bluuebunny/crossref_metadata_2025 huggingface dataset:
Acquisition and Colonization Stability of
Actinobacillus actinomycetemcomitans
and
Porphyromonas gingivalis
in Children
Authors: Lamell Celeste W. , Griffen Ann L. , McClellan Dawn L. , Leys Eugene J.
Published: December 2019

The presence of
Porphyromonas gingivalis
has been shown to be a risk factor for periodontitis in adults, and
Actinobacillus actinomycetemcomitans
has been implicated as a pathogen in early-onset periodontitis. Both species have been shown to establish stable colonization in adults. In cross-sectional studies, both
A. actinomycetemcomitans
and
P. gingivalis
have been detected in over one-third of apparently healthy children. Information on the stability of colonization with these organisms in children could help to elucidate the natural history of the development of periodontitis. For this purpose, samples previously collected from a cohort of 222 children between the ages of 0 and 18 years and previously examined for the presence of
P. gingivalis
with a PCR-based assay were examined for the presence of
A. actinomycetemcomitans
. It was detected in 48% of subjects and, like
P. gingivalis
, was found at similar frequencies among children of all ages (
P
= 0.53), suggesting very early initial acquisition. One hundred one of the original subjects were recalled after 1 to 3 years to determine the continuing presence of both
A. actinomycetemcomitans
and
P. gingivalis
. The prevalence of both species remained unchanged at resampling. However, in most children both species appeared to colonize only transiently, with random concordance between the results of the first and second sampling. Stability of colonization was unrelated to age for
A. actinomycetemcomitans
, but
P. gingivalis
was more stable in the late teenage years.
Development of a Hypersensitive Detection Method for Human Parvovirus B19 DNA
Authors: Sato Kazuaki , Matsuda Eiji , Kamisango Keiichi , Iwasaki Hiroaki , Matsubara Shuzo , Matsunaga Yasuko
Published: December 2019

A new detection method for human parvovirus B19 DNA was established using PCR coupled with a hybridization protection assay. The amplified product was detected using acridinium ester-labeled DNA probes. By this method, a few copies of B19 DNA were detected in human serum albumin.
Use of Calibrated Viral Load Standards for Group M Subtypes of Human Immunodeficiency Virus Type 1 To Assess the Performance of Viral RNA Quantitation Tests
Authors: Jagodzinski Linda L. , Wiggins Debra L. , McManis Joshua L. , Emery Sandra , Overbaugh Julie , Robb Merlin , Bodrug Sharon , Michael Nelson L.
Published: December 2019

Optimal management of human immunodeficiency virus type 1 (HIV-1) disease requires accurate quantitation of viral RNA concentrations in plasma. Evidence for increasing geographic intermixing of HIV-1 subtypes makes equivalent quantitation of all subtypes essential. The performances of six quantitative viral RNA tests are described, for the first time, with calibrated viral isolates of diverse subtypes.
Rapid Discrimination of Monkey B Virus from Human Herpes Simplex Viruses by PCR in the Presence of Betaine
Authors: Hirano Makoto , Nakamura Shin , Okada Maki , Ueda Masahiro , Mukai Ryozaburo
Published: December 2019

A PCR method to amplify DNA segments of the glycoprotein G gene of monkey B virus (BV) was achieved by adding betaine to the PCR mixture, in spite of the high G+C content of this gene. No product was obtained when DNA of human herpes simplex viruses (HSVs) was used as the template under the same conditions. Thus, this PCR method is useful in discriminating BV from HSVs.
Rapid Subtyping of Dengue Virus Serotypes 1 and 4 by Restriction Site-Specific PCR
Authors: Miagostovich Marize P. , dos Santos Flavia B. , Gutiérrez C. Milena , Riley Lee W. , Harris Eva
Published: December 2019

We previously reported a simple subtyping method, restriction site-specific PCR (RSS-PCR), for dengue virus serotypes 2 and 3; here we describe its application for subtyping dengue virus serotypes 1 and 4. Three major RSS-PCR types were observed for dengue virus serotype 1 and two types were observed for dengue virus serotype 4, in agreement with previous strain classifications based on sequence analysis. Because of its simplicity, this method is amenable to rapid subtyping and application to epidemiological studies of dengue in countries where dengue is endemic.
Accelerated Detection and Identification of Mycobacteria with MGIT 960 and COBAS AMPLICOR Systems
Authors: Katila Marja-Leena , Katila Päivi , Erkinjuntti-Pekkanen Riitta
Published: December 2019

An automated cultivation system for mycobacteria, the MGIT 960 system (MGIT system), was compared in the clinical routine with two variants of Löwenstein-Jensen (L-J) medium. A total of 152 isolates were recovered from 2,015 specimens: 139 (91%) with the MGIT system and 127 (84%) with L-J media (
P
= 0.05). These included 68 isolates of
Mycobacterium tuberculosis
, of which 88% grew in the MGIT system and 93% grew in L-J media (
P
= 0.389), and 84 isolates of mycobacteria other than
M. tuberculosis
(MOTT), of which 94% grew in the MGIT system and 76% grew in L-J media (
P
= 0.003). More
M. avium
complex isolates were detected in the MGIT system (
n
= 65) than in L-J media (
n
= 50) (
P
= 0.001). Growth in the MGIT system was detected in 2 weeks for 78% of the isolates, whereas growth was detected in the two L-J media for 17 and 25% of the isolates, respectively. The mean times to detection of
M. tuberculosis
were 12 days in the MGIT system and 20 days in L-J media, and for
M. avium
complex the mean times to detection were 8 and 22 to 25 days, respectively. The contamination rates were similar (8.7 to 8.9%) in all media. A commercial amplification system (COBAS AMPLICOR) was evaluated for its ability to rapidly identify
M. tuberculosis
,
M. avium
, and
M. intracellulare
directly from 393 samples in MGIT system broth. A correct PCR result, as evaluated by culture or clinical data, was obtained for 96% of the samples, with inhibition being detected for 2% of the samples. Of the 89 results positive for
M. tuberculosis
, 91% were regarded as true positive, 8% were regarded as inconclusive, and 2% were considered false positive. For results positive for
M. avium
and
M. intracellulare
, 97 and 79%, respectively, were regarded as true positive. Increased rapidity and enhanced isolation of MOTT were obtained with the MGIT system. COBAS AMPLICOR was suitable for rapid identification of these three common pathogens from MGIT system broth.
Evaluation of a Capacitance Method for Direct Antifungal Susceptibility Testing of Yeasts in Positive Blood Cultures
Authors: Chang Chang Hsein , Jung Chang Jui , Huey Huang Ay , Chain Chang Tsung
Published: December 2019

The feasibility of using a capacitance method (CM) for direct antifungal susceptibility testing of yeasts in positive blood cultures was evaluated. The CM used the same test conditions as those recommended by the National Committee for Clinical Laboratory Standards. After direct inoculation of positive culture broths into module wells (Bactometer; bioMérieux, Inc., Hazelwood, Mo.), the end-point determination was made by monitoring the capacitance change in the culture broths with Bactometer. The MIC of amphotericin B was the lowest concentration at which yeast growth was completely inhibited, while the MICs of ketoconazole, flucytosine, and fluconazole were the concentrations at which a ≥80% reduction in capacitance change was observed. The MICs of the four drugs against each blood isolate obtained on subculture plates were also determined by the macrodilution method. For 51 positive blood cultures tested, the percent agreement (±2 log
2
dilutions) between the CM and the macrodilution method were as follows: amphotericin B (98%), ketoconazole (92%), flucytosine (84%), and fluconazole (96%). The CM was further used for breakpoint susceptibility testing of fluconazole (8 and 64 μg/ml) and flucytosine (4 and 32 μg/ml) against yeasts in positive blood cultures. After testing of 74 specimens by the CM, flucytosine and fluconazole produced one (1.4%) major error and two (2.8%) minor errors, respectively. All yeasts that displayed resistance to flucytosine or fluconazole were detected within 24 h after direct inoculation of the positive broths into Bactometer. The CM may be useful for the rapid detection of antifungal resistance in positive blood cultures containing yeasts.
Evaluation of Serotype Prediction by
cpsA
-
cpsB
Gene Polymorphism in
Streptococcus pneumoniae
Authors: Lawrence Elliot R. , Arias Cesar A. , Duke Brigid , Beste Dani , Broughton Karen , Efstratiou Androulla , George Robert C. , Hall Lucinda M. C.
Published: December 2019

New pneumococcal conjugate vaccines covering a limited number of serotypes are likely to come into widespread use over the next few years. It is unknown what effect this will have on the relative importance of different serotypes as causes of pneumococcal infection. Hence, it will be important to monitor serotype prevalence before, during, and after the introduction of new vaccines. We have investigated the ability of a PCR method based on polymorphisms in two genes common to the different capsule loci to predict the serotype of 93 clinical isolates of
Streptococcus pneumoniae
submitted to the Central Public Health Laboratory in 1997. Of 70 isolates with vaccine serotypes, 65 were predicted to belong to the correct serotype; this number was improved to 69 with the inclusion of two additional patterns to the database. Of 23 isolates with other serotypes, 19 were correctly predicted as non-vaccine serotypes, the discrepancy lying with four isolates of 6A (non-vaccine serotype) that were indistinguishable from isolates of 6B (vaccine serotype). In situations in which culture of the organism is not feasible, this method could potentially be applicable directly to clinical specimens and could be a valuable aid to the surveillance of pneumococcal serotypes.
A Rapid, Automated Enzymatic Fluorometric Assay for Determination of
d
-Arabinitol in Serum
Authors: Yeo Siew Fah , Zhang Yeyan , Schafer David , Campbell Sheldon , Wong Brian
Published: December 2019

A rapid enzymatic fluorometric assay for measuring
d
-arabinitol in serum was developed using recombinant
d
-arabinitol dehydrogenase from
Candida albicans
(rArDH). rArDH was produced in
Escherichia coli
and purified by dye-ligand affinity chromatography. rArDH was highly specific for
d
-arabinitol, cross-reacting only with xylitol (4.9%) among all polyols tested. A Cobas Fara II centrifugal autoanalyzer (Roche) was used to measure NADH fluorometrically when rArDH and NAD were added to serum extracts, and
d
-arabinitol concentrations were calculated from standard curves derived from pooled human serum containing known amounts of
d
-arabinitol. The method was precise (mean intra-assay coefficients of variation [CVs], 0.8%, and mean interassay CVs, 1.6%) and rapid (3.5 min per assay) and showed excellent recovery of added
d
-arabinitol in serum (mean recovery rate, 101%). The mean and median
d
-arabinitol/creatinine ratios were 2.74 and 2.23 μM/mg/dl, respectively, for the 11 patients with candidemia compared to 1.14 and 1.23 μM/mg/dl, respectively, for 10 healthy controls (
P
< 0.01). These results confirm earlier studies showing that serum
d
-arabinitol measurement may help to promptly diagnose invasive candidiasis. The technique shows a significant improvement in terms of accuracy, cost, simplicity, specificity, and speed compared with gas chromatography, mass spectrometry, and earlier enzymatic assays.
Incidence and Identification of
Klebsiella planticola
in Clinical Isolates with Emphasis on Newborns
Authors: Westbrook Glennis L. , O'Hara Caroline Mohr , Roman Susan B. , Miller J. Michael
Published: December 2019

Studies conducted in France and Germany suggest that up to 19% of clinically identified
Klebsiella
sp. are actually
Klebsiella planticola
, an environmental species that has been attributed to two cases of septicemia, with a rare isolate of
Klebsiella terrigena
(0.4%) being identified. A 1-year survey of newborns on a neonatal ward, also conducted in Germany, reported that 72% of
Klebsiella
sp. were
Klebsiella oxytoca
and 8.7% were
K. planticola
. The tests necessary to identify these species are not found in most clinical identification schemes or in the database matrices of most commercial identification products. To determine the incidence of unrecognized
K. planticola
among the
Klebsiella
sp. isolates in our collection, we used the battery of seven supplemental tests amended from the work of Monnet and Freney to test 352 stock isolates and 84 fresh clinical isolates from four local hospitals. After testing 436 strains of
Klebsiella
, only one strain was identified as a possible
K. planticola
and none was identified as
K. terrigena
. We tested an additional 43 stock strains of
K. oxytoca
isolated from newborns by using eight biochemical tests and found one additional strain of
K. planticola
. The occurrence of
K. planticola
in our collection is far less frequent than that observed in other countries.
High Homogeneity of the
Yersinia pestis
Fatty Acid Composition
Authors: Leclercq Alexandre , Guiyoule Annie , El Lioui Mohamed , Carniel Elisabeth , Decallonne Jacques
Published: December 2019

The cellular fatty acid compositions of 29 strains of
Yersinia pestis
representing the global diversity of this species have been analyzed by gas-liquid chromatography to investigate the extent of fatty acid polymorphism in this microorganism. After culture standardization, all
Y. pestis
strains studied displayed some major fatty acids, namely, the 12:0, 14:0, 3-OH-14:0, 16:0, 16:1ω9cis, 17:0-cyc, and 18:1ω9trans compounds. The fatty acid composition of the various isolates studied was extremely homogeneous (average Bousfield's coefficient, 0.94) and the subtle variations observed did not correlate with epidemiological and genetic characteristics of the strains.
Y. pestis
major fatty acid compounds were analogous to those found in other
Yersinia
species. However, when the ratios for the 12:0/16:0 and 14:0/16:0 fatty acids were plotted together, the genus
Yersinia
could be separated into three clusters corresponding to (i) nonpathogenic strains and species of
Yersinia
, (ii) pathogenic
Yersinia enterocolitica
isolates, and (iii)
Yersinia pseudotuberculosis
and
Y. pestis
strains. The grouping of the two latter species into the same cluster was also demonstrated by their high Bousfield's coefficients (average, 0.89). Therefore, our results indicate that the fatty acid composition of
Y. pestis
is highly homogeneous and very close to that of
Y. pseudotuberculosis
.
Identification of an Epitope Common to Genogroup 1 “Norwalk-Like Viruses”
Authors: Hale Antony D. , Tanaka Tomoyuki N. , Kitamoto Noritoshi , Ciarlet Max , Jiang Xi , Takeda Naokazu , Brown David W. G. , Estes Mary K.
Published: December 2019

A panel of 10 monoclonal antibodies (MAbs) to recombinant Norwalk virus (NV) capsid protein were tested in competition enzyme-linked immunosorbent assays. Patterns of competition indicated that these MAbs recognize six to eight epitopes covering five nonoverlapping regions of the capsid protein. A single epitope, recognized by NV MAbs NV3901, NV3912, and NV2461 was found to occur in the majority of genogroup 1 (G1) but not genogroup 2 (G2) “Norwalk-like viruses” (NLVs). This observation supports the subdivision of human NLVs into two genogroups and provides an assay for the rapid identification of G1 NLVs in fecal specimens.
Relevance of Commercial Diagnostic Tests to Detection of Enteric Adenovirus Infections in South Africa
Authors: Moore P. L. , Steele A. D. , Alexander J. J.
Published: December 2019

The prevalence of enteric adenoviruses detected by an in-house enzyme-linked immunosorbent assay (the RIVM-ELISA) ranged from 13 to 38%, and subgroup F adenoviruses comprised 86%. All subgroup F adenoviruses reacted with both RIVM anti-adenovirus type 40 (Ad40) and anti-adenovirus type 41 (Ad41) monoclonal antibodies but were not detected by Adenoclone Type 40/41 enzyme immunoassay (EIA). The correlation between the Biotrin EIA and RIVM-ELISA results was low (26%). Immunospecific tests suggest that a significant proportion of enteric adenoviruses, possibly comprising previously unidentified or emerging types, are not detected by commercial diagnostic tests in South Africa.
A Field-Suitable, Semisolid Aerobic Enrichment Medium for Isolation of
Campylobacter jejuni
in Small Numbers
Authors: Jeffrey J. S. , Hunter A. , Atwill E. R.
Published: December 2019

The objective of this study was to produce an economical, easy to prepare, field-suitable enrichment medium for detection of
Campylobacter jejuni
in small numbers. A semisolid aerobic enrichment medium was developed. Rates of recovery from inoculated medium, sterile swabs, and mixed cultures of
C. jejuni
and coliform bacteria were tested.
G and P Genotyping of Rotavirus Strains Circulating in France over a Three-Year Period: Detection of G9 and P[6] Strains at Low Frequencies
Authors: Bon F. , Fromantin C. , Aho S. , Pothier P. , Kohli E. ,   The Azay Group
Published: December 2019

Rotavirus G and P types from 716 children with acute diarrhea in Dijon from 1995 to 1998 and throughout France during the winter of 1997-1998 were analyzed by reverse transcription-PCR. P[8],G1 predominated, followed by P[8],G4, which emerged during the last winter. G9 and P[6] strains were detected at low frequencies.
Rapid Phenotypic Characterization Method for Herpes Simplex Virus and Varicella-Zoster Virus Thymidine Kinases To Screen for Acyclovir-Resistant Viral Infection
Authors: Suzutani Tatsuo , Saijo Masayuki , Nagamine Masayoshi , Ogasawara Masahiro , Azuma Masanobu
Published: December 2019

A rapid phenotypic screening method for herpes simplex virus (HSV) and varicella-zoster virus (VZV) thymidine kinase (TK) genes was developed for monitoring acyclovir-resistant viruses. This method determines the biochemical phenotype of the TK polypeptide, which is synthesized in vitro from viral DNA using a procedure as follows. The TK gene of each sample virus strain is amplified and isolated under the control of a T7 promoter by PCR. The PCR products are transcribed with T7 RNA polymerase and translated in a rabbit reticulocyte lysate. Using this method, enzymatic characteristics and the size of the TK polypeptides encoding HSV and VZV DNA were defined in less than 2 days without virus isolation. The assay should be a powerful tool in monitoring drug-resistant viruses, especially in cases in which virus isolation is difficult.
Rapid Identification of
Yersinia enterocolitica
in Blood by the 5′ Nuclease PCR Assay
Authors: Sen Keya
Published: December 2019

Yersinia enterocolitica
accounts for 50% of the clinical sepsis episodes caused by the transfusion of contaminated red blood cells. A 5′ nuclease TaqMan PCR assay was developed to detect
Y. enterocolitica
in blood. Primers and a probe based on the nucleotide sequence of the 16S rRNA gene from
Y. enterocolitica
were designed. Whole-blood samples were spiked with various numbers of
Y. enterocolitica
cells, and total chromosomal DNA was extracted. When the TaqMan PCR assay was performed, as few as six bacteria spiked in 200 μl of blood could be detected. The assay was specific and did not detect other
Yersinia
species. The TaqMan assay is easy to perform, takes 2 h, and has the potential for use in the rapid detection of
Y. enterocolitica
contamination in stored blood units.
Occurrence of
Bacteroides fragilis
Enterotoxin Gene-Carrying Strains in Germany and the United States
Authors: Claros Marina C. , Claros Zaida C. , Tang Yajarayma J. , Cohen Stuart H. , Silva Joseph , Goldstein Ellie J. C. , Rodloff Arne C.
Published: December 2019

Ninety-three
Bacteroides fragilis
isolates from different geographic locations were analyzed for the presence of an enterotoxin-encoding gene. It was shown that blood culture isolates were more likely to carry this gene than strains from other sources. All enterotoxin-positive strains belonged to the PCR fingerprint group I.
Determination of Human Immunodeficiency Virus Type 1 Subtypes in Taiwan by
vpu
Gene Analysis
Authors: Lee Chun-Nan , Wang Wei-Kung , Fan Wen-Sheng , Twu Shing-Jer , Chen Shou-Chien , Sheng Ming-Ching , Chen Mao-Yuan
Published: December 2019

The genetic diversity of human immunodeficiency virus (HIV) type 1 (HIV-1) has been characterized mainly by analysis of the
env
and
gag
genes. Information on the
vpu
genes in the HIV sequence database is very limited. In the present study, the nucleotide sequences of the
vpu
genes were analyzed, and the genetic subtypes determined by analysis of the
vpu
gene were compared with those previously determined by analysis of the
gag
and
env
genes. The
vpu
genes were amplified by nested PCR of proviral DNA extracted from 363 HIV-1-infected individuals and were sequenced directly by use of the PCR products. HIV-1 subtypes were determined by sequence alignment and phylogenetic analysis with reference strains. The strains in all except one of the samples analyzed could be classified as subtype A, B, C, E, or G. The
vpu
subtype of one strain could not be determined. Of the strains analyzed, genetic subtypes of 247 (68.0%) were also determined by analysis of the
env
or
gag
gene. The genetic subtypes determined by
vpu
gene analysis were, in general, consistent with those determined by
gag
and/or
env
gene analysis except for those for two AG recombinant strains. All the strains that clustered with a Thailand subtype E strain in the
vpu
phylogenetic analyses were subtype E by
env
gene analysis and subtype A by
gag
gene analysis. In summary, our genetic typing revealed that subtype B strains, which constituted 73.8% of all strains analyzed, were most prevalent in Taiwan. While subtype E strains constituted about one-quarter of the viruses, they were prevalent at a higher proportion in the group infected by heterosexual transmission. Genetic analysis of
vpu
may provide an alternate method for determination of HIV-1 subtypes for most of the strains, excluding those in which intersubtype recombination has occurred.
Molecular Typing of
Mycobacterium tuberculosis
Based on Variable Number of Tandem DNA Repeats Used Alone and in Association with Spoligotyping
Authors: Filliol Ingrid , Ferdinand Severine , Negroni Laetitia , Sola Christophe , Rastogi Nalin
Published: December 2019

Fingerprinting based on variable numbers of tandem DNA repeats (VNTR), a recently described methodology, was evaluated for molecular typing of
Mycobacterium tuberculosis
in an insular setting. In this study, VNTR fingerprinting was used alone or as a second-line test in association with spoligotyping, double-repetitive-element PCR (DRE-PCR), and IS
6110
restriction fragment length polymorphism (RFLP) analysis, and the discriminatory power for each method or the combination of methods was compared by calculating the Hunter-Gaston discriminative index (HGI). The results obtained showed that in 6 out of 12 (50%) cases, VNTR-defined clusters were further subdivided by spoligotyping, compared to 7 out of 18 (39%) cases where spoligotyping-defined clusters were further subdivided by VNTR. When used alone, VNTR was the least discriminatory method (HGI = 0.863). Although VNTR was significantly more discriminatory when used in association with spoligotyping (HGI = 0.982), the combination of spoligotyping and DRE-PCR (HGI = 0.992) was still the most efficient among rapid, PCR-based methodologies, giving results comparable to IS
6110
RFLP analysis. Nonetheless, VNTR typing may provide additional phylogenetical information that may be helpful to trace the molecular evolution of tubercle bacilli.
Major Change in the Predominant Type of “Norwalk-Like Viruses” in Outbreaks of Acute Nonbacterial Gastroenteritis in Osaka City, Japan, between April 1996 and March 1999
Authors: Iritani Nobuhiro , Seto Yoshiyuki , Haruki Kosuke , Kimura Masatsugu , Ayata Minoru , Ogura Hisashi
Published: December 2019

In Osaka City, Japan, between April 1996 and March 1999, a total of 350 fecal specimens from 64 outbreaks of acute nonbacterial gastroenteritis were examined to investigate infection by “Norwalk-like viruses” (NLVs). By reverse transcription (RT)-PCR, 182 samples (52.0%) from 47 outbreaks (73.4%) were NLV positive. During those three years, the incidence of NLV-associated outbreaks showed seasonality, being higher during January to March (winter to early spring). The ingestion of contaminated oysters was the most common transmission mode (42.6%). The amplicons of the 47 outbreak strains that were NLV positive by RT-PCR were tested using Southern hybridization with four probe sets (Ando et al., J. Clin. Microbiol. 33:64–71, 1995). Forty of the outbreak strains were classified as 4 probe 1-A (P1-A) strains, 6 P1-B strains, 10 P2-A strains, 17 P2-B strains, and 3 untypeable strains, and the other 7 outbreaks were determined to be mixed-probe-type strains. Probe typing and partial sequence analysis of the outbreak strains indicated that a predominant probe type of NLVs in Osaka City had drastically changed; P2-B strains (77.8%) with multiple genetic clusters were observed during the 1996–97 season, the P2-A common strain (81.3%) related to the Toronto virus cluster was observed during the 1997–98 season, and P1-B strains (75.0%) with a genetic similarity were observed during the 1998–99 season. For the three untypeable outbreak strains (96065, 97024, and 98026), the 98026 outbreak strain had Southampton virus (SOV)-like sequences, and each of the other outbreak strains had a unique 81-nucleotide sequence. Newly designed probes (SOV probe for the 98026 outbreak strain and the 96065 probe for the 96065 and 97024 outbreak strains) were hybridized with relative strains and without other probe type strains. The prevalent NLV probe types in Osaka City during those three years were classified in six phylogenetic groups: P1-A, P1-B, P2-A, P2-B, SOV, and 96065 probe types.
Performance Characteristics of the QUANTIPLEX HIV-1 RNA 3.0 Assay for Detection and Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma
Authors: Erice Alejo , Brambilla Donald , Bremer James , Jackson J. Brooks , Kokka Robert , Yen-Lieberman Belinda , Coombs Robert W.
Published: December 2019

The QUANTIPLEX HIV-1 RNA assay, version 3.0 (a branched DNA, version 3.0, assay [bDNA 3.0 assay]), was evaluated by analyzing spiked and clinical plasma samples and was compared with the AMPLICOR HIV-1 MONITOR Ultrasensitive (ultrasensitive reverse transcription-PCR [US-RT-PCR]) method. A panel of spiked plasma samples that contained 0 to 750,000 copies of human immunodeficiency virus type 1 (HIV-1) RNA per ml was tested four times in each of four laboratories (1,344 assays). Negative results (<50 copies/ml) were obtained in 30 of 32 (94%) assays with seronegative samples, 66 of 128 (52%) assays with HIV-1 RNA at 50 copies/ml, and 5 of 128 (4%) assays with HIV-1 RNA at 100 copies/ml. The assay was linear from 100 to 500,000 copies/ml. The within-run standard deviation (SD) of the log
10
estimated HIV-1 RNA concentration was 0.08 at 1,000 to 500,000 copies/ml, increased below 1,000 copies/ml, and was 0.17 at 100 copies/ml. Between-run reproducibility at 100 to 500 copies/ml was <0.10 log
10
in most comparisons. Interlaboratory differences across runs were ≤0.10 log
10
at all concentrations examined. A subset of the panel (25 to 500 copies/ml) was also analyzed by the US-RT-PCR assay. The within-run SD varied inversely with the log
10
HIV-1 RNA concentration but was higher than the SD for the bDNA 3.0 assay at all concentrations. Log-log regression analysis indicated that the two methods produced very similar estimates at 100 to 500 copies/ml. In parallel testing of clinical specimens with low HIV-1 RNA levels, 80 plasma samples with <50 copies/ml by the US-RT-PCR assay had <50 copies/ml when they were retested by the bDNA 3.0 assay. In contrast, 11 of 78 (14%) plasma samples with <50 copies/ml by the bDNA 3.0 assay had ≥50 copies/ml when they were retested by the US-RT-PCR assay (median, 86 copies/ml; range, 50 to 217 copies/ml). Estimation of bDNA 3.0 values of <50 copies/ml by extending the standard curve of the assay showed that these samples with discrepant results had higher HIV-1 RNA levels than the samples with concordant results (median, 34 versus 17 copies/ml;
P
= 0.0051 by the Wilcoxon two-sample test). The excellent reproducibility, broad linear range, and good sensitivity of the bDNA 3.0 assay make it a very attractive method for quantitation of HIV-1 RNA levels in plasma.
Clinical Evaluation of the Automated COBAS AMPLICOR HCV MONITOR Test Version 2.0 for Quantifying Serum Hepatitis C Virus RNA and Comparison to the Quantiplex HCV Version 2.0 Test
Authors: Yu Ming-Lung , Chuang Wan-Long , Dai Chia-Yen , Chen Shinn-Cherng , Lin Zu-Yau , Hsieh Ming-Yuh , Wang Liang-Yen , Chang Wen-Yu
Published: December 2019

A second-generation hepatitis C virus (HCV) quantitative assay (COBAS AMPLICOR HCV MONITOR Test, version 2.0; COBAS HCM-2) has been developed, with the intention of achieving equivalent quantification of all HCV genotypes and improving assay performance. To evaluate the clinical performance of COBAS HCM-2 and its utility in predicting the response to alpha interferon treatment, sera from 215 chronic hepatitis C patients were analyzed and the results were compared with those obtained by the Quantiplex bDNA HCV RNA, version 2.0, assay (bDNA-2). The COBAS HCM-2 had significantly greater sensitivity than bDNA-2 (94.9 versus 88.4%;
P
< 0.001) when performed with sera from chronic hepatitis C patients who were viremic by a qualitative PCR test. The standard deviations for the within-run and between-run reproducibilities of COBAS HCM-2 were <0.1 and <0.2, respectively, and it showed an improved linear range between genotypes with the threefold serial dilutions tested (
r
2
= 0.986 to 0.995). The COBAS HCM-2 results were positively correlated with the bDNA-2 results, but the values for COBAS HCM-2 were on average 0.96 log lower than the values for bDNA-2. The mean difference in quantification values between these two assays did not differ among samples with different genotypes (0.70 to 1.00 log). No genotype-dependent difference in viral load was observed. The pretreatment viral load was significantly lower in complete responders. By using multivariate analysis, the viral load 2 weeks after the initiation of alpha interferon treatment was the strongest predictor of a complete response. In conclusion, COBAS HCM-2 demonstrated good sensitivity, linearity, and reproducibility and efficiency equal to that of bDNA-2 for the quantification of HCV genotypes 1 and 2. Hence, this assay provides a rapid and reliable method for the quantification of HCV RNA in serum and is useful for the planning of interferon treatment.
Fluorescence-Based Quantitative Methods for Detecting Human Immunodeficiency Virus Type 1-Induced Syncytia
Authors: Wünschmann Sabina , Stapleton Jack T.
Published: December 2019

Cell fusion induced by human immunodeficiency virus type 1 (HIV-1) is usually assessed by counting multinucleated giant cells (syncytia) visualized by light microscopy. Currently used methods do not allow quantification of syncytia, nor do they estimate the number of cells involved in cell fusion. We describe two fluorescence-based methods for the detection and quantification of HIV-1-induced in vitro syncytium formation. The lymphoblastoid cell lines MT-2 and SupT1 were infected with syncytium-inducing (SI) HIV-1 isolates. Syncytia were detected by DNA staining with propidium iodide using flow cytometry to determine cell size or by two-color cytoplasmic staining of infected cell populations by using fluorescence microscopy. Both methods were able to detect and quantify HIV-induced syncytia. The methods could distinguish between SI and non-SI HIV isolates and could be used with at least two separate types of CD4+T-cell lines. Small syncytia can be readily identified by the two-color cytoplasmic staining method. Both methods were also shown to be useful for evaluating antiretroviral compounds, as demonstrated by the accurate assessment of HIV inhibition by azidothymidine (zidovudine), dideoxycytidine (zalcytibine), and hydroxyurea. These fluorescence-based assays allow a rapid and practical method for measuring HIV replication and anti-HIV activity of potential inhibitory compounds.
Borrelia
Isolates in Northern Colorado Identified as
Borrelia bissettii
Authors: Schneider Bradley S. , Zeidner Nordin S. , Burkot Thomas R. , Maupin Gary O. , Piesman Joseph
Published: December 2019

Previous work described
Borrelia burgdorferi
sensu lato group DN127 as a new genospecies,
Borrelia bissettii
, and prompted the present study to identify the
Borrelia
spp. that exist in northern Colorado. To determine the genospecies present, we analyzed two specific intergenic spacer regions located between the 5S and 23S and the 16S and 23S ribosomal genes. Phylogenetic analysis of the derived sequences clearly demonstrated that these isolates, originating from rodents captured in the foothills of northern Colorado, diverged from
B. burgdorferi
sensu stricto by 5 to 5.5% and were members of the new genospecies
B. bissettii
.
Comparison of Three Different Sensitive Assays for Hepatitis B Virus DNA in Monitoring of Responses to Antiviral Therapy
Authors: Chan Henry L. Y. , Leung Nancy W. Y. , Lau Tracy C. M. , Wong May L. , Sung Joseph J. Y.
Published: December 2019

The aim of our study was to compare the performances of two new hepatitis B virus (HBV) DNA assays, a cross-linking assay (NAXCOR) and a hybrid-capture amplification assay (Digene), versus the widely used branched-DNA (bDNA) assay (Chiron) in the monitoring of HBV DNA levels during antiviral treatment. Serial serum samples from 12 chronically HBV infected patients undergoing a phase II trial of an antiviral drug, 2′,3′-dideoxy-5-fluoro-3′-thiacytidine (FTC), were studied. A total of 96 serum samples were tested for HBV DNA using the cross-linking, hybrid-capture amplification, and bDNA assays. In the comparison of the cross-linking and bDNA assays, concordant results were found in 77 (80.3%) samples, no significant difference was found between the median log10 HBV DNA levels (6.66 versus 7.17 meq/ml), and the results of the two assays were closely correlated (r = 0.95). In the comparison of the hybrid-capture amplification and bDNA assays, concordant results were found in 79 (82.3%) samples, no significant difference was found between the median log10 HBV DNA levels (6.98 versus 6.99 meq/ml), and the results of the two assays were closely correlated (r = 0.99). Six (6.3%) samples by the cross-linking assay and 10 (10.4%) samples by the bDNA assay required retesting because of unacceptably high within-run coefficients of variance. No sample required retesting in the hybrid-capture amplification assay according to the internal validation. In conclusion, the cross-linking and hybrid-capture amplification assays were as sensitive as the bDNA assay for HBV DNA detection and can be recommended for monitoring of HBV DNA levels during antiviral treatment.
PCR-Restriction Fragment Length Polymorphism Analysis for Identification of Bacteroides spp. and Characterization of Nitroimidazole Resistance Genes
Authors: Stubbs Simon L. J. , Brazier Jon S. , Talbot Paul R. , Duerden Brian I.
Published: December 2019

Bacteroides spp. are opportunist pathogens that cause blood and soft tissue infections and are often resistant to antimicrobial agents. We have developed a combined PCR-restriction fragment length polymorphism (RFLP) technique to characterize the 16S rRNA gene for identification purposes and the nitroimidazole resistance (nim) gene for detection of resistance to the major antimicrobial agent used to treat Bacteroidesinfections: metronidazole (MTZ). PCR-RFLP analysis of 16S ribosomal (rDNA) with HpaII and TaqI produced profiles that enabled discrimination of type strains and identification of 70 test strains to the species level. The 16S rDNA PCR-RFLP identification results agreed with routine phenotypic testing for 62 of the strains. The discrepancies between phenotypic and PCR-RFLP methods for eight strains were resolved by 16S rDNA sequencing in three cases, but five strains remain unidentified. The presence of nim genes was indicated by PCR in 25 of 28 strains that exhibited reduced sensitivity to MTZ. PCR-RFLP of the nim gene products identified the four reported genes (nimA, -B, -C, and -D) and indicated the presence of a previously unreported nim gene in 5 strains. This novelnim gene exhibited 75% DNA sequence similarity withnimB. These rapid, accurate, and inexpensive methods should enable improved identification ofBacteroides spp. and the detection of MTZ resistance determinants.
Experimental Model of Progressive Disseminated Trichosporonosis in Mice with Latent Trichosporonemia
Authors: Yamagata Eiji , Kamberi Perparim , Yamakami Yuriko , Hashimoto Atsuro , Nasu Masaru
Published: December 2019

Trichosporon asahii and Trichosporon mucoides are the most common strains of fungi that cause disseminated trichosporonosis, a severe opportunistic infection in immunocompromised hosts. We have previously established a nested PCR assay using serum samples for detection of both strains. Here we describe a new experimental animal model for investigating the underlying mechanisms of disseminated trichosporonosis. T. asahii (OMU239, a clinical isolate from a patient with acute myelogenous leukemia) and 8-week-old ICR male mice were used in all experiments. A suspension of T. asahii (3 × 106 CFU/animal) was injected into the caudal vein of each mouse after immunosuppression with cyclophosphamide (200 mg/kg of body weight/day for 2 days) and prednisolone (30 mg/kg/day for 1 day). Mice were then divided into four subgroups (R0, R1, R2, and R3) based on the time of reimmunosuppression. The latter was performed using the same drugs 1 week (group R1), 2 weeks (group R2), and 3 weeks (group R3) after fungal infection. Reimmunosuppression was not performed in group R0. The 5-week-survival rates of mice after T. asahiiinfection were 0% for group R1, 50% for group R2, 80% for group R3, and 80% for group R0. There was a significant difference in the survival rates between group R1 and either group R0 or R3 (P < 0.05). Fungal clearance in peripheral blood and various organs of group R1 and R2 was delayed relative to that of group R0 but was similar to the control in group R3 in spite of reimmunosuppression. Our results suggest that the critical period for the development of disseminated trichosporonosis in our model is shorter than 3 weeks after T. asahii infection. We concluded that mice during this critical period were in a state of latent trichosporonemia. Comparison of the survival rates suggests that the nested PCR assay was more useful than blood culture and glucuronoxylomannan antigen assay in the detection of this latent trichosporonemia.
Analysis of 16S rRNA and 51-Kilodalton Antigen Gene and Transmission in Mice of Ehrlichia risticii in Virgulate Trematodes from Elimia livescens Snails in Ohio
Authors: Kanter Manuel , Mott Jason , Ohashi Norio , Fried Bernard , Reed Stephen , Lin Young C. , Rikihisa Yasuko
Published: December 2019

Operculate snails (the family Pleuroceridae:Elimia livescens) were collected between June and October 1998 from a river in central Ohio where repeated cases of Potomac horse fever (PHF) have occurred. Of collected snails, consistently 50 to 80% carried a combination of cercariae and sporocysts of digenetic virgulate trematodes. The trematodes obtained from each snail were pooled and tested for Ehrlichia risticii, the agent of PHF, by nested PCR using primers specific to the 16S rRNA gene. Out of a total of 209 trematode pools, 50 pools were found to be positive by PCR. The DNA sequence of the 16S rRNA gene identified in one trematode pool was identical to that of the type strain of E. risticii, and the sequence of the gene identified in another pool differed from that of the type strain by 1 nucleotide. Comparison of the deduced amino acid sequence of the partial 51-kDa antigen gene from various sources revealed that Maryland, Ohio (except Ohio 081), and Kentucky strains are in a cluster distinct from the sequences obtained from sources in California and Oregon. Ohio 081 was shown previously by antigenic composition analysis to be distinct from other groups. However, all sequences examined were not segregated according to their sources: horse blood or infected trematodes. E. risticiiwas found to be transmittable from trematodes to mice and was subsequently passaged from infected mice to additional mice, as determined by PCR analysis. Our findings suggest the evolution ofE. risticii in the natural reservoir in separate geographic regions and persistent infection of trematode populations with E. risticii during summer and early fall. The study also suggests that the mouse can be used to isolate E. risticii from the infected trematode.
Genotyping of the Capsule Gene Cluster (cps) in Nontypeable Group B Streptococci Reveals Two Majorcps Allelic Variants of Serotypes III and VII
Authors: Sellin Mats , Olofsson Carin , Håkansson Stellan , Norgren Mari
Published: December 2019

Forty group B Streptococcus (GBS) isolates obtained from Europe and the United States previously reported to be nontypeable (NT) by capsule serotype determination were subjected to buoyant density gradient centrifugation. From nearly half of the isolates capsule-expressing variants could be selected. For characterization of the remaining NT-GBS isolates, the capsule operon (cps) was amplified by the long-fragment PCR technique and compared by restriction fragment length polymorphism (RFLP) analysis. The patterns from serotype reference isolates (n = 32) were first determined and used as a comparison matrix for the NT-GBS isolates. Using two restriction enzymes, SduI and AvaII, cluster analysis revealed a high degree of similarity within serotypes but less than 88% similarity between serotypes. However, serotypes III and VII were each split in two distant RFLP clusters, which were designated III1 and III2 and VII1and VII2, respectively. Among the isolates that remained NT after repeated Percoll gradient selections, two insertional mutants were revealed. Both were found in blood isolates and harbored insertion sequence (IS) elements within cpsD: one harbored IS1548, and the other harbored IS861. All other NT-GBS isolates could, by cluster analysis, be referred to different serotypes by comparison to the RFLP reference matrix. In pulsed-field gel electrophoresis of SmaI-restricted chromosomal DNA, patterns from allelic type 1 and 2 isolates were essentially distributed in separate clusters in serotypes III and VII. A covariation with insertion sequence IS1548 in thehylB gene was suggested for serotype III, since allelic type III1 harboring IS1548 in hylB, clustered separately. The variation in serotype VII was not dependent on the presence of IS1548, which was not detected at any position in the type VII chromosome.
Analysis of Genetic Heterogeneity in Chlamydia trachomatis Clinical Isolates of Serovars D, E, and F by Amplified Fragment Length Polymorphism
Authors: Morré Servaas A. , Ossewaarde Jacobus M. , Savelkoul Paul H. M. , Stoof Jeroen , Meijer Chris J. L. M. , van den Brule Adriaan J. C.
Published: December 2019

Amplified fragment length polymorphism (AFLP) fingerprinting of clinical isolates of Chlamydia trachomatis serovars D, E, and F showed a low percentage of genetic heterogeneity, but clear differences were found. Isolates from index patients and partners had identical AFLP patterns and AFLP markers. Characterization of these AFLP markers could give more insight into the differences in virulence and clinical course of C. trachomatis infections.
Identification and Type Distribution of Astroviruses among Children with Gastroenteritis in Colombia and Venezuela
Authors: Medina Sandra M. , Gutierrez María F. , Liprandi Ferdinando , Ludert Juan E.
Published: December 2019

Astrovirus infections were detected by enzyme immunoassay in 12 (5%) of 251 stool samples from children with gastroenteritis from Bogota, Colombia. In addition, astroviruses were detected by reverse transcription-PCR in 3 (10%) of 29 stool samples negative for other enteric pathogens collected in Caracas, Venezuela, from children with gastroenteritis. Astrovirus type 1 was the most frequently detected virus.
The Tripartite Leader Sequence of Subgroup C Adenovirus Major Late mRNAs Can Increase the Efficiency of mRNA Export
Authors: Huang W. , Flint S. J.
Published: December 2019

The subgroup C human adenoviruses induce selective export of newly synthesized viral mRNA from the nucleus to the cytoplasm, with concomitant inhibition of export of the majority of cellular mRNA species. Such posttranscriptional regulation of viral and cellular gene expression in infected cells requires viral E1B and E4 proteins. To facilitate the investigation of parameters that govern selective export in adenovirus-infected cells, we constructed a marked human β-actin minigene under the control of the glucocorticoid-inducible enhancer-promoter of mouse mammary tumor virus and introduced it into the left end of the adenovirus type 5 (Ad5) genome. Transcription of this reporter gene (designated MA) as well as of a sibling, which differed only in the inclusion of a cDNA copy of the Ad2 major late tripartite leader sequence upstream of β-actin sequences (termed MtplA), in recombinant virus-infected cells was strictly dependent on the addition of dexamethasone to the medium. When transcription of the MA gene was induced during the late phase of infection, newly synthesized MA RNA entered the cytoplasm. These transcripts, which contain no viral sequences, therefore reproduce the behavior of exceptional cellular mRNA species observed when transcription of their genes is activated during the late phase of infection (U.-C. Yang, W. Huang, and S. J. Flint, J. Virol. 70:4071–4080, 1996). Unexpectedly, however, higher concentrations of newly synthesized RNA accumulated in the cytoplasm when the tripartite leader sequence was present in the reporter RNA, despite equal rates of transcription of the two reporter genes. Examination of the partitioning of both newly synthesized and steady-state populations of MA and MtplA RNAs between nuclear and cytoplasmic compartments indicated that the tripartite leader sequence did not increase RNA stability in the cytoplasm. Comparison of nuclear and cytoplasmic reporter RNA species by Northern blotting, primer extension, and reverse transcription-PCR provided no evidence for altered processing induced by the tripartite leader sequence. We therefore conclude that the tripartite leader sequence, long known to facilitate the translation of mRNAs during the late phase of adenovirus infection, can also modulate mRNA export from the nucleus.