bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assessment of Hepatitis B Virus DNA Stability in Serum by the Chiron Quantiplex Branched-DNA Assay
Authors: Krajden Mel , Comanor Lorraine , Rifkin Oretta , Grigoriew Anna , Minor James M. , Kapke Gordon F. 
Published: December 2019

ABSTRACT

Quantification of hepatitis B virus (HBV) DNA in serum is used to establish eligibility for treatment and to monitor therapeutic response. With the trend toward centralized testing, defining the conditions that preserve sample integrity is of paramount importance. We therefore evaluated the stability of HBV DNA in 26 previously frozen (PF) and 5 fresh, never previously frozen serum specimens. PF specimens, covering a 3-log
10
HBV DNA dynamic range, were thawed and stored at −70, 4, 23, 37, and 45°C (±1.5°C) for 0, 24, 72, and 120 h (±2 h) and were refrozen at −70°C prior to testing. Five fresh specimens were split into two groups. Both group FG1 and group FG2 specimens were handled as described above; however, group FG1 specimens were subsequently maintained at 4°C and were never frozen prior to testing. Linear regression analysis of PF specimens demonstrated no significant HBV DNA degradation at ≤4°C over 5 days; however, HBV DNA levels decreased by 1.8, 3.4, and 20% per day at 23, 37, and 45°C, respectively. Three independent statistical methods confirmed that the probability of specimen failure, defined as a loss of 20% or more of HBV DNA and/or coagulation of serum, was lowest at ≤4°C and increased with temperature. Because only 10 to 20% of individual patient specimens demonstrated losses of HBV DNA of ≥20% at 23 or 37°C, sufficient numbers of serum specimens must be evaluated to determine overall statistical trends. In conclusion, HBV DNA integrity in separated serum specimens is preserved for at least 5 days when the specimens are stored at −70 or 4°C.
Rapid Detection of
Candida albicans
in Clinical Samples by DNA Amplification of Common Regions from
C. albicans
-Secreted Aspartic Proteinase Genes
Authors: Flahaut M. , Sanglard D. , Monod M. , Bille J. , Rossier M. 
Published: December 2019

ABSTRACT

Laboratory diagnosis based on genomic amplification methods such as PCR may provide an alternative and more sensitive method than conventional culture for the early detection of deep-seated candidiasis, an increasing cause of morbidity and mortality among immunocompromised patients. A novel method of DNA extraction from clinical samples based on treatment with proteinase K and isolation of DNA on a silica membrane was developed. The targets used for DNA amplification were the
Candida albicans
-secreted aspartic proteinase (
SAP
) genes, a multiple-gene family of at least seven members in
C. albicans
. A single pair of primers was designed in order to detect six of these
SAP
genes and, subsequently, to increase the sensitivity of the test. Detection of the PCR product by enzyme-linked immunosorbent assay was found to be as sensitive as Southern blotting with an
SAP
-labeled probe. The sensitivity of the assay was 1 cell/ml from serially diluted
Candida
cultures and 1 to 4 cells/ml from seeded blood specimens. The sensitivity and specificity of the present assay were tested in a retrospective study performed blindly with 156 clinical samples and were 100 and 98%, respectively, compared with the results of culture. For the subset of blood culture samples (
n
= 124), the sensitivity and the specificity were 100%. The two false-positive PCR samples came from patients treated with azole antifungal agents, indicating that PCR was probably able to detect damaged organisms that could not be recovered by culture.
Impact of Strain Heterogeneity on Lyme Disease Serology in Europe: Comparison of Enzyme-Linked Immunosorbent Assays Using Different Species of
Borrelia burgdorferi
Sensu Lato
Authors: Hauser Ulrike , Krahl Heide , Peters Helmut , Fingerle Volker , Wilske Bettina 
Published: December 2019

ABSTRACT

For the standardization of serological tests for Lyme borreliosis (LB) in Europe, the influence of the heterogeneity of
Borrelia burgdorferi
sensu lato must be assessed in detail. For this study four immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assays (ELISAs) with octyl-β-
d
-glucopyranoside extracts of strains PKo (
Borrelia afzelii
), PBi (
Borrelia garinii
), and PKa2 and B31 (both
B. burgdorferi
sensu stricto) were compared. Strains PKo, PBi, and PKa2 at the passages used for antigen preparations abundantly expressed outer surface protein C (OspC), whereas strain B31 at the passage used for antigen preparation did not express OspC. Sera (all from Germany) from 222 patients with clinically defined LB of all stages, 133 blood donors, and 458 forest workers were tested. None of the forest workers had symptoms consistent with LB at the time that the samples were collected. For IgM tests, receiver operating characteristic curves demonstrated that discrimination between sera from patients and blood donors was best with strain PKo and worst with strain B31. The discriminatory abilities of the four IgG ELISAs were similar in a diagnostically reasonable specificity range (90 to 100%). More than 20% of the sera from forest workers reacted strongly in the PKo IgG ELISA (optical density value, >1.5; other assays, less than 8%). Western blots of the sera with the most discrepant ELISA results revealed almost exclusive reactivity with p17. This highly immunogenic antigen is only expressed by strain PKo. This observation might be important for the development of assays enabling discrimination between asymptomatic or previous infection and active disease.
Hepatitis G Virus Infection in Amerindians and Other Venezuelan High-Risk Groups
Authors: Pujol Flor H. , Khudyakov Yury E. , Devesa Marisol , Cong Mian-E. , Loureiro Carmen L. , Blitz Linda , Capriles Freya , Beker Simón , Liprandi Ferdinando , Fields Howard A. 
Published: December 2019

ABSTRACT
Recently, a new virus related to flaviviruses, the hepatitis G virus (HGV), or GBV-C virus, was discovered as a putative blood-borne human pathogen. HGV RNA (NS5 region) was amplified by reverse transcription-nested PCR in the sera of 6 of 64 (9%) hemodialysis patients; 2 of 80 (2.5%) West Yukpa Amerindians, a population with a high rate of HBV infection but negative for HCV infection; and 1 patient with an acute episode of non-A, non-B, non-C hepatitis (NABCH). The patterns of single-strand conformation polymorphism of the amplified products were unique among different specimens and similar on follow-up for hemodialysis patients. All patients tested remained HGV RNA positive 1 and 2 years later, without major sequence variation, except for the NABCH patient, for whom a double infection and an apparent clearance of the original dominant variant was observed after 2 years. The sequences of the NS5 amplified products demonstrated 85 to 90% identity with other reported HGV sequences.
Pooling Urine Samples for Ligase Chain Reaction Screening for Genital
Chlamydia trachomatis
Infection in Asymptomatic Women
Authors: Kacena Katherine A. , Quinn Sean B. , Howell M. René , Madico Guillermo E. , Quinn Thomas C. , Gaydos Charlotte A. 
Published: December 2019

ABSTRACT

The accuracy of pooling urine samples for the detection of genital
Chlamydia trachomatis
infection by ligase chain reaction (LCR) was examined. A model was also developed to determine the number of samples to be pooled for optimal cost savings at various population prevalences. Estimated costs included technician time, laboratory consumables, and assay costs of testing pooled samples and retesting individual specimens from presumptive positive pools. Estimation of population prevalence based on the pooled LCR results was also applied. After individual urine specimens were processed, 568 specimens were pooled by 4 into 142 pools and another 520 specimens were pooled by 10 into 52 pools. For comparison, all 1,088 urine specimens were tested individually. The sample-to-cut-off ratio was lowered from 1.0 to 0.2 for pooled samples, after a pilot study which tested 148 samples pooled by 4 was conducted. The pooling algorithm was 100% (48 of 48) sensitive when samples were pooled by 4 and 98.4% (61 of 62) sensitive when samples were pooled by 10. Although 2.0% (2 of 99) of the negative pools of 4 and 7.1% (1 of 14) of the negative pools of 10 tested presumptive positive, all samples in these presumptive-positive pools were negative when retested individually, making the pooling algorithm 100% specific. In a population with 8% genital
C. trachomatis
prevalence, pooling by four would reduce costs by 39%. The model demonstrated that with a lower prevalence of 2%, pooling eight samples would reduce costs by 59%. Pooling urine samples for detection of
C. trachomatis
by LCR is sensitive, specific, and cost saving compared to testing individual samples.
Stability of Human Immunodeficiency Virus RNA in Blood Specimens as Measured by a Commercial PCR-Based Assay
Authors: Sebire Kimberley , McGavin Kate , Land Sally , Middleton Tracey , Birch Chris 
Published: December 2019

ABSTRACT

We investigated the effects of conditions often encountered during handling, transit, and storage of blood specimens on the quantity of detectable human immunodeficiency virus (HIV) RNA in plasma. HIV RNA copy numbers were measured with a commercially available assay (the Amplicor HIV-1 Monitor test kit). Variables examined were the time to processing of blood and plasma, the holding temperature of blood and plasma prior to processing, the effect of freezing and thawing of plasma, and the use of different anticoagulants. The relationship between the HIV RNA copy number and the HIV isolation rate by peripheral blood mononuclear cell (PBMC) coculture was also examined. We found that RNA copy numbers were maintained to within 0.5 log
10
(approximately threefold) in blood and plasma samples held at room temperature or 4°C for up to 3 days and remained stable despite (limited) freezing and thawing of the plasma. HIV RNA copy numbers were also maintained after long-term storage of plasma at −70°C. The ability to isolate HIV from PBMCs was directly proportional to the HIV RNA copy number.
Comparative Study of Bacteremias Caused by
Enterococcus
spp. with and without High-Level Resistance to Gentamicin
Authors: Caballero-Granado Francisco Javier , Cisneros J. M. , Luque R. , Torres-Tortosa M. , Gamboa F. , Díez F. , Villanueva J. L. , Pérez-Cano R. , Pasquau J. , Merino D. , Menchero A. , Mora D. , López-Ruz M. A. , Vergara A. , Infecciosas for the Grupo Andaluz Para El Estudio De Las Enfermedades 
Published: December 2019

ABSTRACT

A prospective, multicenter study was carried out over a period of 10 months. All patients with clinically significant bacteremia caused by
Enterococcus
spp. were included. The epidemiological, microbiological, clinical, and prognostic features and the relationship of these features to the presence of high-level resistance to gentamicin (HLRG) were studied. Ninety-three patients with enterococcal bacteremia were included, and 31 of these cases were caused by HLRG (33%). The multivariate analysis selected chronic renal failure, intensive care unit stay, previous use of antimicrobial agents, and
Enterococcus faecalis
species as the independent risk factors that influenced the development of HLRG. The strains with HLRG showed lower levels of susceptibility to penicillin and ciprofloxacin. Clinical features (except for chronic renal failure) were similar in both groups of patients. HLRG did not influence the prognosis for patients with enterococcal bacteremia in terms of either the crude mortality rate (29% for patients with bacteremia caused by enterococci with HLRG and 28% for patients not infected with strains with HLRG) or the hospital stay after the acquisition of enterococcal bacteremia. Hemodynamic compromise, inappropriate antimicrobial therapy, and mechanical ventilation were revealed in the multivariate analysis to be the independent risk factors for mortality. Prolonged hospitalization was associated with the nosocomial acquisition of bacteremia and polymicrobial infections.
Evaluation of the RapID CB Plus System for Identification of
Corynebacterium
Species and Other Gram-Positive Rods
Authors: Hudspeth Marie K. , Gerardo Sharon Hunt , Citron Diane M. , Goldstein Ellie J. C. 
Published: December 2019

ABSTRACT

Due to the difficulty of identifying
Corynebacterium
spp. with standard methods, we compared them with the RapID CB Plus system (Remel, Lenexa, Kans. [formerly Innovative Diagnostic Systems, Norcross, Ga.]), which consists of 4 carbohydrate and 14 preformed enzyme tests, for the identification of 98 clinical isolates of
Corynebacterium
sp., other coryneforms,
Listeria monocytogenes
, and 17 ATCC strains. Forty (95%) of 42 strains of
Corynebacterium
spp. were accurately identified to the species level by the RapID CB Plus system, and two additional strains of
C. striatum
were identified with one additional conventional test for lipid requirement. Twenty-seven (75%) of the 36 coryneform strains tested were identified correctly to the species level. However, three of four strains of
Brevibacterium
sp. and all seven of the
L. monocytogenes
strains were identified to the genus level only.
Actinomyces
strains had variable results, and the one strain of
Arcanobacterium haemolyticum
tested was not identified. Overall, the RapID CB Plus system compared favorably with the conventional methods, was easy to inoculate and interpret, and is promising as a new method for identification of gram-positive bacilli.
Genetic Heterogeneity of Strains of
Neisseria meningitidis
Belonging to Serotype 22 Isolated in the Czech Republic
Authors: Musilek Martin , Giorgini Dario , Hamadouche Nora , Kriz Paula , Taha Muhamed-Kheir 
Published: December 2019

ABSTRACT

Strains of
Neisseria meningitidis
of serogroup B isolated in the Czech Republic frequently belong to serotype 22. We analyzed the genetic relationships among strains of this serotype by using the multilocus enzyme electrophoresis technique and the polymorphism of the
pilA
gene. Our results indicate that these strains correspond to a highly heterogeneous population rather than to the expansion of a single clone.
Detection and Characterization of Shiga Toxigenic
Escherichia coli
by Using Multiplex PCR Assays for
stx
1
,
stx
2
,
eaeA
, Enterohemorrhagic
E. coli hlyA
,
rfb
O111
, and
rfb
O157
Authors: Paton Adrienne W. , Paton James C. 
Published: December 2019

ABSTRACT

Shiga toxigenic
Escherichia coli
(STEC) comprises a diverse group of organisms capable of causing severe gastrointestinal disease in humans. Within the STEC family, certain strains appear to be of greater virulence for humans, for example, those belonging to serogroups O111 and O157 and those with particular combinations of other putative virulence traits. We have developed two multiplex PCR assays for the detection and genetic characterization of STEC in cultures of feces or foodstuffs. Assay 1 utilizes four PCR primer pairs and detects the presence of
stx
1
,
stx
2
(including variants of
stx
2
),
eaeA
, and enterohemorrhagic
E. coli hlyA
, generating amplification products of 180, 255, 384, and 534 bp, respectively. Assay 2 uses two primer pairs specific for portions of the
rfb
(O-antigen-encoding) regions of
E. coli
serotypes O157 and O111, generating PCR products of 259 and 406 bp, respectively. The two assays were validated by testing 52 previously characterized STEC strains and observing 100% agreement with previous results. Moreover, assay 2 did not give a false-positive O157 reaction with enteropathogenic
E. coli
strains belonging to clonally related serogroup O55. Assays 1 and 2 detected STEC of the appropriate genotype in primary fecal cultures from five patients with hemolytic-uremic syndrome and three with bloody diarrhea. Thirty-one other primary fecal cultures from patients without evidence of STEC infection were negative.
Corynebacterium riegeliisp. nov., an Unusual Species Isolated from Female Patients with Urinary Tract Infections
Authors: Funke Guido , Lawson Paul A. , Collins Matthew D. 
Published: December 2019

ABSTRACTFour strains of an unknown coryneform bacterium were isolated in pure culture from females with urinary tract infections. Strong urease activity and the ability to slowly ferment maltose but not glucose were the most significant phenotypic features of this catalase-positive, nonmotile, nonlipophilic, rod-shaped bacterium which served to distinguish it from all other presently defined coryneform bacteria. Chemotaxonomic investigations demonstrated that the unknown bacterium belonged to the genusCorynebacterium. Comparative 16S rRNA gene sequence analysis revealed that the isolates were genealogically identical and represented a new subline within the genusCorynebacterium, for which the designationCorynebacterium riegeliisp. nov. is proposed. The type strain ofCorynebacterium riegeliiis CCUG 38180 (DSM 44326, CIP 105310).
Controlled Clinical Laboratory Comparison of Two Supplemented Aerobic and Anaerobic Media Used in Automated Blood Culture Systems To Detect Bloodstream Infections
Authors: Ziegler R. , Johnscher I. , Martus P. , Lenhardt D. , Just H.-M. 
Published: December 2019

ABSTRACT

A 20-ml blood sample was collected from adult patients with suspected bloodstream infections and distributed equally into the four volume-controlled bottles of a blood culture set consisting of aerobic and anaerobic BACTEC Plus/F bottles and aerobic and anaerobic BacT/Alert FAN bottles. All bottles were incubated in their respective instruments for a standard 5-day protocol or until the instruments signalled positivity. Samples in all bottles with negative results by these instruments were terminally subcultured. A total of 8,390 blood culture sets were obtained during the study period, of which 4,402 (52.5%) met the study criteria. Of these, 946 (21.5%) were positive either by instrument signal or by additional terminal subculture of all negative bottles and yielded growth of microorganisms. Five hundred eighty-nine (13.4%) blood culture sets were considered to have recovered 663 clinically significant organisms. When both the BACTEC and the BacT/Alert systems were used, 465 positive sets were detected; BACTEC alone detected 52 positive sets and BacT/Alert alone detected 72 (
P
= 0.09). No differences were found between the two systems in microbial recovery rate from blood cultures obtained from patients on antibiotic therapy. Significantly more members of the family
Enterobacteriaceae
(
P
< 0.01) were detected from patients without antimicrobial therapy by BacT/Alert than by BACTEC. The false-negative rates were 0.20% for BACTEC and 0.32% for BacT/Alert. A significantly higher false-positive rate was found for BACTEC (
P
< 0.0001). Both systems were comparable for the time to detection of microorganisms. However, gram-positive bacteria were detected faster by BACTEC and
Enterobacteriaceae
were detected faster on average by BacT/Alert. We concluded that both systems are comparable in their abilities to recover aerobic and anaerobic organisms from blood cultures and a terminal subculture might not be necessary for either of the two systems. The increased positivity rate when using an anaerobic bottle in a two-bottle blood culture set is due to the additional blood volume rather than to the use of an anaerobic medium.
Rapid Detection of
Mycobacterium paratuberculosis
in Clinical Samples from Ruminants and in Spiked Environmental Samples by Modified BACTEC 12B Radiometric Culture and Direct Confirmation by IS
900
PCR
Authors: Whittington R. J. , Marsh I. , Turner M. J. , McAllister S. , Choy E. , Eamens G. J. , Marshall D. J. , Ottaway S. 
Published: December 2019

ABSTRACT

The suitability of a radiometric culture medium consisting of BACTEC 12B with PANTA PLUS, mycobactin J, and egg yolk was evaluated for detection of
Mycobacterium paratuberculosis
in feces, mesenteric lymph nodes, and intestinal walls from cattle, sheep, and goats. In addition, a simple method that would enable the rapid identification of
Mycobacterium paratuberculosis
by IS
900
PCR in the primary cultures was sought so that subculture to secondary egg-free radiometric medium could be avoided. An ethanol extraction followed by differential centrifugation was used to separate
M. paratuberculosis
from PCR inhibitors in the primary culture. PCR was then undertaken with the pellet, after boiling to lyse the mycobacteria; if this test was negative, the DNA in the lysate was purified with guanidine thiocyanate and silica. Cultures of feces, ilea, and mesenteric lymph nodes from cattle, sheep, and goats known to have or suspected of having Johne’s disease yielded positive PCR results 1 to 7 weeks after inoculation. Similar results were obtained with soil and pasture samples that had been spiked with
M. paratuberculosis
. The results suggested that radiometric culture was more sensitive than histopathology in detecting
M. paratuberculosis
infection in sheep and goats and more sensitive than culture on Herrold’s egg yolk medium for the detection of the infection in cattle. Of 259 individual PCR tests with samples from cultures with growth indices of ≥10,237 (91.5%) were positive, with only 28 (11.8%) requiring both ethanol and silica preparation to yield a positive result. Of the 22 negative PCR results for samples from cultures with growth indices of ≥10, 18 were for samples from cultures that had only just developed evidence of growth. PCR-positive cultures tended to remain PCR positive over successive weeks. Flexibility in the timing of the sampling for PCR is thus possible, facilitating batch processing of samples in large-scale disease control programs for ruminants.
Clinical Comparison of an Enhanced-Sensitivity Branched-DNA Assay and Reverse Transcription-PCR for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma
Authors: Nolte Frederick S. , Boysza Jodi , Thurmond Cathy , Clark W. Scott , Lennox Jeffrey L. 
Published: December 2019

ABSTRACT

The performance characteristics of an enhanced-sensitivity branched-DNA assay (bDNA) (Quantiplex HIV-1 version 2.0; Chiron Corp., Emeryville, Calif.) and a reverse transcription (RT)-PCR assay (AMPLICOR HIV-1 Monitor; Roche Diagnostic Systems, Inc., Branchburg, N.J.) were compared in a molecular diagnostic laboratory. Samples used in this evaluation included linearity and reproducibility panels made by dilution of a human immunodeficiency virus type 1 (HIV-1) stock culture of known virus particle count in HIV-1-negative plasma, a subtype panel consisting of HIV-1 subtypes A through F at a standardized level, and 64 baseline plasma specimens from HIV-1-infected individuals. Plots of log
10
HIV RNA copies per milliliter versus log
10
nominal virus particles per milliliter demonstrated that both assays were linear over the stated dynamic ranges (bDNA,
r
= 0.98; RT-PCR,
r
= 0.99), but comparison of the slopes of the regression lines (bDNA,
m
= 0.96; RT-PCR,
m
= 0.83) suggested that RT-PCR had greater proportional systematic error. The between-run coefficients of variation for bDNA and RT-PCR were 24.3 and 34.3%, respectively, for a sample containing 1,650 nominal virus particles/ml and 44.0 and 42.7%, respectively, for a sample containing 165 nominal virus particles/ml. Subtypes B, C, and D were quantitated with similar efficiencies by bDNA and RT-PCR; however, RT-PCR was less efficient in quantitating subtypes A, E, and F. One non-B subtype was recognized in our clinical specimens based on the ratio of values obtained with the two methods. HIV-1 RNA was quantitated in 53 (83%) baseline plasma specimens by bDNA and in 55 (86%) specimens by RT-PCR. RT-PCR values were consistently greater than bDNA values, with population means of 142,419 and 67,580 copies/ml, respectively (
P
< 0.01). The results were highly correlated (
r
= 0.91), but the agreement was poor (mean difference in log
10
copies per milliliter ± 2 standard deviations, 0.45 ± 0.61) for the 50 clinical specimens that gave discrete values with both methods.