bluuebunny/crossref_metadata_2025_split huggingface dataset:
Safe Determination of Susceptibility of
Mycobacterium tuberculosis
to Antimycobacterial Agents by Flow Cytometry
Authors: Moore Andrea V. , Kirk Scott M. , Callister Steven M. , Mazurek Gerald H. , Schell Ronald F. 
Published: December 2019

ABSTRACT

We showed previously that susceptibility testing for
Mycobacterium tuberculosis
labeled with fluorescein diacetate could be accomplished rapidly by using flow cytometry. However, safety was a major concern because mycobacteria were not killed prior to flow cytometric analysis. In this study, we developed a biologically safe flow cytometric susceptibility test that depends on detection and enumeration of actively growing
M. tuberculosis
organisms in drug-free and antimycobacterial agent-containing medium. The susceptibilities of 17 clinical isolates of
M. tuberculosis
to ethambutol, isoniazid, and rifampin were tested by the agar proportion and flow cytometric methods. Subsequently, all flow cytometric susceptibility test samples were inactivated by exposure to paraformaldehyde before analysis with a flow cytometer. Agreement between the results from the two methods was 98%. In addition, the flow cytometric results were available 72 h after the initiation of testing. The flow cytometric susceptibility assay is safe, simple to perform, and more rapid than conventional test methods, such as the BACTEC system and the proportion method.
Prospective Comparison of Whole-Blood- and Plasma-Based Hepatitis C Virus RNA Detection Systems: Improved Detection Using Whole Blood as the Source of Viral RNA
Authors: Stapleton Jack T. , Klinzman Donna , Schmidt Warren N. , Pfaller Michael A. , Wu Ping , LaBrecque Douglas R. , Han Jian-qiu , Phillips Mary Jeanne Perino , Woolson Robert , Alden Beth 
Published: December 2019

ABSTRACT

We previously demonstrated that whole blood contains significantly more hepatitis C virus (HCV) RNA than plasma. To validate the whole-blood-based HCV RNA detection method, a prospective comparison of HCV RNA detection in whole blood and plasma from 50 patients with chronic liver disease was undertaken. Whole-blood and plasma aliquots were independently tested for HCV RNA by reverse transcriptase (RT) PCR assay, and plasma was tested by the Roche Amplicor assay. HCV RNA was detected in 35 of 50 (70%) whole-blood samples by RT-PCR but in only 26 of 50 (52%) plasma samples tested by the Amplicor assay (
P
< 0.01). HCV RNA was detected in 85% of HCV antibody-positive patients by the whole-blood method compared with 74% of plasma samples by the Amplicor method. The five HCV antibody-positive subjects who were negative by whole-blood-based RT-PCR assay were all receiving interferon therapy and had normal transaminases at the time of testing. HCV RNA was detected in 38% of HCV antibody-negative subjects by the whole-blood-based RT-PCR assay compared with 6.25% of these patients by the Amplicor assay (
P
< 0.05). There were nine samples in which HCV RNA was detected in whole blood but the Amplicor test was negative. Eight of the nine RNAs prepared from these whole-blood samples tested positive in the Amplicor assay, thus confirming the specificity of our results. This study demonstrates that whole-blood-based HCV RNA detection is more sensitive than currently available commercial tests and that whole-blood RNA is suitable for use in commercial assays.
Dissemination of Two Methicillin-Resistant
Staphylococcus aureus
Clones Exhibiting Negative Staphylase Reactions in Intensive Care Units
Authors: Hsueh Po-Ren , Teng Lee-Jene , Yang Pan-Chyr , Pan Hui-Ju , Chen Yu-Chi , Wang Li-Hua , Ho Shen-Wu , Luh Kwen-Tay 
Published: December 2019

ABSTRACT

From December 1997 to March 1998, 25 methicillin-resistant
Staphylococcus aureus
(MRSA) isolates exhibiting negative Staphylase (Oxoid Ltd., Basingstoke, England) reactions were identified from various clinical specimens from 13 patients in six intensive care units (ICUs) or in wards following a stay in an ICU at the National Taiwan University Hospital. The characteristics of these isolates have not been previously noted in other MRSA isolates from this hospital. Colonies of all these isolates were grown on Trypticase soy agar supplemented with 5% sheep blood and were nonhemolytic and unpigmented. Seven isolates, initially reported as
Staphylococcus haemolyticus
(5 isolates) and
Staphylococcus epidermidis
(2 isolates) by the routine identification scheme and with the Vitek GPI system (bioMerieux Vitek, Inc., Hazelwood, Mo.), were subsequently identified as
S. aureus
by positive tube coagulase tests, standard biochemical reactions, and characteristic cellular fatty acid chromatograms. The antibiotypes obtained by the E test, coagulase types, restriction fragment length polymorphism profiles of the staphylococcal coagulase gene, and random amplified polymorphic DNA patterns generated by arbitrarily primed PCR of the isolates disclosed that two major clones disseminated in the ICUs. Clone 1 (16 isolates) was resistant to clindamycin and was susceptible to trimethoprim-sulfamethoxazole (TMP-SMZ) and was coagulase type II. Clone 2 (eight isolates) was resistant to clindamycin and TMP-SMZ and was coagulase type IV. These two epidemic clones from ICUs are unique and underline the need for caution in identifying MRSA strains with colonial morphologies not of the typical type and with negative Staphylase reactions.
Typing of Clinical
Mycobacterium avium
Complex Strains Cultured during a 2-Year Period in Denmark by Using IS
1245
Authors: Bauer Jeanett , Andersen Åse B. , Askgaard Dorthe , Giese Sten B. , Larsen Birger 
Published: December 2019

ABSTRACT

In the present study restriction fragment length polymorphism analyses with the recently described insertion sequence IS
1245
as a probe was performed with clinical
Mycobacterium avium
complex strains cultured in Denmark during a 2-year period. The overall aim of the study was to disclose potential routes of transmission of these microorganisms. As a first step, the genetic diversity among isolates from AIDS patients and non-human immunodeficiency virus (HIV)-infected patients was described. In addition, a number of isolates from nonhuman sources cultured during the same period were analyzed and compared to the human isolates. A total of 203 isolates from AIDS patients (
n
= 90), non-HIV-infected patients (
n
= 91), and nonhuman sources (
n
= 22) were analyzed. The presence of IS
1245
was restricted to
Mycobacterium avium
subsp.
avium
isolates. The majority of human isolates had large numbers of IS
1245
copies, while nonhuman isolates could be divided into a high-copy-number group and a low-copy-number group. Groups of identical strains were found to be geographically widespread, comprising strains from AIDS patients as well as strains from non-HIV-infected patients. Samples of peat (to be used as potting soil) and veterinary samples were found to contain viable
M. avium
isolates belonging to genotypes also found in humans.
TechLab and Alexon
Giardia
Enzyme-Linked Immunosorbent Assay Kits Detect Cyst Wall Protein 1
Authors: Boone James H. , Wilkins Tracy D. , Nash Theodore E. , Brandon Jill E. , Macias Elizabeth A. , Jerris Robert C. , Lyerly David M. 
Published: December 2019

ABSTRACT

A
Giardia lamblia
antigen detected by the TechLab
Giardia Test
(TechLab, Inc., Blacksburg, Va.) and the Alexon ProSpecT
Giardia
microplate assay (Alexon, Inc., Sunnyvale, Calif.) was purified by immunoaffinity chromatography from supernatant fluids of encystment cultures. Two major proteins (
M
r
22,000 and 26,000) were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie staining that did not resemble the GSA65 antigen reportedly detected by the Alexon test. These proteins reacted intensely with the monoclonal antibodies used in both commercial enzyme-linked immunosorbent assays (ELISAs). Both proteins had identical N-terminal amino acid sequences and were identified as cyst wall protein 1 (CWP1). The 26-kDa form appeared early during encystment followed by the appearance of the 22-kDa form. Recombinant CWP1 (
M
r
26,000) was strongly positive in both commercial tests. CWP1 was stable in human stool specimens, resistant to degradation by proteases and
N
- and
O
-glycanases, and unaffected by oxidation with sodium periodate. Two minor proteins with
M
r
s of 32,000 and 39,000 were detected in CWP1 preparations by using a sensitive fluorescent protein stain. Both were identified as CWP2, and neither reacted with the monoclonal antibodies from the commercial tests. We analyzed 535 stool specimens for CWP1 by using both commercial ELISAs and resolved discrepant results by using routine ova and parasite examination (O&P) and on immunofluorescence antibody assay. The presence of CWP1 correlated well between both ELISAs (98.7% correlation). Our results demonstrate that both commercial ELISAs detect CWP1, which is a useful diagnostic marker because it is highly stable, is secreted in large amounts by encysting trophozoites, and correlates well with O&P.
Application of
pbp1A
PCR in Identification of Penicillin-Resistant
Streptococcus pneumoniae
Authors: du Plessis Mignon , Smith Anthony M. , Klugman Keith P. 
Published: December 2019

ABSTRACT

A seminested PCR assay, based on the amplification of the pneumococcal
pbp1A
gene, was developed for the detection of penicillin resistance in clinical isolates of
Streptococcus pneumoniae
. The assay was able to differentiate between intermediate (MICs = 0.25 to 0.5 μg/ml) and higher-level (MICs = ≥1 μg/ml) resistance. Two species-specific primers, 1A-1 and 1A-2, which amplified a 1,043-bp region of the
pbp1A
penicillin-binding region, were used for pneumococcal detection. Two resistance primers, 1A-R1 and 1A-R2, were designed to bind to altered areas of the
pbp1A
gene which, together with the downstream primer 1A-2, amplify DNA from isolates with penicillin MICs of ≥0.25 and ≥1 μg/ml, respectively. A total of 183 clinical isolates were tested with the
pbp1A
assay. For 98.3% (180 of 183) of these isolates, the PCR results obtained were in agreement with the MIC data. The positive and negative predictive values of the assay were 100 and 91%, respectively, for detecting strains for which the MICs were ≥0.25 μg/ml and were both 100% for strains for which the MICs were ≥1 μg/ml.
Rapid Detection of Epidemic Strains of Methicillin-ResistantStaphylococcus aureus
Authors: Wichelhaus Thomas A. , Kern Sylvia , Schäfer Volker , Brade Volker 
Published: December 2019

ABSTRACTFifty methicillin-resistantStaphylococcus aureus(MRSA) initial isolates obtained from patients hospitalized in the orthopedic clinic of the Frankfurt University Hospital and 150 methicillin-sensitiveStaphylococcus aureus(MSSA) isolates were investigated in this study to determine whether the Slidex Staph-Kit is capable of differentiating between MRSA and MSSA owing to its unique performance characteristics. The Slidex Staph-Kit is a combined latex hemagglutination test designed to detect clumping factor, protein A, and a specific surface immunogen forS. aureus. Clumping factor-positive strains cause erythrocytes sensitized with fibrinogen to hemagglutinate, thereby resulting in visible red clumps.S. aureusstrains deficient in clumping factor agglutinate latex particles sensitized with specific antibodies against surface proteins ofS. aureus, thereby resulting in visible white clumps. Our results demonstrate that white clumping has a 99% specificity as well as a 98% positive predictive value for MRSA. Clumping factor-negative MRSA, which have been reported to occur in several countries, are epidemic in the Frankfurt area and account for 80% of all MRSA initial isolates in the orthopedic clinic of the Frankfurt University Hospital. Genotyping of all MRSA isolates by macrorestriction analysis of chromosomal DNA revealed that 83% of clumping factor-negative MRSA are closely related to the “southern-German” epidemic strain. This is the first study demonstrating the Slidex Staph-Kit’s capability for identifying epidemic clumping factor-negativeS. aureusstrains as methicillin resistant even prior to antimicrobial susceptibility testing.
Lactobacillus
Species Identification, H
2
O
2
Production, and Antibiotic Resistance and Correlation with Human Clinical Status
Authors: Felten Annie , Barreau Claude , Bizet Chantal , Lagrange Philippe Henri , Philippon Alain 
Published: December 2019

ABSTRACT

Lactobacilli recovered from the blood, cerebrospinal fluid, respiratory tract, and gut of 20 hospitalized immunocompromised septic patients were analyzed. Biochemical carbohydrate fermentation and total soluble cell protein profiles were used to identify the species. Hydrogen peroxide production was measured. Susceptibility to 19 antibiotics was tested by a diffusion method, and the MICs of benzylpenicillin, amoxicillin, imipenem, erythromycin, vancomycin, gentamicin, and levofloxacin were determined. A small number of species produced H
2
O
2
, and antibiotic susceptibilities were species related. Eighteen (90%) of the isolates were
L. rhamnosus
, one was
L. paracasei
subsp.
paracasei
, and one was
L. crispatus
.
L. rhamnosus
,
L. paracasei
subsp.
paracasei
isolates, and the type strains were neither H
2
O
2
producers nor vancomycin susceptible (MICs, ≥256 μg/ml).
L. crispatus
, as well as most of the type strains of lactobacilli which belong to the
L. acidophilus
group, was an H
2
O
2
producer and vancomycin susceptible (MICs, <4 μg/ml).
Isolation of
Abiotrophia adiacens
from a Brain Abscess Which Developed in a Patient after Neurosurgery
Authors: Biermann C. , Fries G. , Jehnichen P. , Bhakdi S. , Husmann M. 
Published: December 2019

ABSTRACT

We report the case of a patient who developed a large brain abscess after neurosurgery. Cerebrospinal fluid from the abscess drainage yielded
Abiotrophia adiacens
-specific PCR products and microorganisms that were identified by conventional microbiological methods and by 16S ribosomal DNA analysis as
Abiotrophia adiacens
, which was formerly classified as a member of nutritionally variant streptococci.
Clonal Diversity of Chilean Isolates of Enterohemorrhagic
Escherichia coli
from Patients with Hemolytic-Uremic Syndrome, Asymptomatic Subjects, Animal Reservoirs, and Food Products
Authors: Rios Maritza , Prado Valeria , Trucksis Michele , Arellano Carolina , Borie Consuelo , Alexandre Marcela , Fica Alberto , Levine Myron M. 
Published: December 2019

ABSTRACT

To determine clonal relationship among Chilean enterohemorrhagic
Escherichia coli
(EHEC) strains from different sources (clinical infections, animal reservoirs, and food), 54 EHEC isolates (44 of
E. coli
O157, 5 of
E. coli
O111, and 5 of
E. coli
O26) were characterized for virulence genes by colony blot hybridization and by pulsed-field gel electrophoresis (PFGE). By colony blotting, 12 different genotypes were identified among the 44
E. coli
O157 isolates analyzed, of which the genetic profile
stx
1
+
stx
2
+

hly
+
eae

+
was the most prevalent. All human O157 strains that were associated with sporadic cases of hemolytic-uremic syndrome (HUS) carried both the
stx
1
and
stx
2
toxin-encoding genes and were
eaeA
positive. Only 9 of 13 isolates from human controls were
stx
1
+
stx
2
+
, and 8 carried the
eaeA
gene. Comparison of profiles obtained by PFGE of
Xba
I-digested genomic DNA showed a great diversity among the
E. coli
O157 isolates, with 37 different profiles among 39 isolates analyzed. Cluster analysis of PFGE profiles showed a wide distribution of clinical isolates obtained from HUS cases and asymptomatic individuals and a clonal relationship among O157 isolates obtained from HUS cases and pigs. Analysis of virulence genes showed that a correlation exists among strains with the genotype
stx
1
+
stx
2
+
eae
+
and pathogenic potential. A larger difference in the PFGE restriction patterns was observed among the EHEC strains of serogroups O26 and O111. These results indicate that several different EHEC clones circulate in Chile and suggest that pigs are an important animal reservoir for human infections by EHEC. Guidelines have been proposed for better practices in the slaughter of animals in Chile.
Variation in Structure and Location of VanA Glycopeptide Resistance Elements among Enterococci from a Single Patient
Authors: Tremlett Catherine H. , Brown Derek F. J. , Woodford Neil 
Published: December 2019

ABSTRACT
Forty-six VanA glycopeptide-resistant enterococci (GRE) from a single patient were investigated for variation in structure and location of VanA resistance elements. Together with identification to species level and pulsed-field gel electrophoresis, these data divided the GRE into 10 groups and subgroups. Combining data in this manner appears helpful when investigating the epidemiology of GRE.
Identification of Contaminating Fungal DNA Sequences in Zymolyase
Authors: Rimek Dagmar , Garg Amar P. , Haas Walter H. , Kappe Reinhard 
Published: December 2019

ABSTRACT

When different preparations of Zymolyase were included in the pretreatment protocol of a panfungal PCR assay using a primer system for the 18S rRNA gene, an amplification product occurred in negative controls. The amplified fragment showed 100.0% sequence identity to the
Saccharomyces
sensu stricto complex and
Kluyveromyces lodderae
. Lyticase, lysing enzymes, and proteinase K appeared to be free from fungal DNA.
Quality Control in Nucleic Acid Amplification Methods: Use of Elementary Probability Theory
Authors: Shapiro Daniel S. 
Published: December 2019

ABSTRACT
Since it is not possible to state with certainty that contamination has occurred during a nucleic acid amplification assay in the absence of a positive result for a negative control, methods of elementary probability theory are used to illustrate how to identify those runs in which the possibility of contamination should be considered. The use of binomial and Poisson distributions and an analysis of clusters are presented with illustrative examples to demonstrate their use.
Identification of Seven
Treponema
Species in Health- and Disease-Associated Dental Plaque by Nested PCR
Authors: Willis S. G. , Smith K. S. , Dunn V. L. , Gapter L. A. , Riviere K. H. , Riviere G. R. 
Published: December 2019

ABSTRACT

Species-specific nested PCR was used to detect
Treponema amylovorum
,
Treponema denticola
,
Treponema maltophilum
,
Treponema medium
,
Treponema pectinovorum
,
Treponema socranskii
, and
Treponema vincentii
in dental plaque. Subjects with periodontitis harbored all species, but
T. pectinovorum
and
T. vincentii
were not found in plaque from disease-free subjects.
Comparison of Large Restriction Fragments of
Mycobacterium avium
Isolates Recovered from AIDS and Non-AIDS Patients with Those of Isolates from Potable Water
Authors: Aronson T. , Holtzman A. , Glover N. , Boian M. , Froman S. , Berlin O. G. W. , Hill H. , Stelma G. 
Published: December 2019

ABSTRACT

We examined potable water in Los Angeles, California, as a possible source of infection in AIDS and non-AIDS patients. Nontuberculous mycobacteria were recovered from 12 (92%) of 13 reservoirs, 45 (82%) of 55 homes, 31 (100%) of 31 commercial buildings, and 15 (100%) of 15 hospitals. Large-restriction-fragment (LRF) pattern analyses were done with
Ase
I. The LRF patterns of
Mycobacterium avium
isolates recovered from potable water in three homes, two commercial buildings, one reservoir, and eight hospitals had varying degrees of relatedness to 19 clinical isolates recovered from 17 patients. The high number of
M. avium
isolates recovered from hospital water and their close relationship with clinical isolates suggests the potential threat of nosocomial spread. This study supports the possibility that potable water is a source for the acquisition of
M. avium
infections.
Human Papillomavirus (HPV) DNA Copy Number Is Dependent on Grade of Cervical Disease and HPV Type
Authors: Swan David C. , Tucker Ruth Ann , Tortolero-Luna Guillermo , Mitchell Michele Follen , Wideroff Louise , Unger Elizabeth R. , Nisenbaum Rosane A. , Reeves William C. , Icenogle Joseph P. 
Published: December 2019

ABSTRACT

The association between human papillomavirus (HPV) DNA copy number and cervical disease was investigated. Viral DNA copy number for the most common high-risk HPV types in cervical cancer (types 16, 18, 31, and 45) was determined in cervical cytobrush specimens from 149 women with high-grade cervical intraepithelial neoplasias (CIN II-CIN III), 176 with low-grade CIN (CIN I), and 270 with normal cytology. Quantitative, PCR-based fluorescent assays for each of the HPV genotypes and for the β-globin gene were used. The amount of cellular DNA increased significantly with increasing disease; thus, HPV was expressed as copies per microgram of cellular DNA. The assay had a dynamic range of >10
7
, allowing documentation for the first time of the wide range of HPV copy numbers seen in clinical specimens. Median HPV DNA copy number varied by more than 10
4
among the viral types. HPV16 was present in the highest copy number; over 55% of HPV16-positive samples contained more than 10
8
copies/μg. Median copy number for HPV16 showed dramatic increases with increasing epithelial abnormality, an effect not seen with the other HPV types. HPV16 increased from a median of 2.2 × 10
7
in patients with normal cytology, to 4.1 × 10
7
in CIN I patients, to 1.3 × 10
9
copies/μg in CIN II-III patients. Even when stratified by cervical disease and viral type, the range of viral DNA copies per microgram of cellular DNA was quite large, precluding setting a clinically significant cutoff value for “high” copy numbers predictive of disease. This study suggests that the clinical usefulness of HPV quantitation requires reassessment and is assay dependent.
Use of PCR and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Techniques for Differentiation of
Prevotella intermedia
Sensu Stricto and
Prevotella nigrescens
Authors: Premaraj Thyagaseely , Kato Naoki , Fukui Katsuhito , Kato Haru , Watanabe Kunitomo 
Published: December 2019

ABSTRACT

Primers were designed from 16S rRNA sequences of
Prevotella intermedia
sensu stricto and
Prevotella nigrescens
and were used to discriminate these two species by PCR. The results were compared with those from the PCR technique using primers designed from arbitrarily primed PCR products by Guillot and Mouton (E. Guillot and C. Mouton, J. Clin. Microbiol. 35:1876–1882, 1997). The specificities of both assays were studied by using
P. intermedia
ATCC 25611,
P. nigrescens
ATCC 33563, 174 clinical isolates of
P. intermedia
sensu lato, and 59 reference strains and 58 clinical isolates of other
Prevotella
species and/or common oral flora. In addition, the usefulness and reliability of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the differentiation of the two species were examined by comparing the results with those from PCR assays. The controversial lipase test for distinguishing these species was also carried out. Unambiguous differentiation was made by both PCR assays, and the results matched each other. The SDS-PAGE assay was found to misidentify a few strains tested, compared with the results of PCR assays. The lipase test was positive for both species, including the reference strains of
P. intermedia
and
P. nigrescens
. We conclude that both PCR assays are simple, rapid, reliable, and specific methods which could be used in clinical studies and that the lipase test is not valuable in the differentiation. The reliable discrimination of the two species by SDS-PAGE is questionable.