bluuebunny/crossref_metadata_2025_split huggingface dataset:
Simple DNA Extraction Method for Dried Blood Spots and Comparison of Two PCR Assays for Diagnosis of Vertical Human Immunodeficiency Virus Type 1 Transmission in Rwanda
Authors: Fischer A. , Lejczak C. , Lambert C. , Servais J. , Makombe N. , Rusine J. , Staub T. , Hemmer R. , Schneider F. , Schmit J. C. , Arendt V. 
Published: January 2004

ABSTRACT

Dried blood spots (DBS) on filter paper facilitate the collection, transport, and storage of blood samples for laboratory use. A rapid and simple DNA extraction procedure from DBS was developed and evaluated for the diagnosis of human immunodeficiency virus type 1 (HIV-1) infection in children by an in-house nested-PCR assay on three genome regions and by the Amplicor HIV-1 DNA prototype assay version 1.5 (Roche Molecular Systems). A total of 150 samples from children born to HIV-1-infected mothers were collected in Kigali, Rwanda, in parallel as DBS and as peripheral blood mononuclear cell (PBMC) pellets. The results obtained on DBS by the two PCR assays were compared to the results of nested PCR on PBMCs. Of 150 PBMC samples, 10 were positive, 117 were negative, and 23 were indeterminate for HIV-1 infection. In DNA extracted from filter papers and amplified by using the in-house nested PCR, 9 of these 10 positive samples (90%) were found to be positive, and 1 was found to be indeterminate (only the
pol
region could be amplified). All of the negative samples and all of the 23 indeterminate samples tested negative for HIV-1 infection. When we used the Amplicor DNA test on DBS, all of the 10 PBMC-positive samples were found to be positive and all of the 23 indeterminate samples were found to be negative. Of the PBMC-negative samples, 115 were found to be negative and 2 were found to be indeterminate. We conclude that this simple rapid DNA extraction method on DBS in combination with both detection methods gave a reliable molecular diagnosis of HIV-1 infection in children born to HIV-infected mothers.
Increased Sensitivity of Mannanemia Detection Tests by Joint Detection of α- and β-Linked Oligomannosides during Experimental and Human Systemic Candidiasis
Authors: Sendid Boualem , Jouault Thierry , Coudriau Richard , Camus Daniel , Odds Frank , Tabouret Marc , Poulain Daniel 
Published: January 2004

ABSTRACT

An enzyme immunoassay (EIA)—the commercially available Platelia
Candida
antigen test—developed for the diagnosis of systemic candidiasis is based on the detection of α-linked oligomannose residues (α-Man) released from
Candida
cells into the serum. This test has good specificity but has to be repeated frequently because of the rapid clearance of detectable mannanemia. We have developed a second EIA based on detection of beta-linked oligomannoses (β-Man), since β-Man are linked to different
Candida
molecules and interact differently with the host immune system and endogenous lectins and should therefore present different kinetics of serum clearance. In a guinea pig model of
Candida albicans
systemic infection, the relative amounts of detectable α- and β-Man differed considerably according to the virulence of the strain, the infecting dose, and the time after challenge that serum samples were drawn. Detection of α-Man was more sensitive per serum sample than that of β-Man, and the sensitivity for the combination reached 90%. The same tests were applied to 90 sera from 26 patients selected retrospectively for having been infected with the most-pathogenic
Candida
species:
C. albicans
(19),
C. tropicalis
(4), and
C. glabrata
(3). A total of 22 patients had positive antigenemia, 4 had α-mannanemia, 4 had β-mannanemia, and 14 showed the presence of both. For the patients showing the presence of both forms of mannanemia, the use of both tests enhanced the duration of the detection of mannanemia. Mannanemia was correlated with early clinical symptoms and isolation of
Candida
in culture, which occurred in 55% of the patients at an average of 4.7 days after the first positive mannanemia test result. A combination of the two tests had a cumulated specificity of 95%, and positive and negative predictive values were 79 and 97%, respectively. These findings provide evidence for different kinetics of β- and α-Man circulation during experimental and human candidiasis and suggest the joint detection of both types of epitopes as a rational approach contributing to increases in the sensitivity and earliness of diagnosis.
Decision Support Tools for Clinical Diagnosis of Disease in Cows with Suspected Bovine Spongiform Encephalopathy
Authors: Saegerman C. , Speybroeck N. , Roels S. , Vanopdenbosch E. , Thiry E. , Berkvens D. 
Published: January 2004

ABSTRACT
Reporting of clinically suspected cattle is currently the most common method for detecting cases of bovine spongiform encephalopathy (BSE). Improvement of clinical diagnosis and decision-making remains crucial. A comparison of clinical patterns, consisting of 25 signs, was made between all 30 BSE cases, confirmed in Belgium before October 2002, and 272 suspected cases that were subsequently determined to be histologically, immunohistochemically, and scrapie-associated-fiber negative. Seasonality in reporting suspected cases was observed, with more cases being reported during wintertime when animals were kept indoors. The median duration of illness was 30 days. The 10 most relevant signs of BSE were kicking in the milking parlor, hypersensitivity to touch and/or sound, head shyness, panic-stricken response, reluctance to enter in the milking parlor, abnormal ear movement or carriage, increased alertness behavior, reduced milk yield, teeth grinding, and temperament change. Ataxia did not appear to be a specific sign of BSE. A classification and regression tree was constructed by using the following four features: age of the animal, year of birth, number of relevant BSE signs noted, and number of clinical signs, typical for listeriosis, noted. The model had a sensitivity of 100% and a specificity of 85%. This approach allows the use of an interactive decision-support tool, based entirely on odds ratios, a statistic independent of disease prevalence.
Detection of the
Bacillus anthracis gyrA
Gene by Using a Minor Groove Binder Probe
Authors: Hurtle William , Bode Elizabeth , Kulesh David A. , Kaplan Rebecca Susan , Garrison Jeff , Bridge Deanna , House Michelle , Frye Melissa S. , Loveless Bonnie , Norwood David 
Published: January 2004

ABSTRACT

Identification of chromosomal markers for rapid detection of
Bacillus anthracis
is difficult because significant chromosomal homology exists among
B. anthracis
,
Bacillus cereus
, and
Bacillus thuringiensis
. We evaluated the bacterial
gyrA
gene as a potential chromosomal marker for
B. anthracis
. A real-time PCR assay was developed for the detection of
B. anthracis
. After analysis of the unique nucleotide sequence of the
B. anthracis gyrA
gene, a fluorescent 3′ minor groove binding probe was tested with 171 organisms from 29 genera of bacteria, including 102
Bacillus
strains. The assay was found to be specific for all 43 strains of
B. anthracis
tested. In addition, a test panel of 105 samples was analyzed to evaluate the potential diagnostic capability of the assay. The assay showed 100% specificity, demonstrating the usefulness of the
gyrA
gene as a specific chromosomal marker for
B. anthracis
.
Characterization of Group C and G Streptococcal Strains That Cause Streptococcal Toxic Shock Syndrome
Authors: Hashikawa Shinnosuke , Iinuma Yoshitsugu , Furushita Manabu , Ohkura Teruko , Nada Toshi , Torii Keizo , Hasegawa Tadao , Ohta Michio 
Published: January 2004

ABSTRACT

Twelve strains (the largest number ever reported) of group C and G
1
streptococci (GCS and GGS, respectively) that caused streptococcal toxic shock syndrome (STSS) were collected and characterized. Eleven strains were identified as
Streptococcus dysgalactiae
subsp.
equisimilis
, and one strain was identified as
Streptococcus equi
subsp.
zooepidemicus
. We found that it was the first reported case of STSS caused by
S. equi
subsp.
zooepidemicus
. Cluster analysis according to the 16S rRNA gene (rDNA) sequences revealed that the
S. dysgalactiae
strains belonged to clusters I and II, both of which were closely related. The
emm
types and the restriction patterns of chromosomal DNA measured by pulsed-field gel electrophoresis were highly variable in these strains except BL2719 and N1434. The 16S rDNA sequences and other characteristics of these two strains were indistinguishable, suggesting the clonal dissemination of this particular
S. dysgalactiae
strain in Japan. As the involvement of superantigens in the pathogenesis of group A streptococcus-related STSS has been suggested, we tried to detect known streptococcal superantigens in GCS and GGS strains. However, only the
spegg
gene was detected in seven
S. dysgalactiae
strains, with none of the other superantigen genes being detected in any of the strains. However, the
sagA
gene was detected in all of the strains except Tokyo1291. In the present study no apparent factor(s) responsible for the pathogenesis of STSS was identified, although close genetic relationships of GCS and GGS strains involved in this disease were suggested.
Development of a Dipstick Assay for Detection of
Leishmania
-Specific Canine Antibodies
Authors: Schallig Henk D. F. H. , Cardoso Luís , Hommers Marieke , Kroon Nel , Belling Guus , Rodrigues Manuela , Semião-Santos Saul J. , Vetter Hans 
Published: January 2004

ABSTRACT

A dipstick assay, based on
Leishmania infantum
antigen, for the rapid detection of
Leishmania
-specific antibodies in canine serum samples was developed and evaluated. After determination of optimal dipstick test conditions, test performance was compared with two existing serological tests, i.e., the direct agglutination test (DAT) and the fast agglutination screening test (FAST). In the present study the dipstick test had a sensitivity of 99.2% and a specificity of 87.9%. The DAT had a sensitivity of 97.7% and a specificity of 95.2%, whereas the FAST had also a sensitivity of 97.7% and a specificity of 93.0%. High degrees of agreement were observed between the dipstick test and DAT (93.7%; κ value, 0.86), between the dipstick test and FAST (91.8%; κ value, 0.82), and between the DAT and FAST (95.2%; κ value, 0.90). The high sensitivity and ease of performance make the dipstick test very suitable for surveillance surveys.
Demonstration of
Streptococcus mutans
with a Cell Wall Polysaccharide Specific to a New Serotype,
k
, in the Human Oral Cavity
Authors: Nakano Kazuhiko , Nomura Ryota , Nakagawa Ichiro , Hamada Shigeyuki , Ooshima Takashi 
Published: January 2004

ABSTRACT

Streptococcus mutans
organisms are occasionally isolated from the blood of patients with infective endocarditis, though the mechanisms of invasion and survival remain to be elucidated. Two of four blood isolates from patients with bacteremia or infective endocarditis (strains TW295 and TW871) were serologically untypeable by immunodiffusion testing, which was due to a lack of the glucose side chain of the serotype-specific polysaccharide antigen of
S. mutans
. Immunodiffusion analyses using antisera against these strains demonstrated that 2 of 100 isolates from 100 subjects showed a positive reaction, while further analysis of 2,500 isolates from 50 subjects revealed that all 50 isolates from a single subject were not reactive with anti-
c
, -
e
, and -
f
antisera, though they were reactive with anti-TW295 and -TW871 antisera. The oral isolates showed biological properties similar to those of the reference
S. mutans
strain MT8148, including high levels of sucrose-dependent adhesion and cellular hydrophobicity, along with expression of glucosyltransferases and a protein antigen, PA. We designated these organisms serotype
k
. A glucose side chain-defective mutant strain was then constructed by insertional inactivation of the
gluA
gene of strain MT8148, which showed biological properties similar to those of serotype
k
of
S. mutans
. Serotype
k
oral isolates were less susceptible to phagocytosis, as were the
gluA
-inactivated mutant of strain MT8148 and blood isolates. These results indicate that
S. mutans
serotype
k
strains are present in the oral cavity in humans and may be able to survive longer in blood owing to their low susceptibility to phagocytosis.
Diagnostic Assessment of
Mycoplasma genitalium
in Culture-Positive Women
Authors: Baseman Joel B. , Cagle Marianna , Korte Jeffrey E. , Herrera Caleb , Rasmussen Wanda G. , Baseman Janet G. , Shain Rochelle , Piper Jeanna M. 
Published: January 2004

ABSTRACT

Detection of
Mycoplasma genitalium
-mediated, chlamydia-negative nongonococcal urethritis and other
M. genitalium
-linked infectious etiologies has been very challenging. Although
M. genitalium
is considered a leading cause of genitourinary symptoms in men and women, extreme difficulties in its cultivation due to its highly fastidious nature and the lack of routine and effective diagnostic tests have slowed the generation of clinical data which directly implicate the presence of
M. genitalium
in disease pathogenesis. In this study, we compared enzyme-linked immunosorbent assays (ELISAs) and immunoblot and PCR assays in
M. genitalium
culture-positive women over 1 to 3 years of clinical visits to determine the usefulness of independent diagnostic strategies. Furthermore, the value of combinatorial diagnostic assessments is described, which provides insights into the dynamics of
M. genitalium
-host interactions. Overall, we show that neither ELISA nor PCR, alone or in combination, provides the sensitivity required to confidently predict the existence of viable
M. genitalium
organisms in cervical and vaginal samples. Additionally, culture-positive women exhibited a range of antibody responsiveness to
M. genitalium
based upon ELISA and immunoblot assessments, indicating immune diversity among this high-risk population.
Multicenter Evaluation of a New, Automated Enzyme-Linked Immunoassay for Detection of Human Immunodeficiency Virus-Specific Antibodies and Antigen
Authors: Sickinger Eva , Stieler Myriam , Kaufman Boris , Kapprell Hans-Peter , West Daniel , Sandridge Arnold , Devare Sushil , Schochetman Gerald , Hunt J. C. , Daghfal David 
Published: January 2004

ABSTRACT

A collaborative multicenter study was conducted to evaluate the sensitivity, specificity, and precision of a three-step, fully automated, qualitative microparticle-based enzyme-linked immunoassay (AxSYM HIV Ag/Ab Combo; Abbott Laboratories), designed to simultaneously detect (i) antibodies against human immunodeficiency virus type 1 (HIV-1) and/or type 2 (HIV-2) and (ii) HIV p24 antigen. A significant reduction in the HIV seroconversion window was achieved by combining detection of HIV antibodies and antigen into a single assay format. For 22 selected, commercial HIV seroconversion panels, the mean time of detection with the combined-format HIV antigen-antibody assay was reduced by 6.15 days compared to that with a similar third-generation single-format HIV antibody assay. The quantitative sensitivity of the combination assay for the p24 antigen (17.5 pg/ml by use of the p24 quantitative panel VIH SFTS96′) was nearly equivalent to that of single-format antigen tests. The combination assay demonstrated sensitive (100%) detection of anti-HIV immunoglobulin in specimens from individuals in CDC stages A, B, and C and from individuals infected with different HIV-1 group M subtypes, group O, or HIV-2. The apparent specificity for hospitalized patients (
n
= 1,938) was 99.90%. In a random population of 7,900 volunteer blood donors, the specificity (99.87%) was comparable to that of a third-generation single-format HIV antibody assay (99.92%) on the same donor specimens. In addition, the combination assay was robust to potential interfering specimens. The precision of the combination was high, with intra- and interrun variances of ≤9.3% for each precision panel specimen or assay control and ≤5.3% for the negative assay control.
IS
6110
Mediates Increased Transcription of the
phoP
Virulence Gene in a Multidrug-Resistant Clinical Isolate Responsible for Tuberculosis Outbreaks
Authors: Soto Carlos Y. , Menéndez M. Carmen , Pérez Esther , Samper Sofía , Gómez Ana B. , García María J. , Martín Carlos 
Published: January 2004

ABSTRACT

Drug resistance in
Mycobacterium tuberculosis
complex strains is solely due to chromosomal mutations that could affect bacterial virulence. Molecular epidemiology studies have shown that resistant strains are less likely to be clustered than susceptible strains. However, a few multidrug-resistant (MDR)
M. tuberculosis
complex strains have been described as causing outbreaks, suggesting that they have restored virulence or increased transmission. One of the biggest MDR tuberculosis outbreaks documented to date was caused by the B strain of
M. bovis
. Restriction fragment length polymorphism fingerprinting revealed that the B strain contains two copies of IS
6110
. Here, we mapped and sequenced the regions flanking the two copies of IS
6110
in the B strain. Ligation-mediated PCR showed that one of these IS
6110
copies is located within the promoter region of
phoP
, a transcriptional regulator that is essential for
M. tuberculosis
virulence. We used PCR to screen 219 MDR
M. tuberculosis
complex strains (90.4% of all MDR isolates) isolated in Spain between 1998 and 2002 and found that the B strain was the only strain that contained a copy of IS
6110
in the
phoP
promoter. To determine whether IS
6110
affects
phoP
promoter activity in the B strain, we individually cloned the
phoP
gene and its promoter region (including IS
6110
from the B strain and the equivalent region from
M. tuberculosis
without IS
6110
as a control) into a mycobacterial replicative plasmid and transformed
M. smegmatis
with the resulting plasmid. Primer extension analysis showed that
phoP
transcription was strongly upregulated when the promoter region contained IS
6110
, as in the case of the B strain.
Typing of Clinical and EnvironmentalAeromonassp. Strains by Random Amplified Polymorphic DNA PCR, Repetitive Extragenic Palindromic PCR, and Enterobacterial Repetitive Intergenic Consensus Sequence PCR
Authors: Szczuka Ewa , Kaznowski Adam 
Published: January 2004

ABSTRACTA collection of 120 strains isolated from stool specimens collected from humans suffering from gastroenteritis and from environmental samples were analyzed by random amplified polymorphic DNA PCR (RAPD), repetitive extragenic palindromic PCR (REP-PCR), and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR). Species ofAeromonoas hydrophila,A. bestiarum,A. salmonicida,A. caviae,A. media, andA. veroniirevealed clonal structure. There was no dominant clone causing gastroenteritis in humans. Moreover, there was no genetic similarity between clinical and environmental strains ofAeromonassp. isolated from different geographical areas as well as from the same geographical area. Some clones colonized specific ecosystems, e.g., drinking water distribution systems. RAPD and ERIC-PCR methods had the same discriminatory power and proved to be useful for epidemiological investigation and population genetic analysis ofAeromonasspp., whereas REP-PCR was less effective for differentiating the isolates ofAeromonasspp.
Fluorescent Amplified Fragment Length Polymorphism Genotyping of
Campylobacter jejuni
and
Campylobacter coli
Strains and Its Relationship with Host Specificity, Serotyping, and Phage Typing
Authors: Hopkins Katie L. , Desai Meeta , Frost Jennifer A. , Stanley John , Logan Julie M. J. 
Published: January 2004

ABSTRACT

Fluorescent amplified fragment length polymorphism (FAFLP) analysis was applied to 276
Campylobacter jejuni
strains and 87
Campylobacter coli
strains isolated from humans, pigs, cattle, poultry, and retail meats to investigate whether certain FAFLP genotypes of
C. jejuni
and
C. coli
are associated with a particular host and to determine the degree of association between FAFLP-defined genotypes and heat-stable serotypes and/or phage types. Within
C. coli
, the poultry strains clustered separately from those of porcine origin. In contrast, no evidence of host specificity was detected among
C. jejuni
strains. While
C. coli
strains show host specificity by FAFLP genotyping, C
. jejuni
strains that are genotypically similar appear to colonize a range of hosts, rather than being host adapted. Some serotypes and/or phage types (
C. jejuni
serotype HS18, phage type PT6, and serophage type HS19/PT2 and
C. coli
HS66, PT2, and HS56/PT2) were the most homogeneous by FAFLP genotyping, while others were more heterogeneous (
C. jejuni
HS5 and PT39, and
C. coli
HS24 and PT44) and therefore poor indicators of genetic relatedness between strains. The lack of host specificity in
C. jejuni
suggests that tracing the source of infection during epidemiological investigations will continue to be difficult. The lack of congruence between some serotypes and/or phage types and FAFLP genotype underlines the need for phenotypic testing to be supplemented by genotyping. This study also demonstrates how, in general, FAFLP generates “anonymous” genetic markers for strain characterization and epidemiological investigation of
Campylobacter
in the food chain.
Nonvalue of Culturing Cerebrospinal Fluid for Fungi
Authors: Barenfanger Joan , Lawhorn Jerry , Drake Cheryl 
Published: January 2004

ABSTRACT

No studies have evaluated the efficacy of culturing cerebrospinal fluid (CSF) for fungi. Because of the facts that the most common fungi responsible for meningitis grow well in media utilized for routine bacterial cultures and that cryptococcal antigen tests are commonly ordered, the efficacy of routinely performing fungal cultures specifically to recover fungi has been questioned. We examined data from 1,225 samples of CSF which were cultured for both bacteria and fungi. Twelve specimens yielded fungi, 10 from fungal cultures and 8 from bacterial cultures.
Cryptococcus neoformans
was found in 10 specimens,
Candida albicans
was found in 1, and a
Cladosporium
sp. was found in 1. Eight of 12 positive specimens had concordant culture results. The discordant cases were one specimen that was bacterial culture positive but fungal culture negative and three specimens that were fungal culture positive but bacterial culture negative. Of the latter discrepant cultures, one had fungal contamination only and the other two were positive for cryptococcal antigen. Therefore, omitting the fungal cultures on these specimens would not adversely impact patients. When both cryptococcal antigen tests and bacterial cultures are ordered routinely, eliminating fungal cultures on CSF would have had no impact on the patients in this study. All the clinically significant fungi were detected by the cryptococcal antigen test and/or bacterial culture. With a few exceptions, the combined use of cryptococcal antigen test and bacterial cultures of CSF could replace routine fungal cultures of CSF. Exceptions include settings where fungal pathogens other than
Cryptococcus
and
Candida
remain important causes of meningitis.