bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative Detection of Hepatitis B Virus by Transcription-Mediated Amplification and Hybridization Protection Assay
Authors: Kamisango Keiichi , Kamogawa Chieko , Sumi Mayumi , Goto Susumu , Hirao Akihide , Gonzales Frank , Yasuda Kiyomi , Iino Shiro 
Published: December 2019

ABSTRACT

We have developed a sensitive and quantitative assay using transcription-mediated amplification and hybridization protection assay for the detection of hepatitis B virus (HBV) DNA in serum. The transcription-mediated amplification was carried out in a single tube. The hybridization protection assay was carried out in a microtiter plate with two probes with different specific activities to obtain a broad detection range. As a result, the assay had a detection range of 5 × 10
3
to 5 × 10
8
genome equivalents (GE)/ml and good quantitative accuracy on a logarithmic scale. A moderately sized manual assay run can be completed within 5 h. Measurements of the amounts of HBV DNA in clinical samples by the assay showed the amounts under various disease conditions to be widely distributed (more than 5 logs, from approximately 5 × 10
3
to 5 × 10
8
GE/ml). It was also shown that the amount of HBV DNA in one chronic hepatitis patient varied widely, with a range of more than 5 logs during long-term monitoring. Our assay has the potential to be used to monitor and determine the prognosis of HBV patients and carriers, especially during interferon treatment.
Molecular Genotyping of
Staphylococcus aureus
Strains: Comparison of Repetitive Element Sequence-Based PCR with Various Typing Methods and Isolation of a Novel Epidemicity Marker
Authors: van der Zee Anneke , Verbakel Harold , van Zon Johan-Carlo , Frenay Ine , van Belkum Alex , Peeters Marcel , Buiting Anton , Bergmans Anneke 
Published: December 2019

ABSTRACT

Repetitive sequence-based (Rep)-PCR genotyping as described here is based on the presence of homologues of
Mycoplasma pneumoniae
repeat-like elements in
Staphylococcus
. In this study we comparatively evaluated the usefulness of rep-PCR typing with two sets of well-defined collections of
Staphylococcus aureus
strains. Rep-PCR analysis of the first collection of
S. aureus
strains (
n
= 59) and one
Staphylococcus intermedius
strain showed 14 different rep-PCR patterns, with each pattern harboring 6 to 15 DNA fragments. The discriminatory power of rep-PCR typing compared well to those of arbitrarily primed PCR (average of 20 types) and pulsed-field gel electrophoresis (11 types).
S. aureus
strain collection I comprised four outbreak-related groups of isolates. The isolates in only one group were found to have identical rep-PCR profiles. However, in an analysis of isolates from three additional independent local outbreaks (
n
for outbreaks 1 and 2 = 5,
n
for outbreak 3 = 12), identical rep-PCR types were found among strains isolated during each outbreak. Therefore, we conclude that rep-PCR genotyping may be an easy and fast method for monitoring of the epidemiology of nosocomial
Staphylococcus
infections. Rep-PCR analysis of strain collection II, which consisted of epidemic and nonepidemic methicillin-resistant
S. aureus
(MRSA) strains, revealed that a cluster of similar rep-PCR profiles was found among MRSA isolates which were more frequently isolated and which were most often associated with outbreaks.
Rapid Method for Screening Dried Blood Samples on Filter Paper for Human Immunodeficiency Virus Type 1 DNA
Authors: Panteleeff Dana DeVange , John Grace , Nduati Ruth , Mbori-Ngacha Dorothy , Richardson Barbra , Kreiss Joan , Overbaugh Julie 
Published: December 2019

ABSTRACTPCR is a highly sensitive method for the detection of human immunodeficiency virus type 1 (HIV-1) nucleic acids in blood mononuclear cells and plasma. However, blood separation techniques require extensive laboratory support systems and are difficult when a limited volume of blood is available, which is often the case for infants. The use of blood samples stored on filter paper has many advantages for the detection of perinatal HIV-1 infection, but current methods require extraction and purification of target DNA prior to PCR amplification. We report a highly sensitive and rapid method for the extraction and detection of HIV-1 DNA in infant blood samples stored on filter papers. Because this rapid protocol does not involve steps for the removal of potential inhibitors of the PCR, the highest sensitivity is achieved by testing the filter paper lysate in quadruplicate. Assays for HIV-1 DNA were done by using nested PCR techniques that amplify HIV-1gagDNA from blood spot samples on filter paper and from corresponding viably frozen mononuclear cells separated from venous blood samples obtained from 111 infants born to HIV-1-seropositive mothers. PCR results with blood from filter papers showed 100% specificity (95% confidence internal [CI] 93.1 to 100%) and 96% (95% CI, 88.65 to 98.9%) and 88% (95% CI, 79.2 to 94.5%) sensitivity (for quadruplicate and duplicate tests, respectively) compared to PCR results with blood mononuclear cells. Moreover, this method could detect HIV-1 sequences of multiple subtypes.
Prevalence of β2-ToxigenicClostridium perfringensin Horses with Intestinal Disorders
Authors: Herholz Cornelia , Miserez Raymond , Nicolet Jacques , Frey Joachim , Popoff Michel , Gibert Maryse , Gerber Heinz , Straub Reto 
Published: December 2019

ABSTRACTThe incidence of a new, yet unassigned toxin type ofClostridium perfringenscontaining the genes for the α-toxin and the recently described β2-toxin in horses with intestinal disorders is reported. The study included 18 horses suffering from typical typhlocolitis, 7 horses with atypical typhlocolitis, 16 horses with other intestinal disorders, and 58 horses without intestinal disease. In total, 20 samples of ingesta of the small and large intestines, five biopsy specimens of the intestinal wall, and 74 fecal samples were analyzed bacteriologically.C. perfringensisolates were typed for the presence of the α-, β-, β2-, and ɛ-toxin and enterotoxin genes by PCR, including a newly developed PCR for the detection of the β2-toxin genecpb2. β2-ToxigenicC. perfringenswas detected in samples from 13 of 25 (52%) horses with typical or atypical typhlocolitis, with a particularly high incidence in specimens of ingesta and biopsy specimens (75%), whereas only 6 of 16 specimens from horses with other intestinal diseases yielded β2-toxigenicC. perfringens. No β2-toxigenicC. perfringenswas found in the samples from the 58 control horses, of which only one fecal sample containedC. perfringenstype A. Among the samples from the 15 horses with fatal cases of typical and atypical typhlocolitis 9 (60%) were positive for β2-toxigenicC. perfringens, whereas samples from only 4 of the 10 (40%) animals with nonfatal cases of infection were positive. We found an interesting correlation between the antibiotic-treated horses which were positive for β2-toxigenicC. perfringensand lethal progression of the disease. NoC. perfringensstrains isolated in this study contained genes for the β- and ɛ-toxins and enterotoxin. The high incidence of β2-toxigenicC. perfringensin samples of ingesta, biopsy specimens of the intestinal wall, and feces from horses suffering or dying from typhlocolitis together with the absence of this organism in healthy horses provides strong evidence that β2-toxigenicC. perfringensplay an important role in the pathogenesis of typhlocolitis.
Relatedness of
Streptococcus suis
Serotype 2 Isolates from Different Geographic Origins as Evaluated by Molecular Fingerprinting and Phenotyping
Authors: Chatellier Sonia , Gottschalk Marcelo , Higgins Robert , Brousseau Roland , Harel Josee 
Published: December 2019

ABSTRACT

The genetic diversity of 88
Streptococcus suis
serotype 2 isolates which were recovered from various countries was examined by randomly amplified polymorphic DNA (RAPD) analysis with three primers. This bacterial collection included 80 isolates of porcine origin and 8 of human origin. This investigation allowed the identification of 23 RAPD types containing 1 to 30 isolates originating from one to six countries. Common RAPD patterns were found between human and pig isolates. The isolates were also tested for the production of virulent factors such as hemolysin, muramidase-released protein (MRP), and extracellular factor (EF). All isolates exhibiting the virulent phenotype hemolysin
+
MRP
+
EF
+
clearly clustered on the basis of fingerprinting by RAPD analysis. In a similar way, most of isolates with the hemolysin
−
MRP
−
EF
−
phenotype were assigned to one RAPD cluster. Therefore, RAPD clusters are more related to the phenotype defined with hemolysin, MRP, and EF than to the geographic origin of the isolates. These data indicate that RAPD analysis used in conjunction with phenotypic methods provides a reliable method for the assessment of the clonal relationship between
S. suis
isolates responsible for infections in pigs or humans, especially for those exhibiting the classic “virulent” phenotype hemolysin
+
MRP
+
EF
+
.
Performance Characteristics of an Enzyme-Linked Immunosorbent Assay for Determining Salivary Immunoglobulin G Response to
Helicobacter pylori
Authors: De Pascalis R. , Del Pezzo M. , Nardone G. , Budillon G. , Lavitola A. 
Published: December 2019

ABSTRACT

We evaluated the salivary immunoglobulin G (IgG) immune response to
Helicobacter pylori
in 70 subjects by enzyme-linked immunosorbent assay (ELISA). Subjects with a positive
H. pylori
culture showed significantly higher titers of antibodies than subjects with no detectable
H. pylori
: the overall sensitivity and specificity of the test were 84 and 90%, respectively. The detection of salivary anti-
H. pylori
IgG antibodies may be considered as an alternative to serum IgG detection for ease of sample collection or when blood samples are not available in screening of patients with dyspepsia.
PCR Targeted to the 16S-23S rRNA Gene Intergenic Spacer Region of
Clostridium difficile
and Construction of a Library Consisting of 116 Different PCR Ribotypes
Authors: Stubbs Simon L. J. , Brazier Jon S. , O’Neill Gael L. , Duerden Brian I. 
Published: December 2019

ABSTRACT

A reference library of types of
Clostridium difficile
has been constructed by PCR ribotyping isolates (
n
= 2,030) from environmental (
n
= 89), hospital (
n
= 1,386), community practitioner (
n
= 395), veterinary (
n
= 27), and reference (
n
= 133) sources. The library consists of 116 distinct types identified on the basis of differences in profiles generated with PCR primers designed to amplify the 16S-23S rRNA gene intergenic spacer region. Isolates from 55% of infections in hospitals in the United Kingdom belonged to one ribotype (type 1), but this type was responsible for only 7.5% of community infections.
Genetic Diversity of
Borrelia burgdorferi
in Lyme Disease Patients as Determined by Culture versus Direct PCR with Clinical Specimens
Authors: Liveris Dionysios , Varde Shobha , Iyer Radha , Koenig Seth , Bittker Susan , Cooper Denise , McKenna Donna , Nowakowski John , Nadelman Robert B. , Wormser Gary P. , Schwartz Ira 
Published: December 2019

ABSTRACT

Two hundred seventeen isolates of
Borrelia burgdorferi
originally cultured from skin biopsy samples or blood of early Lyme disease patients were genetically characterized by PCR-restriction fragment length polymorphism (RFLP) typing of the 16S-23S ribosomal DNA intergenic spacer. Three major RFLP types were observed. Of the cultured isolates, 63 of 217 (29.0%) were type 1, 85 of 217 (39.2%) were type 2, and 58 of 217 (26.7%) were type 3; mixtures of two RFLP types were obtained in 6.0% (13 of 217) of the cultures. Comparison of typing of
B. burgdorferi
performed directly on 51 patient skin specimens with typing of cultures originally isolated from the same tissue revealed that a much larger proportion of direct tissue samples had mixtures of RFLP types (43.1% by direct typing versus 5.9% by culture [
P
< 0.001). In addition, identical RFLP types were observed in only 35.5% (11 of 31) of the paired samples. RFLP type 3 organisms were recovered from blood at a significantly lower rate than were either type 1 or type 2 strains. These studies demonstrate that the genetic diversity of
B. burgdorferi
patient isolates as determined by cultivation differs from that assessed by PCR performed directly on patient tissue.
Nested Duplex PCR To Detect
Bordetella pertussis
and
Bordetella parapertussis
and Its Application in Diagnosis of Pertussis in Nonmetropolitan Southeast Queensland, Australia
Authors: Farrell D. J. , Daggard G. , Mukkur T. K. S. 
Published: December 2019

ABSTRACT

A duplex PCR to detect
Bordetella pertussis
and
Bordetella parapertussis
was developed with the insertion sequences IS
481
(
B. pertussis
) and IS
1001
(
B. parapertussis
) and evaluated with specimens from 520 consecutive patients presenting with possible pertussis. No culture-positive–PCR-negative results occurred, giving the method a sensitivity of 100%. For
B. pertussis
, 58 of 520 patients (11.2%) were positive by PCR compared to 17 of 520 patients positive (3.3%) by culture. For
B. parapertussis
, 7 of 520 patients (1.3%) were positive by PCR compared to 2 of 520 patients positive (0.4%) by culture. Two patients were positive for both
B. pertussis
and
B. parapertussis
. Patient records were reviewed to determine the validity of PCR-positive–culture-negative results. Forty-two of 49 patients who could be evaluated fulfilled the criteria for a case definition of pertussis, with 32 patients being <1 year of age and having classical pertussis symptoms. The seven patients who did not fulfil the criteria were aged 7 to 55 years and had a persistent cough for >2 weeks. The method was also used to investigate a classroom outbreak in which
B. pertussis
culture was positive for 5 of 28 patients. All five culture-positive specimens were confirmed by PCR, and an additional eight were positive by PCR. Of 25 patients from a suspected pertussis outbreak in a girls’ dormitory, seven of seven specimens were negative for
B. pertussis
, although 13 of 25 patients were positive for
B. pertussis
immunoglobulin M (IgM) (2 of which produced equivocal IgA results, with 23 of 25 patients being negative). Five symptomatic patients were subsequently found to be positive (by IgM and particle agglutination assays) for
Mycoplasma pneumoniae
, demonstrating the value of PCR in rapidly excluding
B. pertussis
infection in an outbreak situation. Twenty-two of 71 (30.1%) throat swabs were positive by PCR compared to 2 of 71 (2.8%) throat swabs positive by culture, indicating that a reassessment of the use of throat swabs should be considered, particularly for older patients, in contact tracing, and in situations in which specimen collection is difficult.
Improved Silica-Guanidiniumthiocyanate DNA Isolation Procedure Based on Selective Binding of Bovine Alpha-Casein to Silica Particles
Authors: Boom René , Sol Cees , Beld Marcel , Weel Jan , Goudsmit Jaap , Wertheim-van Dillen Pauline 
Published: December 2019

ABSTRACT
DNA purified from clinical cerebrospinal fluid and urine specimens by a silica-guanidiniumthiocyanate procedure frequently contained an inhibitor(s) of DNA-processing enzymes which may have been introduced by the purification procedure itself. Inhibition could be relieved by the use of a novel lysis buffer containing alpha-casein. When the novel lysis buffer was used, alpha-casein was bound by the silica particles in the first step of the procedure and eluted together with DNA in the last step, after which it exerted its beneficial effects for DNA-processing enzymes. In the present study we have compared the novel lysis buffer with the previously described lysis buffer with respect to double-stranded DNA yield (which was nearly 100%) and the performance of DNA-processing enzymes.
PCR Detection of DNA Specific for
Trichosporon
Species in Serum of Patients with Disseminated Trichosporonosis
Authors: Nagai Hiroyuki , Yamakami Yuriko , Hashimoto Atsuro , Tokimatsu Issei , Nasu Masaru 
Published: December 2019

ABSTRACT

Deep-seated trichosporonosis is a lethal opportunistic infection that disseminates rapidly and widely in immunocompromised patients, and early diagnosis is crucial for the treatment of this infection. We developed a novel nested-PCR assay that detects DNA specific for clinically important strains of
Trichosporon
in serum samples from patients with disseminated trichosporonosis. In this assay, two sets of oligonucleotide primers were derived from the sequence of 26S rRNA genes of
Trichosporon asahii
. The specific fragment was amplified from
T. asahii
and
T. mucoides
, but not from other microorganisms, including some other basidiomycetous fungi (Cryptococcus,
Malassezia
,
Rhodotorula
, and
Sporobolomyces
). Target DNA was detected by the nested PCR with as little as 5 fg of the extracted DNA of
T. asahii
. In a study using 11 clinical samples, the specific fragment was detected by the nested PCR in 64% (7 of 11) of sera from patients with histologically diagnosed disseminated trichosporonosis, while glucuronoxylomannan antigen was detected in only 54% (6 of 11) of the samples. Our new nested-PCR assay using serum samples can be performed repeatedly throughout the course of the disease. In addition, not only can it be used for early diagnosis of trichosporonosis, but it may also be beneficial for monitoring its progress or response to therapy.
Genetic Multilocus Studies of Different Strains of
Cryptococcus neoformans
: Taxonomy and Genetic Structure
Authors: Bertout S. , Renaud F. , Swinne D. , Mallié M. , Bastide J.-M. 
Published: December 2019

ABSTRACT

The genotypes of 107 strains of
Cryptococcus
isolated from the environment or from patients from various geographical areas were determined by multilocus enzyme electrophoresis (MLEE). We analyzed the relationships between genotype structure and serotype and between genotype structure and strain origin. Twelve of the 14 enzyme-encoding loci studied were polymorphic, giving rise to 48 electrophoretic types. The genotypes of
C. neoformans
and
C. laurentii
were very similar. MLEE could not distinguish between these two pathogenic species. A correlation between the genetic multilocus structure and the origin of the sample (from the environment or patients) existed. A second analysis detected a correlation between genotype distribution and serotype. The second analysis considered three serotype groups (B, C, and A plus D plus A/D), proving that serotypes A, D, and A/D are closely related. MLEE is a useful epidemiological tool for improving our understanding of the biology of this fungus.
Cholera in Vietnam: Changes in Genotypes and Emergence of Class I Integrons Containing Aminoglycoside Resistance Gene Cassettes in
Vibrio cholerae
O1 Strains Isolated from 1979 to 1996
Authors: Dalsgaard A. , Forslund A. , Tam N. V. , Vinh D. X. , Cam P. D. 
Published: December 2019

ABSTRACT

The number of cholera cases and the mortality rates reported from different regions of Vietnam varied considerably in the period from 1979 to 1996, with between 2,500 and 6,000 cases reported annually from 1992 to 1995. Annual mortality rates ranged from 2.0 to 9.6% from 1979 to 1983 to less than 1.8% after 1983. Major cholera outbreaks were reported from the High Plateau region for the first time in 1994 and 1995; this is an area with limited access to health services and safe drinking-water supplies. All cases were associated with
Vibrio cholerae
O1. Using ribotyping, cholera toxin (CT) genotyping, and characterization of antibiotic susceptibility patterns and antibiotic resistance genes by PCR, we show that strains isolated after 1990 were clearly different from strains isolated before 1991. In contrast to strains isolated before 1991, 94% of 104 strains isolated after 1990 showed an identical ribotype R1, were resistant to sulfamethoxazole and streptomycin, and showed a different CT genotype. Furthermore, PCR analysis revealed that sulfamethoxazole-resistant strains harbored class I integrons containing a gene cassette
ant(3")-1a
encoding resistance to streptomycin and spectinomycin. This is, to our knowledge, the first report of class I integrons in
V. cholerae
. The development of cholera and the changes in the phenotypic and genotypic properties of
V. cholerae
O1 shown in the present study highlight the importance of monitoring
V. cholerae
O1 in Vietnam as in other parts of the world. In particular, the emergence of the new ribotype R1 strain containing class I integrons should be further studied.
Impact of Microbiology Practice on Cumulative Prevalence of Respiratory Tract Bacteria in Patients with Cystic Fibrosis
Authors: Shreve Michael R. , Butler Steven , Kaplowitz Haley J. , Rabin Harvey R. , Stokes Dennis , Light Michael , Regelmann Warren E. ,   Investigators for the Epidemiologic Study of Cystic Fibrosis
Published: December 2019

ABSTRACT

Investigators participating in the Epidemiologic Study of Cystic Fibrosis project began to collect microbiological, pulmonary, and nutritional data on cystic fibrosis (CF) patients at 180 North American sites in 1994. Part of this study was a survey undertaken in August 1995 to determine microbiology laboratory practices with regard to pulmonary specimens from CF patients. The survey included a section on test ordering, completed by a site clinician, and a section on test performance and reporting, completed by each site’s clinical microbiology laboratory staff. Seventy-nine percent of the surveys were returned. There was intersite consistency of microbiology laboratory practices in most cases. The majority of sites follow most of the CF Foundation consensus conference recommendations. There were differences in the frequency at which specimens for culture were obtained, in the use of selective media for
Staphylococcus aureus
and
Haemophilus influenzae
, and in the use of a prolonged incubation for
Burkholderia cepacia
. These variations in practice contribute to prevalence differences among sites and may result in differences in clinical care.
Comparison of CHROMagar Salmonella Medium and Hektoen Enteric Agar for Isolation of Salmonellae from Stool Samples
Authors: Gaillot Olivier , Di Camillo Patrick , Berche Patrick , Courcol René , Savage Colette 
Published: December 2019

ABSTRACT

CHROMagar Salmonella (CAS), a new chromogenic medium, was retrospectively compared to Hektoen enteric agar (HEA) with 501
Salmonella
stock isolates and was then prospectively compared to HEA for the detection and presumptive identification of
Salmonella
spp. with 508 stool samples before and after enrichment. All stock cultures (100%), including cultures of H
2
S-negative isolates, yielded typical mauve colonies on CAS, while 497 (99%) isolates produced typical lactose-negative, black-centered colonies on HEA. Following overnight incubation at 37°C, a total of 20
Salmonella
strains were isolated from the 508 clinical samples. Sensitivities for primary plating and after enrichment were 95% (19 isolates) and 100% (20 isolates), respectively, for CAS and 80% (16 isolates) and 100% (20 isolates), respectively, for HEA. The specificity of CAS (88.9%) was significantly higher than that of HEA (78.5%;
P
< 0.0001). On the basis of its good sensitivity and specificity, CAS medium can be recommended for use for primary plating when human stool samples are screened for
Salmonella
spp.