C. parvum
genotypes were developed. The first method used the same β-tubulin amplification primers and two fluorescently labeled antisense oligonucleotide probes spanning a 49-bp polymorphic sequence diagnostic for
C. parvum
type 1 and type 2. The second genotyping method used SYBR Green I fluorescence and targeted a polymorphic coding region within the GP900/poly(T) gene. Both methods discriminated between type 1 and type 2
C. parvum
on the basis of melting curve analysis. To our knowledge, this is the first report describing the application of melting curve analysis for genotyping of
C. parvum
oocysts.
Dichotomy of Glycoprotein G Gene in Herpes Simplex Virus Type 1 Isolates
Authors: Rekabdar Elham , Tunbäck Petra , Liljeqvist Jan-Åke , Lindh Magnus , Bergström Tomas 
Published: August 2002

ABSTRACT
Herpes simplex virus type 1 (HSV-1) encodes 11 envelope glycoproteins, of which glycoprotein G-1 (gG-1) induces a type-specific antibody response. Variability of the gG-1 gene among wild-type strains may be a factor of importance for a reliable serodiagnosis and typing of HSV-1 isolates. Here, we used a gG-1 type-specific monoclonal antibody (MAb) to screen for mutations in the immunodominant region of this protein in 108 clinical HSV-1 isolates. Of these, 42 isolates showed no reactivity to the anti-gG-1 MAb. One hundred five strains were further examined by DNA sequencing of the middle part of the gG-1 gene, encompassing 106 amino acids including the immunodominant region and epitope of the anti-gG-1 MAb. By phylogenetic comparisons based on the sequence data, we observed two (main) genetic variants of the gG-1 gene among the clinical isolates corresponding to reactivity or nonreactivity to the anti-gG-1 MAb. Furthermore, four strains appeared to be recombinants of the two gG-1 variants. In addition, one strain displayed a gG-1-negative phenotype due to a frameshift mutation, in the form of insertion of a cytosine nucleotide. When immunoglobulin G reactivity to HSV-1 in sera from patients infected with either of the two variants was investigated, no significant differences were found between the two groups, either in a type-common enzyme-linked immunosorbent assay (ELISA) or in a type-specific gG-1 antigen-based ELISA. Despite the here-documented existence of two variants of the gG-1 gene affecting the immunodominant region of the protein, other circumstances, such as early phase of infection, might be sought for explaining the seronegativity to gG-1 commonly found in a proportion of the HSV-1-infected patients.
Detection and Characterization of Hepatitis C Virus RNA in Seminal Plasma and Spermatozoon Fractions of Semen from Patients Attempting Medically Assisted Conception
Authors: Bourlet Thomas , Levy Rachel , Maertens Anne , Tardy Jean-Claude , Grattard Florence , Cordonier Hélène , Laurent Jean-Louis , Guerin Jean-François , Pozzetto Bruno 
Published: August 2002

ABSTRACT

To investigate the risk of transmission of hepatitis C virus (HCV) via semen in assisted reproduction techniques, semen samples from 32 men chronically infected with HCV attending a center for assisted procreation were tested for HCV RNA by a reverse transcription-PCR protocol by using a modified version of the Cobas AMPLICOR HCV assay (version 2.0; Roche Diagnostics). The sensitivity of the test was 40 copies/ml. Four of 32 seminal plasma samples (12.5%) were found to be positive for the presence of HCV RNA. The median HCV load in blood was significantly higher in patients who were found to be positive for the presence of HCV RNA in semen than in those who tested negative (
P
= 0.02). In one man, seven consecutive seminal plasma samples tested positive for HCV RNA, as did two consecutive motile spermatozoon fractions; the corresponding fractions obtained after migration of the spermatozoa remained negative. Despite the absence of the proven infectivity of virus in semen samples that test positive for HCV RNA, these findings highlight the fact that seminal fluid may exhibit prolonged HCV RNA excretion. The usefulness of HCV RNA detection in both seminal plasma and spermatozoon fractions before the start of a program of medically assisted reproduction in couples in whom the male partner is chronically infected with HCV would need to be evaluated prospectively with a larger population of subjects exhibiting HCV RNA in their semen.
Development of a Real-Time Reverse Transcriptase PCR Assay for Type A Influenza Virus and the Avian H5 and H7 Hemagglutinin Subtypes
Authors: Spackman Erica , Senne Dennis A. , Myers T. J. , Bulaga Leslie L. , Garber Lindsey P. , Perdue Michael L. , Lohman Kenton , Daum Luke T. , Suarez David L. 
Published: August 2002

ABSTRACT

A real-time reverse transcriptase PCR (RRT-PCR) assay based on the avian influenza virus matrix gene was developed for the rapid detection of type A influenza virus. Additionally, H5 and H7 hemagglutinin subtype-specific probe sets were developed based on North American avian influenza virus sequences. The RRT-PCR assay utilizes a one-step RT-PCR protocol and fluorogenic hydrolysis type probes. The matrix gene RRT-PCR assay has a detection limit of 10 fg or approximately 1,000 copies of target RNA and can detect 0.1 50% egg infective dose of virus. The H5- and H7-specific probe sets each have a detection limit of 100 fg of target RNA or approximately 10
3
to 10
4
gene copies. The sensitivity and specificity of the real-time PCR assay were directly compared with those of the current standard for detection of influenza virus: virus isolation (VI) in embryonated chicken eggs and hemagglutinin subtyping by hemagglutination inhibition (HI) assay. The comparison was performed with 1,550 tracheal and cloacal swabs from various avian species and environmental swabs obtained from live-bird markets in New York and New Jersey. Influenza virus-specific RRT-PCR results correlated with VI results for 89% of the samples. The remaining samples were positive with only one detection method. Overall the sensitivity and specificity of the H7- and H5-specific RRT-PCR were similar to those of VI and HI.
Distribution of Environmentally Regulated Genes of
Streptococcus suis
Serotype 2 among
S. suis
Serotypes and Other Organisms
Authors: de Greeff Astrid , Buys Herma , Verhaar Robin , van Alphen Loek , Smith Hilde E. 
Published: August 2002

ABSTRACT

The occurrence of 36 environmentally regulated genes of
Streptococcus suis
strain 10 among all 35
S. suis
serotypes was determined by using hybridization with the amplified genes as probes. In addition, the distribution of these genes among the virulence phenotypes of serotypes 1 and 2 was assessed. Hybridization was also performed with various other streptococcal species and nonstreptococcal bacterial species which may be present in pigs. Interestingly, probe ivs-25/iri-1, similar to
agrA
and
sapR
, hybridized only with
S. suis
serotype 1 and 2 strains with virulent phenotypes and is therefore suitable as a diagnostic parameter. Only one probe was specific for
S. suis
. This probe's sequence was identical to the
epf
gene, a putative virulence factor of
S. suis
. Probe ivs-31 was similar to a virulence factor of
S. suis
, namely, a gene encoding a fibronectin- and fibrinogen-binding protein. This probe hybridized only with oral streptococci. Nearly half of the probes (45%) hybridized with the oral streptococci (
S. oralis
,
S. milleri
,
S. sanguis
,
S. gordonii
, and
S. mitis
) and with
Streptococcus pneumoniae
. This indicates a close relationship between
S. suis
, the oral streptococci, and
S. pneumoniae
with respect to the selected environmentally regulated genes. One probe only hybridized with gram-negative species and therefore seems to be obtained by
S. suis
from a gram-negative organism by horizontal transfer.
Effects of Various Test Media on the Activities of 21 Antimicrobial Agents against
Haemophilus influenzae
Authors: Jacobs Michael R. , Bajaksouzian Saralee , Windau Anne , Appelbaum Peter C. , Lin Gengrong , Felmingham David , Dencer Christine , Koeth Laura , Singer Mendel E. , Good Caryn E. 
Published: August 2002

ABSTRACT

As considerable variation in the antimicrobial susceptibility of
Haemophilus influenzae
has been reported, the effects of various test media on the susceptibility of
H. influenzae
were studied. MICs were determined by three laboratories for 21 antimicrobial agents against a panel of 100 selected isolates. Testing was performed using a reference NCCLS frozen broth microdilution method with
Haemophilus
test medium (HTM) broth and dried commercial MIC trays rehydrated with the following media: in-house and commercially prepared HTM broth, Mueller-Hinton broth with 2% lysed horse blood and NAD, IsoSensitest broth with 2% lysed horse blood and NAD, and IsoSensitest broth-based HTM. Overall, all results were very reproducible, with the MIC at which 50% of the isolates tested are inhibited (MIC
50
), MIC
90
, and geometric mean MIC being within one doubling dilution by all six methods and at all three testing centers for 15 of the 21 agents tested. Interlaboratory differences were more marked than intralaboratory differences or differences among media. Cefprozil, cefaclor, and trimethoprim-sulfamethoxazole results differed the most, while results for ampicillin, amoxicillin-clavulanic acid, cefdinir, cefixime, ceftriaxone, and clarithromycin were the most reproducible. However, these variations in results caused considerable differences in susceptibility rates for agents for which NCCLS susceptible breakpoints were close to the geometric mean MIC, particularly for cefaclor and cefprozil. This was much less of a problem when pharmacokinetic-pharmacodynamic breakpoints were used. Reproducible susceptibility results were obtained for a wide range of agents against
H. influenzae
in three laboratories using a variety of media that support the growth of this fastidious species.
Detection of
Trichomonas vaginalis
on Modified Columbia Agar in the Routine Laboratory
Authors: Stary Angelika , Kuchinka-Koch Angelika , Teodorowicz Lilianna 
Published: August 2002

ABSTRACT

Broth culture of
Trichomonas vaginalis
is considered the “gold standard” for the diagnosis of trichomoniasis. Two studies were carried out to evaluate modified Columbia agar (MCA) for the isolation of
T. vaginalis
from clinical samples. Study I compared isolation on MCA to that on liquid medium with 889 vaginal samples. Out of 63 samples positive for
T. vaginalis
(7.1% of total), MCA identified 62 (98.4%) and broth identified 58 (92.1%). In study II, trichomoniasis was diagnosed within the scope of a screening program for a total of 39,585 men and women by culture on MCA and direct microscopy. Culture on MCA detected 199 (98.5%) and Gram staining detected 163 (80.7%) of 202 positive specimens. Wet-mount preparations used for symptomatic patients identified 103 (92.8%) of 111 cases. Culture of
T. vaginalis
from clinical samples on MCA is highly sensitive and reliable, as well as timesaving, and therefore suitable for screening of symptomatic and asymptomatic individuals.
Mycobacterium microti
Infection (Vole Tuberculosis) in Wild Rodent Populations
Authors: Cavanagh Rachel , Begon Michael , Bennett Malcolm , Ergon Torbjørn , Graham Isla M. , de Haas Petra E. W. , Hart C. A. , Koedam Marianne , Kremer Kristin , Lambin Xavier , Roholl Paul , Soolingen Dick van 
Published: August 2002

ABSTRACT

Mycobacterium microti
(vole tuberculosis) infections in small wild mammals were first described more than 60 years ago in several populations in Great Britain. Few studies of vole tuberculosis have been undertaken since then, and little is known about the relationship between
M. microti
isolates originating from different populations or at different times or of the prevalence of this infection in wild rodent populations, despite human cases of
M. microti
infections being increasingly reported. In this study, field voles (
Microtus agrestis
), bank voles (
Clethrionomys glareolus
), and wood mice (
Apodemus sylvaticus
) were found to be infected, with up to 8% having external tuberculous signs, in wild populations in Northumberland and Cheshire, England. Spoligotyping applied directly to the clinical material simultaneously detected and typed
M. microti
bacteria in skin lesions, lymph glands, and internal abcesses. IS
6110
restriction fragment length polymorphism typing of cultured bacteria was used to compare these isolates with previously isolated strains from both animals and humans. This demonstrated that although the current rodent isolates were distinct from those isolated from voles in the 1930s in Great Britain, they had a high degree of similarity to these strains and were distinct from the
M. microti
isolates from humans, a pig, and a ferret from The Netherlands. Thus,
M. microti
infection seems to be widespread in wild rodent populations, but more studies are needed to understand how
M. microti
might be transmitted from animals to humans and to determine better the zoonotic risk posed.
Simultaneous Detection of
Anaplasma marginale
and a New
Ehrlichia
Species Closely Related to
Ehrlichia chaffeensis
by Sequence Analyses of 16S Ribosomal DNA in
Boophilus microplus
Ticks from Tibet
Authors: Wen Bohai , Jian Rui , Zhang Youzhi , Chen Rong 
Published: August 2002

ABSTRACT

To identify ehrlichial agents in
Boophilus microplus
ticks, DNA samples of
B. microplus
collected from the Tibet Autonomous Region and Sichuan Province of China were screened by a nested PCR. Sixteen of 43 (37%) DNA samples of
B. microplus
from Tibet were positive in nested PCR analysis. All 27 samples from Sichuan were negative. The screen identified two ehrlichial agents based on different 16S rRNA genes that were found after amplifying and sequencing the 5′-end fragments of the 16S rRNA genes. One sequence was identical to that of the gene of
Anaplasma marginale
, an etiological agent of animal anaplasmosis. The other sequence was most similar to that of the gene of
Ehrlichia chaffeensis
, an etiological agent of human monocytic ehrlichiosis. The sequence of 1,501 bases from the novel ehrlichial agent was obtained and showed the greatest levels of sequence similarity (97 to 98%) to 16S rRNA gene sequences of the members of the
E. canis
group of the genus
Ehrlichia
. Sequence comparison of the 16S rRNA gene with the members of the genus
Ehrlichia
reveals that the novel ehrlichial agent detected in
B. microplus
ticks is a new species of the genus
Ehrlichia
and is most closely related to
E. chaffeensis
.
Rapid Detection of Methicillin Resistance in Coagulase-Negative Staphylococci with the VITEK 2 System
Authors: Horstkotte Matthias A. , Knobloch Johannes K.-M. , Rohde Holger , Dobinsky Sabine , Mack Dietrich 
Published: August 2002

ABSTRACT

The aim of the present study was to evaluate the accuracy of the new VITEK 2 system (bioMérieux, Marcy l' Etoile, France) for the detection of methicillin resistance in coagulase-negative staphylococci (CoNS) by using AST-P515 and AST-P523 test cards. Analyses of the VITEK 2 oxacillin MIC determination evaluated according to the actual breakpoint (≥0.5 μg/ml) of the National Committee for Clinical Laboratory Standards resulted in a high sensitivity of 99.2% but a moderate specificity of 80%. The newly included oxacillin resistance (OR) test of the VITEK 2 system displayed a high sensitivity and a high specificity of 97.5 and 98.7%, respectively. Concordance between the results of the
mecA
PCR and the VITEK 2 oxacillin MIC was observed for almost all
Staphylococcus epidermidis
strains, but the reduced specificity was attributable to higher oxacillin MICs for
mecA
-negative non-
S. epidermidis
strains, especially
S. saprophyticus
,
S. lugdunensis
, and
S. cohnii
. Evaluation of alternative oxacillin MIC breakpoints of 1, 2, or 4 μg/ml resulted in improved degrees of specificity of 84, 90.7, and 97.3%, respectively. Only minor changes occurred in the corresponding sensitivity values, which were 98.4, 97.5, and 97.5%, respectively. Methicillin resistance in CoNS was detected after 7 and 8 h in 91.1 and 93.5% of the
mecA
-positive strains, respectively, by the VITEK 2 OR test and in 86.3 and 89.5% of the
mecA
-positive strains, respectively, by VITEK 2 oxacillin MIC determination. After 7 and 8 h the VITEK 2 OR test classified 59.2 and 78.9% of the
mecA
-negative strains, respectively, as susceptible to oxacillin, whereas comparable values were obtained 2 h later by VITEK 2 oxacillin MIC determination. The results of our study encourage the use of the VITEK 2 system, which proved to be a highly reliable and rapid phenotypic method for the detection of methicillin resistance in CoNS.