bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid Solid-Phase Immunoassay for Detection of Methicillin-Resistant
Staphylococcus aureus
Using Cycling Probe Technology
Authors: Fong Whalley K. , Modrusan Zora , Mcnevin John P. , Marostenmaki Johanna , Zin Ben , Bekkaoui Faouzi 
Published: December 2019

ABSTRACT

A Cycling Probe Technology (CPT) assay with a lateral-flow device (strip) was developed for the detection of the
mecA
gene from methicillin-resistant
Staphylococcus aureus
(MRSA) cultures. The assay uses a
mecA
probe (DNA-RNA-DNA) labeled with fluorescein at the 5′ terminus and biotin at the 3′ terminus. The CPT reaction occurs at a constant temperature, which allows the probe to anneal to the target DNA. RNase H cuts the RNA portion of the probe, allowing the cleaved fragments to dissociate from the target DNA, making the target available for further cycling. The strip detection step uses a nitrocellulose membrane with streptavidin and immunoglobulin G antibody impregnated on the surface. In the absence of the
mecA
gene, the uncut probe is bound to an antifluorescein-gold conjugate and is then captured by the streptavidin to form a test line. In the presence of the
mecA
gene, the probe is cut and no test line is formed on the strip. A screen of 324
S. aureus
clinical isolates by the CPT-strip assay showed a 99.4% sensitivity and a 100% specificity compared to the results of PCR for the detection of the
mecA
gene. Specificity testing showed that the CPT-strip assay did not exhibit any cross-reactivity with a panel of
mecA
-negative non-
S. aureus
isolates. The CPT-strip assay is simple and does not require sophisticated equipment. Furthermore, the assay takes 1.5 h starting from a primary culture to the time to detection of the
mecA
gene in
S. aureus
isolates.
Use of Pooled Fecal Culture for Sensitive and Economic Detection ofMycobacterium aviumsubsp.paratuberculosisInfection in Flocks of Sheep
Authors: Whittington R. J. , Fell S. , Walker D. , McAllister S. , Marsh I. , Sergeant E. , Taragel C. A. , Marshall D. J. , Links I. J. 
Published: December 2019

ABSTRACTOvine Johne's disease, or paratuberculosis, occurs in many countries. In Australia, surveillance using serology is used as part of a control program, but the testing regime is costly relative to its sensitivity. For this reason, culturing ofMycobacterium aviumsubsp.paratuberculosisin fecal samples pooled from a number of sheep was evaluated. Initially, the effect of pooling on the sensitivity of fecal culture was evaluated using samples from 20 sheep with multibacillary paratuberculosis and 20 sheep with paucibacillary paratuberculosis, each confirmed histologically. All multibacillary cases and 50% of paucibacillary cases were detected by culturing of feces at a pooling rate of 1 infected plus 49 uninfected sheep. In a pilot-scale study in 1997,M. aviumsubsp.paratuberculosiswas detected by pooled fecal culture on 93% of 27 infected farms which were identified originally based on history, clinical signs, and one or more rounds of testing using serologic and histopathologic examinations. Pooled fecal culture was compared with serologic examination for submissions from 335 farms where both tests had been conducted on the same sheep and was significantly more sensitive (P< 0.001). Computer simulation of random sampling indicated that the testing of 6 pools of 50 sheep would provide 95% confidence in detecting ≥2% prevalence of infection. The estimated laboratory cost of pooled fecal culture when applied as a flock test is approximately 30% that of serologic examination, and sample collection costs are lower. It is recommended that pooled fecal culture replace serologic examination for detection ofM. aviumsubsp.paratuberculosisinfection at the flock level.
Quantitation of Varicella-Zoster Virus DNA in Whole Blood, Plasma, and Serum by PCR and Electrochemiluminescence
Authors: de Jong Menno D. , Weel Jan F. L. , Schuurman Tim , Wertheim-van Dillen Pauline M. E. , Boom René 
Published: December 2019

ABSTRACT
We describe a highly sensitive assay for quantitation of varicella-zoster virus (VZV) DNA in blood, involving PCR amplification, solution hybridization with Tris-(2,2′-bipyridine)-ruthenium(II) chelate-labeled probes, and measurement by electrochemiluminescence (ECL). Extraction and amplification efficiencies were monitored by the inclusion of internal control (IC) DNA, mimicking the VZV target, in the DNA extraction. Viral DNA load was calculated from the ratio of VZV and IC ECL signals. The lower limit of sensitivity was 20 VZV DNA copies/ml of plasma or serum and 80 copies/ml of whole blood. In reconstruction experiments, expected and calculated VZV DNA loads were in excellent accordance. Blood specimens from 42 VZV-infected patients were tested for the presence of VZV DNA and showed detection rates of 86% in patients with varicella and 81% in patients with herpes zoster. In specimens obtained during the first week after onset of the rash, detection rates were 100 and 89%, respectively. Viral DNA was detected in all immunocompromised patients with herpes zoster, emphasizing the risk of disseminated disease in this patient group. VZV DNA load was similar in patients with varicella and multidermatomal herpes zoster and lower in patients with unidermatomal zoster. Despite the cell-associated nature of the virus, VZV DNA was detected in serum and plasma at high copy numbers, and at similar frequencies compared to whole-blood specimens. Quantitation of VZV DNA in blood is of potential importance for diagnosis and clinical management of VZV-infected patients. Plasma and serum provide convenient matrices for this purpose.
Prevalence of GB Virus C (Also Called Hepatitis G Virus) Markers in Norwegian Blood Donors
Authors: Nordbø Svein Arne , Krokstad Sidsel , Winge Per , Skjeldestad Finn Egil , Dalen Are B. 
Published: December 2019

ABSTRACT
GB virus C (GBV-C), also called hepatitis G virus (HGV), occurs worldwide, but the clinical significance of this virus is still unclear. Plasma samples from 1,001 blood donors were tested by reverse transcription PCR using primers from the NS5 region and by a commercial enzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin G antibodies against the putative envelope of HGV (anti-HGV E2). GBV-C/HGV RNA was present in the plasma from 2.5% of the blood donors, and anti-HGV E2 antibodies could be detected in 10.5% of the samples. Only one of the blood donors with viremia had elevated levels of alanine aminotransferase. Among ELISA-positive donors, there was a significantly higher percentage (16.5%) of individuals who had been treated by acupuncture than individuals who had not been given this treatment (9.4%). No other variables showed significant differences. Screening of medical records from 401 recipients of blood from PCR-positive donors revealed no association with liver disease. Four of 12 partners (33%) were HGV RNA positive, and sequence analyses of the strains showed that four of the couples probably were infected with the same strains, while strains from different couples were not identical. Anti-HGV E2 antibodies were detected in serum samples from four other partners. The prevalence of GBV-C/HGV among blood donors in our region is dramatically higher than the prevalence of hepatitis C virus (0.03%).
Analytical Sensitivity, Reproducibility of Results, and Clinical Performance of Five PCR Assays for Detecting
Chlamydia pneumoniae
DNA in Peripheral Blood Mononuclear Cells
Authors: Mahony J. B. , Chong S. , Coombes B. K. , Smieja M. , Petrich A. 
Published: December 2019

ABSTRACT

Chlamydia pneumoniae
has been associated with atherosclerosis and coronary artery disease (CAD), and its DNA has been detected in atheromatous lesions of the aorta, carotid, and coronary arteries by a variety of PCR assays. The objective of this study was to compare the performances of five published PCR assays in the detection of
C. pneumoniae
in peripheral blood mononuclear cells (PBMCs) from patients with coronary artery disease. The assays included two conventional PCRs, one targeting a cloned
Pst
I fragment and one targeting the 16S rRNA gene; two nested PCRs, one targeting the 16S rRNA gene and one targeting
ompA
; and a touchdown enzyme time release (TETR) PCR, targeting the 16S rRNA gene. All PCRs had similar analytical sensitivities and detected a minimum of 0.005 inclusion-forming units (IFU) of
C. pneumoniae
; the
ompA
nested PCR and the TETR PCR were slightly more sensitive and detected 0.001 IFU. Assay reproducibility was examined by testing 10 replicates of
C. pneumoniae
DNA by each assay. All five assays showed excellent reproducibility at high levels of DNA, with scores of 10 out of 10 for 0.01 IFU, but exhibited decreased reproducibility for smaller numbers of
C. pneumoniae
IFU for all tests. Pairwise comparison of test results indicated that there was a significant difference between tests (Cochran Q = 32.0,
P
< 0.001), with the
Pst
I fragment (
P
< 0.001) and 16S rRNA (
P
= 0.002) assays having lower reproducibility than the nested
ompA
and TETR assays. To further analyze assay sensitivity,
C. pneumoniae
-infected U-937 mononuclear cells were added to whole blood, and extracted mononuclear-cell DNA was tested by each assay. All five assays showed similar sensitivities, detecting 15 infected cells; three assays detected 3 infected cells, while all assays were negative at the next dilution (1.5 infected cells). A striking difference in performance of the five assays was seen, however, when PBMCs from CAD patients were tested for
C. pneumoniae
DNA. The
ompA
nested PCR detected
C. pneumoniae
DNA in 11 of 148 (7.4%) specimens, the 16S rRNA nested PCR detected 2 positives among the 148 specimens (1.4%) (
P
< 0.001), and the other 3 assays detected no positive specimens (
P
< 0.001, compared with the
ompA
assay). These results indicate that analytical sensitivity alone does not predict the ability of an assay to detect
C. pneumoniae
in whole-blood-derived PBMCs. Before standardized assays can be used in wide-scale epidemiological studies, further characterization of these assays will be required to improve our understanding of their performance in the detection of
C. pneumoniae
in clinical material.
Characterization of United Kingdom Isolates ofCorynebacterium pseudotuberculosisUsing Pulsed-Field Gel Electrophoresis
Authors: Connor Kathleen M. , Quirie Malcolm M. , Baird Graham , Donachie William 
Published: December 2019

ABSTRACTCaseous lymphadenitis is a chronic suppurative disease caused byCorynebacterium pseudotuberculosisand is responsible for serious economic losses to the sheep and goat industry. Caseous lymphadenitis was first reported for goats in the United Kingdom in 1990 and for sheep in 1991. Recent evidence suggests that the prevalence of the disease within the national flock is increasing. Fifty isolates ofC. pseudotuberculosisfrom the United Kingdom comprising sheep and horse isolates, the original goat outbreak strain, and the type strain were characterized by biotyping, antimicrobial susceptibility, production of phospholipase D, and genotyping by pulsed-field gel electrophoresis usingSfiI andSmaI. All of the isolates were confirmed asC. pseudotuberculosis, and all produced phospholipase D but none reduced nitrate. Restriction withSfiI generated 16 to 18 bands between 48.5 and 290 kb and differentiated six pulsotypes. We conclude that 80% of the strains tested were epidemiologically related to the outbreak strain and that the equine profile was distinct both phenotypically and genotypically.
Diagnosis and Clinical Virology of Lassa Fever as Evaluated by Enzyme-Linked Immunosorbent Assay, Indirect Fluorescent-Antibody Test, and Virus Isolation
Authors: Bausch D. G. , Rollin P. E. , Demby A. H. , Coulibaly M. , Kanu J. , Conteh A. S. , Wagoner K. D. , McMullan L. K. , Bowen M. D. , Peters C. J. , Ksiazek T. G. 
Published: December 2019

ABSTRACTThe Lassa virus (an arenavirus) is found in West Africa, where it sometimes causes a severe hemorrhagic illness called Lassa fever. Laboratory diagnosis has traditionally been by the indirect fluorescent-antibody (IFA) test. However, enzyme-linked immunosorbent assays (ELISAs) for Lassa virus antigen and immunoglobulin M (IgM) and G (IgG) antibodies have been developed that are thought to be more sensitive and specific. We compared ELISA and IFA testing on sera from 305 suspected cases of Lassa fever by using virus isolation with a positive reverse transcription-PCR (RT-PCR) test as the “gold standard.” Virus isolation and RT-PCR were positive on 50 (16%) of the 305 suspected cases. Taken together, Lassa virus antigen and IgM ELISAs were 88% (95% confidence interval [CI], 77 to 95%) sensitive and 90% (95% CI, 88 to 91%) specific for acute infection. Due to the stringent gold standard used, these likely represent underestimates. Diagnosis could often be made on a single serum specimen. Antigen detection was particularly useful in providing early diagnosis as well as prognostic information. Level of antigenemia varied inversely with survival. Detection by ELISA of IgG antibody early in the course of illness helped rule out acute Lassa virus infection. The presence of IFA during both acute and convalescent stages of infection, as well as significant interobserver variation in reading the slides, made interpretation difficult. However, the assay provided useful prognostic information, the presence of IFA early in the course of illness correlating with death. The high sensitivity and specificity, capability for early diagnosis, and prognostic value of the ELISAs make them the diagnostic tests of choice for the detection of Lassa fever.
18S Ribosomal DNA-Based PCR for Diagnosis of
Trichomonas vaginalis
Authors: Mayta Holger , Gilman Robert H. , Calderon Maritza M. , Gottlieb Aren , Soto Giselle , Tuero Iskra , Sanchez Sixto , Vivar Aldo 
Published: December 2019

ABSTRACT

Trichomonas vaginalis
remains the most common sexually transmitted parasite in the world and is considered a major risk factor in the transmission of the human immunodeficiency virus. A PCR technique using primers targeting a specific region of the 18S rRNA gene of
T. vaginalis
was developed. The PCR test was standardized using 15 reference strains, giving a single product of 312 bp in all strains. No amplification was observed when DNA from related organisms or human DNA was used as a target. The test was evaluated on 372 vaginal swab specimens and 361 urine samples from women attending infertility and obstetric clinics at two separate hospitals in Lima, Peru. Compared to
T. vaginalis
culture, the overall sensitivity and specificity of PCR of vaginal swab samples was 100% and 98%, respectively. The PCR of urine samples was 100% sensitive and 99.7% specific compared to culture of vaginal swab, but the sensitivity drops to 83.3% when compared to PCR of vaginal swabs. All culture-positive samples were found to be positive by PCR in either urine or vaginal secretion. None of the PCR-negative samples were positive by culture. The origin of the amplification was confirmed by digestion of PCR products with
Hae
III. This PCR assay, which is easy to perform and has a high sensitivity and specificity, should be useful for routine diagnosis of
T. vaginalis
infection.
Recognition of Enteropathogenic
Escherichia coli
Virulence Determinants by Human Colostrum and Serum Antibodies
Authors: Parissi-Crivelli Aurora , Parissi-Crivelli Joaquín M. , Girón Jorge A. 
Published: December 2019

ABSTRACT

Human colostra and sera collected from Mexican mothers and their children at birth and 6 months thereafter were studied for the presence of antibodies against the bundle-forming pilus and several chromosomal virulence gene products (intimin and secreted proteins EspA and EspB) of enteropathogenic
Escherichia coli
(EPEC). Among 21 colostrum samples studied, 76, 71.5, 57, and 47% of them contained immunoglobulin A (IgA) antibodies against EspA, intimin, EspB, and BfpA, respectively. Interestingly, there was a difference in IgG response to EPEC antigens between the sera from neonates and sera from the same children 6 months later. While the number of neonates reacting to Esps and intimin diminished when they reached 6 months of age, those reacting with BfpA increased from 9 to 71%. Intimin from an enterohemorrhagic
E. coli
strain was also recognized by most of the samples reacting with EPEC intimin. These data suggest that Bfp and Esps elicit an antibody response during the early days of life of neonates and support the value of breast-feeding in areas of the world where bacterial diarrheal infections are endemic.
Comparison of Sequencing by Hybridization and Cycle Sequencing for Genotyping of Human Immunodeficiency Virus Type 1 Reverse Transcriptase
Authors: Hanna George J. , Johnson Victoria A. , Kuritzkes Daniel R. , Richman Douglas D. , Martinez-Picado Javier , Sutton Lorraine , Hazelwood J. Darren , D'Aquila Richard T. 
Published: December 2019

ABSTRACT
The performances of two methods of nucleotide sequencing were compared for the detection of drug resistance mutations in human immunodeficiency virus type 1 reverse transcriptase (RT) in viruses isolated from highly RT inhibitor-experienced individuals. Of 11,677 amino acids deduced from population PCR products by both cycle sequencing and sequencing by hybridization to high-density arrays of oligonucleotide probes, 97.4% were concordant by both methods, 0.8% were discordant, and 1.7% had an ambiguous determination by at least one method. A higher rate of discordance (3.9%) was observed among RT inhibitor resistance-associated codons. In 45% of the isolates, RT codon 67 was deduced as the wild-type Asp by hybridization sequencing but as the zidovudine resistance-associated Asn by cycle sequencing. In other resistance-associated codon discordances, cycle sequencing also more commonly called a known resistance-associated amino acid than hybridization sequencing did. The nucleotide sequence in the vicinity of several codons with discordant calls influenced population-based hybridization sequencing. For isolates evaluated by additional sequencing of molecular clones of PCR products by both methods, the discordance between methods was less frequent (0.4% of all 5,994 amino acids and 0 of 494 drug resistance-associated codons). At positions which were discordant or ambiguous in the population sequences, the results of sequencing of clones by both methods were usually in agreement with the population cycle sequencing result. In summary, most RT codons were highly concordant by both methods of population-based sequencing, with discordances due in large part to genetic mixtures within or adjacent to discordant codons.
Detection of Human Herpesvirus 6 and Varicella-Zoster Virus in Tear Fluid of Patients with Bell's Palsy by PCR
Authors: Pitkäranta A. , Piiparinen H. , Mannonen L. , Vesaluoma M. , Vaheri A. 
Published: December 2019

ABSTRACT
Human herpesvirus 6 DNA was detected by PCR in the tear fluid of 7 (35%) of 20 patients with Bell's palsy and of 1 (5%) of 20 healthy controls. Varicella-zoster virus was detected by PCR in the tear fluid of 2 of 20 Bell's palsy patients but in none of the tear fluids from 20 healthy controls. These findings suggest an association between human herpesviruses and Bell's palsy.
Fluorescent Whole-Cell Hybridization with 16S rRNA-Targeted Oligonucleotide Probes To Identify
Brucella
spp. by Flow Cytometry
Authors: Fernández-Lago Luis , Vallejo F. Javier , Trujillano Ignacio , Vizcaíno Nieves 
Published: December 2019

ABSTRACT

A whole-cell hybridization assay with fluorescent oligonucleotide probes derived from the 16S rRNA sequence of
Brucella abortus
in combination with flow cytometry has been developed. With the three fluorescent probes selected, a positive signal was observed with all the representative strains of the species and biovars of
Brucella
and with a total of nine different
Brucella
clinical isolates. Using the B9 probe in the hybridization assay, it was possible to discriminate between
Brucella suis
biovars 2, 3, 4, and 5 and almost all the other
Brucella
spp. On the basis of differences in fluorescence intensities, no discrimination was established between
Brucella
spp. and other phylogenetically related microorganisms. No positive fluorescence signals were detected with any of the bacteria showing serological cross-reactions with
Brucella
spp. and with a total of 17 clinical isolates not belonging to the genus
Brucella
. These results suggest that the 16S rRNA whole-cell hybridization technique could be a valuable diagnostic tool for the detection and identification of
Brucella
spp.
Bacteriological and Serological Findings in a Further Case of Transfusion-MediatedYersinia enterocoliticaSepsis
Authors: Strobel E. , Heesemann J. , Mayer G. , Peters J. , Müller-Weihrich S. , Emmerling P. 
Published: December 2019

ABSTRACTA 13-year-old patient developed severe shock due to administration of aYersinia enterocolitica-contaminated red blood cell concentrate.Y. enterocolitica(serotype O:9, biotype II) was cultivated from the residual blood in the blood bag and from a stool sample of the blood donor. In the donor's plasma immunoglobulin M (IgM), IgA, and IgG antibodies againstYersiniaouter proteins (YopM, -H, -D, and -E) were found. Since the donor remembered a short-lasting, mild diarrhea 14 days prior to blood donation, a transient attack ofYersiniaenteritis may be associated with a longer than expected period of asymptomatic bacteremia that causes contamination of donor blood. Serological screening for IgM antibodies againstYersiniaouter proteins might offer a way to reduce the risk of transfusion-associatedY. enterocoliticasepsis.
Comparison and Application of a Novel Genotyping Method, Semiautomated Primer-Specific and Mispair Extension Analysis, and Four Other Genotyping Assays for Detection of Hepatitis C Virus Mixed-Genotype Infections
Authors: Hu Yu-Wen , Balaskas Evan , Furione Milena , Yen Pei-Hua , Kessler Garry , Scalia Vito , Chui Linda , Sher Graham 
Published: December 2019

ABSTRACT
To date the true prevalence of hepatitis C virus (HCV) mixed-genotype infections has not been established mainly because currently available methods are not suitable for the detection of mixed genotypes in a viral population. A novel semiautomated genotyping method, primer-specific and mispair extension analysis (S-PSMEA), which is more reliable than other genotyping assays was developed for detection of HCV mixed-genotype infections. A genotype present at levels as low as 0.8% in a defined mix of HCV genotypes was detected, showing a 20-fold increase in sensitivity over that of direct DNA sequencing. A total of 434 HCV isolates were genotyped and analyzed for a comparative study of the accuracy between S-PSMEA and four current genotyping methods. The results showed that viruses in approximately 40% of the samples from this group determined to be infected with mixed genotypes by S-PSMEA were undetected by direct DNA sequencing due to its low sensitivity. Type-specific PCR, line probe assay, and restriction fragment length polymorphism analysis performed poorly, being able to identify only 38.5, 16.1, and 15.4% of mixed-genotype infections, respectively, that were detected by direct DNA sequencing. The prevalence of mixed-genotype infections detected by S-PSMEA was 7.9% (12 of 152 donors) among HCV-infected blood donors, 14.3% (15 of 105) among patients with chronic hepatitis C, and 17.1% (6 of 36) among thalassemia patients who had received multiple transfusions. The data lead us to conclude that HCV mixed-genotype infections are more common than previously estimated and that S-PSMEA may be the method of choice when detection of genotypes present at low levels in mixed-genotype infections is required due to its higher level of sensitivity.