bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of the Biostar Chlamydia OIA Assay with Specimens from Women Attending a Sexually Transmitted Disease Clinic
Authors: Pate Mitchell S. , Dixon Paula B. , Hardy Kim , Crosby Mark , Hook E. W. 
Published: December 2019

ABSTRACT

Chlamydia trachomatis
infections are the most prevalent sexually transmitted diseases (STDs) in the United States. In acute-care settings such as clinics and emergency rooms, a desirable chlamydia screening assay should exhibit good sensitivity and good specificity and should provide test results while the patient is still present. The Biostar Chlamydia OIA (Biostar, Inc., Boulder, Colo.) is an optical immunoassay (OIA) that provides test results in less than 30 min and that uses a test format that allows office-based testing. This assay is performed entirely at room temperature without the need for rotators or other specialized equipment. The goal of this study was to compare the performance of the Biostar Chlamydia OIA for the detection of
C. trachomatis
with the performance of cell culture, direct fluorescent-antibody (DFA) assay (Syva MicroTrak; Syva Co., Palo Alto, Calif.), and PCR (Roche Amplicor
Chlamydia trachomatis
; Roche, Branchburg, N.J.) for the detection of
C. trachomatis
infections in women attending an urban STD clinic. For calculations of relative test performance (sensitivity, specificity, and positive and negative predictive values), patient specimens that yielded positive results by two or more of the four assays (cell culture, DFA assay, PCR, and OIA) were classified as “true infections.” By these criteria, 42 of 306 total specimens were classified as positive for
C. trachomatis
(positive prevalence, 13.7%), 11 (3.6%; 10 by PCR and 1 by DFA assay) were positive by a single assay, and 253 (82.7%) were negative by all four tests. All culture-positive specimens were also positive by at least one other assay. Among the culture-negative specimens, 14 (5%) specimens were positive by two of the three non-culture-based assays used. By using the criterion that positivity by at least two of the tests indicated a true infection, the relative sensitivities were as follows: culture and PCR, 92.9% each; Biostar Chlamydia OIA, 73.8%; and DFA assay, 59.5%.
Emergence of Vancomycin-Resistant Enterococci in Australia: Phenotypic and Genotypic Characteristics of Isolates
Authors: Bell Jan M. , Paton James C. , Turnidge John 
Published: December 2019

ABSTRACT

Enterococci with resistance to glycopeptides have recently emerged in Australia. We developed multiplex PCR assays for
vanA
,
vanB
,
vanC1
, and
vanC2
or
vanC3
in order to examine the genetic basis for vancomycin resistance in Australian isolates of vancomycin-resistant
Enterococcus faecium
and
E. faecalis
(VRE). The predominant genotype from human clinical
E. faecium
isolates was
vanB
. The PCR
van
genotype was consistent with the resistance phenotype in all but six cases. One
vanA E. faecalis
isolate had a VanB phenotype, one
vanB E. faecium
isolate had a VanA phenotype, and four
E. faecalis
isolates were consistently negative for
vanA
,
vanB
,
vanC1
, and
vanC2
or
vanC3
, even though they exhibited a VanB phenotype. These four isolates were subsequently examined for the presence of
vanD
by published methods and were found to be negative. No vancomycin-susceptible strains produced a PCR product. On the basis of our findings the epidemiology of VRE in Australia appears to be different from that in either the United States or Europe. Our multiplex PCR assays gave a rapid and accurate method for determining the genotype and confirming the identification of glycopeptide-resistant enterococci. Rapid and accurate methods are essential, because laboratory-based surveillance is critical in programs for the detection, control, and prevention of the transmission of glycopeptide-resistant enterococci.
Direct Identification of
Coxiella burnetii
Plasmids in Human Sera by Nested PCR
Authors: Zhang G. Q. , Hotta A. , Mizutani M. , Ho T. , Yamaguchi T. , Fukushi H. , Hirai K. 
Published: December 2019

ABSTRACT

Nested PCR assays were used for the direct identification of
Coxiella burnetii
plasmids in human sera. A total of 81 serum samples from 81 patients with Q fever were tested by nested PCR with four sets of primers. The first set of primers was used to detect the genomic sequences. The second set of primers was used to detect the conserved sequences of the plasmids. Another two sets of primers were used to identify the QpH1 and QpRS plasmids. QpH1 and QpRS plasmid-specific sequences were identified in 40 (49.4%) and 24 (29.6%) of the serum samples, respectively. Both of the QpH1 and QpRS plasmid-specific sequences were detected in 5 (8.6%) of the serum samples but were not found in 12 (20.7%) of the serum samples. Furthermore, all of the 23 acute-phase serum samples were positive for the QpH1 plasmid and negative for the QpRS plasmid. Nested PCR with plasmid-specific primers appears to be a useful method for the direct typing of
C. burnetii
plasmids in human sera.
Variants of the 3′ Region of the
cagA
Gene in
Helicobacter pylori
Isolates from Patients with Different
H. pylori
-Associated Diseases
Authors: Yamaoka Yoshio , Kodama Tadashi , Kashima Kei , Graham David Y. , Sepulveda Antonia R. 
Published: December 2019

ABSTRACT

The CagA protein of
Helicobacter pylori
is an immunogenic antigen of variable size and unknown function that has been associated with increased virulence as well as two mutually exclusive diseases, duodenal ulcer and gastric carcinoma. The 3′ region of the
cagA
gene contains repeated sequences. To determine whether there are structural changes in the 3′ region of
cagA
that predict outcome of
H. pylori
infection, we examined 155
cagA
gene-positive
H. pylori
isolates from Japanese patients including 50 patients with simple gastritis, 40 with gastric ulcer, 35 with duodenal ulcer, and 30 with gastric cancer. The 3′ region of the
cagA
gene was amplified by PCR followed by sequencing. CagA proteins were detected by immunoblotting using a polyclonal antibody against recombinant CagA. One hundred forty-five strains yielded PCR products of 642 to 651 bp; 10 strains had products of 756 to 813 bp. The sequence of the 3′ region of the
cagA
gene in Japan differs markedly from the primary sequence of
cagA
genes from Western isolates. Sequence analysis of the PCR products showed four types of primary gene structure (designated types A, B, C, and D) depending on the type and number of repeats. Six of the seven type C strains were found in patients with gastric cancer (
P
< 0.01 in comparison to noncancer patients). Comparison of type A and type C strains from patients with gastric cancer showed that type C was associated with higher levels of CagA antibody and more severe degrees of atrophy. Differences in
cagA
genotype may be useful for molecular epidemiology and may provide a marker for differences in virulence among
cagA
-positive
H. pylori
strains.
Detection and Differentiation of
vanC-1
,
vanC-2
, and
vanC-3
Glycopeptide Resistance Genes in Enterococci
Authors: Clark Nancye C. , Teixeira Lucia M. , Facklam Richard R. , Tenover Fred C. 
Published: December 2019

ABSTRACT

The VanC phenotype, as found in
Enterococcus gallinarum
,
E. casseliflavus
, and
E. flavescens
, is characterized by intrinsic low-level resistance to vancomycin. The nucleotide sequences of the
vanC-1
gene in
E. gallinarum
, the
vanC-2
gene in
E. casseliflavus
, and the
vanC-3
gene in
E. flavescens
have been reported, although there is some disagreement as to whether
E. flavescens
is a legitimate enterococcal species. Previous attempts to differentiate the
vanC-2
and
vanC-3
genes by PCR analysis have been unsuccessful. The purpose of the present study was to detect and differentiate the three
vanC
determinants and examine the distribution of these genes in a collection of both typical and atypical enterococci. The 796-bp
vanC-1
PCR product was amplified only from
E. gallinarum
isolates. As expected, due to the extensive homology in the
vanC-2
and
vanC-3
gene sequences, all of the
E. casseliflavus
and
E. casseliflavus/flavescens
isolates produced the 484-bp
vanC-2
PCR product, although the
E. gallinarum
isolates were negative. Only the
E. casseliflavus/flavescens
isolates produced the 224-bp
vanC-3
product. Using the three sets of primers, we were able to detect and distinguish the
vanC-1
,
vanC-2
, and
vanC-3
genes from both typical and atypical enterococci strains. Antimicrobial susceptibility tests and analysis of genomic DNA by pulsed-field gel electrophoresis were also performed, but the results indicated that they were not able to distinguish among strains possessing the three
vanC
genotypes.
Isolation ofPorphyromonas gingivalisand Detection of Immunoglobulin A Specific to Fimbrial Antigen in Gingival Crevicular Fluid
Authors: Condorelli Francesca , Scalia Guido , Calì Giuditta , Rossetti Bruno , Nicoletti Giuseppe , Lo Bue Anna M. 
Published: December 2019

ABSTRACTThe present study evaluated the prevalence ofPorphyromonas gingivalisand the correlation between the bacterial culture method and the detection of immunoglobulin A (IgA) specific to theP. gingivalisfimbrial antigen in gingival crevicular fluid (GCF).P. gingivaliswas isolated from 78.3% of subgingival plaque samples obtained from active sites and 34.7% of those from inactive sites of periodontal patients.P. gingivaliswas isolated from only 4.7% of healthy subjects (control group). Immunoglobulins specific to theP. gingivalisfimbrial antigen were detected by enzyme-linked immunosorbent assay (ELISA). The overall agreement between the results of theP. gingivalisculture method and the results of specific IgA detection in periodontal patients was 71.7% for active sites and 58.7% for inactive sites. IgA specific toP. gingivaliswas absent in GCF from all of the sites of healthy subjects. The results suggest thatP. gingivalisis associated with the local production of specific IgA. The detection of IgA antibodies specific toP. gingivalisin GCF by ELISA may be used as a predictive parameter to reveal the early phase of the activation of recurrent periodontal infections.
Identification of a Rough Strain of
Escherichia coli
O157:H7 That Produces No Detectable O157 Antigen
Authors: Feng Peter , Sandlin Robin C. , Park Choong H. , Wilson Richard A. , Nishibuchi Mitsuaki 
Published: December 2019

ABSTRACT

MA6, an O157:H7-like strain, did not react with most anti-O157 kits examined; however, it had the
rfbE
gene that is essential for O157 expression and carried O157:H7 virulence factors. Lipopolysaccharide analysis showed that MA6 is a rough strain that does not produce the O157 antigen, but genetically, it belongs in the O157:H7 clonal group.
Inhibition of PCR in Genital and Urine Specimens Submitted for
Chlamydia trachomatis
Testing
Authors: Toye B. , Woods W. , Bobrowska M. , Ramotar K. 
Published: December 2019

ABSTRACT

We determined the frequency of PCR inhibition in genital and urine specimens submitted for
Chlamydia trachomatis
testing using the internal control DNA provided with the COBAS AMPLICOR
C. trachomatis
test and assessed methods to remove it. Inhibition occurred in 65 of 906 (7%) cervical swabs, 23 of 51 (45%) urethral swabs, and 2 of 175 (1.1%) urine samples. Overall, inhibition was eliminated in processed specimens after storage at 4°C in 77 of 90 specimens (86%), freezing at −70°C in 59 of 82 specimens (72%), storage at 4°C followed by either 1:100 dilution in 37 of 43 specimens (86%) or 1:10 dilution in 42 of 47 specimens (89%), and phenol-chloroform extraction in 79 of 80 specimens (99%). No positive specimens were missed due to inhibition. We conclude that PCR inhibition is rare with urine specimens and infrequent with endocervical swabs but occurs frequently with urethral swabs. The frequency of PCR inhibition may be significantly reduced by methods which can be easily incorporated into the processing of specimens.
Purification of
Encephalitozoon
Cultures Contaminated by Mycoplasmas by Murine Intraperitoneal Inoculation
Authors: Ridoux Olivier , Foucault Cédric , Drancourt Michel 
Published: December 2019

ABSTRACT

Encephalitozoon
species are strict intracellular microsporidia. Cocultures with eukaryotic cell lines can become accidently contaminated by mycoplasmas. We propose a decontamination protocol based on differential cell targeting after intraperitoneal inoculation in mice. Mycoplasma-free microsporidia were isolated from the brains and spleens of inoculated mice 24 h postinoculation by using the centrifugation shell vial system. Identification was confirmed by direct sequencing of PCR-amplified 16S rRNA.
Colony Size Can Be Used To Determine the MIC of Fluconazole for Pathogenic Yeasts
Authors: Xu Jianping , Vilgalys Rytas , Mitchell Thomas G. 
Published: December 2019

ABSTRACT
This report describes a new statistical method for estimating the MIC of fluconazole for yeasts pathogenic to humans. This method is based on comparison of the colony sizes on solid media containing different concentrations of fluconazole. By this method, the MICs of fluconazole for 10 yeast strains were comparable to results obtained by the standard method recommended by the National Committee for Clinical Laboratory Standards. This method is simple to perform and easy to interpret. The turnaround time is faster than other methods. The method should be applicable to the determination MICs of other antifungal drugs for yeasts.
Detection by PCR of Enteroviruses in Cerebrospinal Fluid during a Summer Outbreak of Aseptic Meningitis in Switzerland
Authors: Gorgievski-Hrisoho Meri , Schumacher Jean-Daniel , Vilimonovic Nevenka , Germann Daniel , Matter Lukas 
Published: December 2019

ABSTRACTEnteroviruses (EV) are among the most common causes of aseptic meningitis. Standard diagnostic techniques are often too slow and lack sensitivity to be of clinical relevance. EV RNA can be detected within 5 h by a commercially available reverse transcription-PCR (RT-PCR) test kit. Cerebrospinal fluid (CSF) samples from 68 patients presenting with aseptic meningitis during a summer outbreak in Switzerland were examined in parallel with cell culture and commercial RT-PCR. RT-PCR was positive in all 16 CSF specimens positive by cell culture (100%). In addition, 42 of 52 (80%) CSF samples negative by cell culture were PCR positive. In 26 of these 42 (62%) patients, viral culture from other sites (throat swab or stool) was also positive. The CSF virus culture took 3 to 7 days to become positive. Echovirus 30 was the type most often isolated in this outbreak. The sensitivity of CSF RT-PCR based on clinical diagnosis during this aseptic meningitis outbreak in patients with negative bacterial culture results was 85%, i.e., considerably higher than the sensitivity of CSF virus culture (24%). We conclude that this commercial RT-PCR assay allows a positive diagnosis with minimal delay and may thus influence clinical decisions.
Cooperative Oligonucleotides Mediating Direct Capture of Hepatitis C Virus RNA from Serum
Authors: O’Meara Deirdre , Yun Zhibing , Sönnerborg Anders , Lundeberg Joakim 
Published: December 2019

ABSTRACT
A novel method for direct capture of hepatitis C virus (HCV) RNA from clinical samples has been developed. This approach takes advantage of the cooperative interactions between adjacently hybridized oligonucleotides. Here, this cooperative effect was combined with solid-phase technology, whereby a capture probe was covalently coupled to magnetic beads and a second probe, which anneals adjacent to the capture probe site, was prehybridized in solution to the target. When these contiguously hybridized probes were used for the extraction of HCV RNA from clinical samples, the capture efficiency was increased up to 25-fold in comparison to capture with a single probe. The applicability of this sample preparation assay was further investigated by performing a comparative study with both a conventional guanidinium extraction method and a commercial quantitative assay.
Necessity of Molecular Techniques To Distinguish between
Neisseria meningitidis
Strains Isolated from Patients with Meningococcal Disease and from Their Healthy Contacts
Authors: Vogel Ulrich , Morelli Giovanna , Zurth Kerstin , Claus Heike , Kriener Eugen , Achtman Mark , Frosch Matthias 
Published: December 2019

ABSTRACT

Serogroup C strains of
Neisseria meningitidis
were isolated from a Germany patient with severe meningococcal disease after a trip to the Czech Republic. These strains (case isolates) were characterized by classical and molecular techniques, as were other strains (carrier isolates) isolated from healthy contacts. Five of 10 carrier isolates had switched off the expression of capsular polysaccharide, as demonstrated by a serogroup-specific PCR. The two case isolates were indistinguishable by multilocus sequence typing and belonged to the ET-37 complex. The carrier isolates belonged to four different sequence types, all unrelated to that of the case strains. Pulsed-field gel electrophoresis showed that the case isolates differed from reference ET-37 complex strains from the Czech Republic and Canada as well as from all the carrier isolates. The isolate from the patient’s nasopharynx was indistinguishable from the blood isolate except for a 40,000-bp chromosomal deletion that had occurred during systemic spread.
Determination of Copy Number of rRNA Genes in
Pneumocystis carinii
f. sp.
hominis
Authors: Tang Xing , Bartlett Marilyn S. , Smith James W. , Lu Jang-Jih , Lee Chao-Hung 
Published: December 2019

ABSTRACT

Differential PCR was performed to determine the copy number of rRNA genes in
Pneumocystis carinii
f. sp.
hominis
. Two different reference genes, thymidylate synthase (TS) and beta-tubulin (BTU) genes, were used. Primers for the internal transcribed spacer (ITS) region of nuclear rRNA genes and either the TS or BTU gene were mixed together to perform PCR on seven different bronchoalveolar lavage specimens from patients with
P. carinii
pneumonia. The radioactivity derived from the incorporated radioactive nucleotides of each PCR product band was then used to calculate the copy number of the ITS relative to that of the TS or BTU gene. The copy number ratio between the ITS and the TS gene was determined to be 0.8, and that between the ITS and the BTU gene was also 0.8. These results suggest that the ITS has the same copy number as the TS or BTU gene. Since the copy number of the TS or BTU gene is presumed to be 1, the results also suggest that
P. carinii
f. sp.
hominis
has only one copy of the ITS and thus one copy of the nuclear rRNA genes. Therefore, two types of ITS sequences derived from a specimen would indicate that the patient is infected by two types of
P. carinii
f. sp.
hominis
.
Comparison of NucliSens and Amplicor Monitor Assays for Quantification of Human Immunodeficiency Virus Type 1 (HIV-1) RNA in Plasma of Persons with HIV-1 Subtype A Infection in Abidjan, Côte d’Ivoire
Authors: Nkengasong John N. , Kalou Mirielle , Maurice Chantal , Bile Celestin , Borget Marie-Yolande , Koblavi Stéphania , Boateng Emmanuel , Sassan-Morokro Madeleine , Anatole-Ehounou Ekpini , Ghys Peter , Greenberg Alan E. , Wiktor Stefan Z. 
Published: December 2019

ABSTRACT

We compared the sensitivity and accuracy of the NucliSens assay and those of both the standard and modified (addition of a new primer set, primer mix 1, supplied by Roche) Amplicor HIV Monitor assays to quantify human immunodeficiency virus type 1 (HIV-1) RNA in persons infected with HIV-1 subtype A in Abidjan, Côte d’Ivoire. Seventy-one plasma samples from HIV-1-seropositive persons at different stages of HIV infection and 15 samples from HIV antibody-negative persons were analyzed. The HIV-1 genetic subtype was determined either by DNA sequencing or by a restriction fragment length polymorphism assay. Of the 71 samples, 70 (98%) were subtype A and 1 was subtype G. Of the 70 subtype A samples, the proportion of RNA-positive plasma samples and mean HIV-1 RNA levels were significantly higher by the modified HIV Monitor assay (
n
= 67 [96%]; mean RNA levels, 5.2 log
10
HIV-1 RNA copies/ml) than the NucliSens assay (
n
= 56 [80%]; 4.3 log
10
HIV-1 RNA copies/ml) or the standard HIV Monitor assay (
n
= 44 [63%]; mean RNA levels, 3.8 log
10
HIV-1 RNA copies/ml) (all
P
values were <0.05). The HIV-1 RNA levels by the modified HIV Monitor assay correlated significantly with those by the NucliSens assay (
r
= 0.76;
P
< 0.001) and the standard HIV Monitor assay (
r
= 0.57;
P
< 0.001), as did the RNA levels by the NucliSens and the standard HIV Monitor assays (
r
= 0.60;
P
< 0.001). Lower CD4 cell counts were significantly correlated with higher HIV-1 RNA levels by all three assays (
r
= −0.47 for the NucliSens assay, −0.45 for the standard HIV Monitor assay, and −0.62 for the modified HIV Monitor assay). These results indicate that the modified HIV Monitor assay has the highest sensitivity and efficiency at quantifying the levels of RNA in persons infected with HIV-1 subtype A and thus constitutes a valuable tool for the monitoring of RNA levels in areas of Africa were HIV-1 subtype A is predominant.
Antigenic Characterization of Hantaan and Seoul Virus Nucleocapsid Proteins Expressed by Recombinant Baculovirus: Application of a Truncated Protein, Lacking an Antigenic Region Common to the Two Viruses, as a Serotyping Antigen
Authors: Morii Mayuko , Yoshimatsu Kumiko , Arikawa Jiro , Zhou Guizen , Kariwa Hiroaki , Takashima Ikuo 
Published: December 2019

ABSTRACT

Hantaan virus (HTN) and Seoul virus (SEO) are members of the genus
Hantavirus
in the family
Bunyaviridae
and are causative agents of hemorrhagic fever with renal syndrome. The complete and truncated nucleocapsid proteins (NP) of HTN and SEO were expressed by a recombinant baculovirus system. Antigenic characterization of the NP using monoclonal antibodies (MAbs) indicated that the binding sites for the serotype-specific MAbs were located between amino acids (aa) 155 and 429. A Western blot assay indicated that the serotype-specific epitopes were conformation dependent. An indirect immunofluorescence antibody (IFA) assay with the truncated NP (aa 155 to 429) was able to distinguish convalescent-phase sera from HTN and SEO patients. However, the antibody titers with the truncated NP were lower than those with the whole NP. The truncated NP of SEO (aa 155 to 429) could be used as an enzyme-linked immunosorbent assay (ELISA) antigen, but the truncated NP from HTN lost its reactivity when used for ELISA. The IFA assay using baculovirus-expressed truncated NP as an antigen is a rapid, simple, and safe test for distinguishing between HTN and SEO infections by serotype.
Expanding Allelic Diversity of
Helicobacter pylori vacA
Authors: van Doorn Leen-Jan , Figueiredo Céu , Sanna Ricardo , Pena Salvador , Midolo Peter , Ng Enders K. W. , Atherton John C. , Blaser Martin J. , Quint Wim G. V. 
Published: December 2019

ABSTRACT

The diversity of the gene encoding the vacuolating cytotoxin (
vacA
) of
Helicobacter pylori
was analyzed in 98 isolates obtained from different geographic locations. The studies focused on variation in the previously defined s and m regions of
vacA
, as determined by PCR and direct sequencing. Phylogenetic analysis revealed the existence of four distinct types of s-region alleles: aside from the previously described s1a, s1b, and s2 allelic types, a novel subtype, designated s1c, was found. Subtype s1c was observed exclusively in isolates from East Asia and appears to be the major s1 allele in that part of the world. Three different allelic forms (m1, m2a, and m2b) were detected in the m region. On the basis of sequence alignments, universal PCR primers that allow effective amplification of the s and m regions from
H. pylori
isolates from all over the world were defined. Amplimers were subsequently analyzed by reverse hybridization onto a line probe assay (LiPA) that allows the simultaneous and highly specific hybridization of the different
vacA
s- and m-region alleles and tests for the presence of the cytotoxin-associated gene (
cagA
). This PCR-LiPA method permits rapid analysis of the
vacA
and
cagA
status of
H. pylori
strains for clinical and epidemiological studies and will facilitate identification of any further variations.