bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of
Pneumocystis carinii
f. sp.
hominis
Gene Sequences in Filtered Air in Hospital Environments
Authors: Olsson Mats , Lidman Christer , Latouche Sophie , Björkman Anders , Roux Patricia , Linder Ewert , Wahlgren Mats 
Published: December 2019

ABSTRACT

To evaluate the risk of a nosocomial spread of
Pneumocystis carinii
f. sp.
hominis
(
P. carinii hominis
), air filter samples from rooms of
P. carinii
pneumonia (PCP) patients, adjacent corridors, and other hospital environments have been investigated for the presence of
P. carinii hominis
. Amplified DNA from air filters and sputum or bronchoalveolar lavage samples from the PCP patients have been genotyped with the
P. carinii hominis
genes of the mitochondrial large-subunit (mtLSU) rRNA and the internal transcribed spacers (ITS1 and ITS2) of the rRNA. Genotypes of the two loci were identified by direct sequencing, and for site 85 of the mtLSU locus, three allele-specific PCR assays were used.
P. carinii hominis
DNA was identified in the air of five of seven PCP patient rooms and in the air of two of four air filtrations from the ward corridors. The
P. carinii hominis
genotypes were the same in four of the five room air samples as those in the corresponding patients, suggesting a risk of person-to-person transmission of
P. carinii hominis
from PCP patients. Three of 16 air samples collected in infectious disease wards without the presence of PCP patients and one sample from a cardiology unit in a separate hospital building were also positive, which further strengthens the possibility of acquisition of
P. carinii hominis
from the environment.
Low-Level Vancomycin Resistance in
Clostridium innocuum
Authors: Mory Francine , Lozniewski Alain , David Veronique , Carlier Jean Philippe , Dubreuil Luc , Leclercq Roland 
Published: December 2019

ABSTRACT

Low-level vancomycin resistance was observed for 28 clinical
Clostridium innocuum
isolates and
C. innocuum
NCIB 10674, whereas teicoplanin was active. DNA from three clinical isolates and the type strain could not be amplified by PCR with primers specific for the genes
vanA
,
vanB
, and
vanC
, suggesting that
C. innocuum
is intrinsically resistant to vancomycin.
Molecular Epidemiological Study of Nosocomial
Enterobacter aerogenes
Isolates in a Belgian Hospital
Authors: Jalaluddin Sheikh , Devaster Jeanne-Marie , Scheen Robert , Gerard Michele , Butzler Jean-Paul 
Published: December 2019

ABSTRACT

In 1995, the rate of isolation of
Enterobacter aerogenes
in the Saint-Pierre University Hospital in Brussels, Belgium, was higher than that in the preceding years. A total of 45 nosocomial
E. aerogenes
strains were collected from 33 patients of different units during that year, and they were isolated from 19 respiratory specimens, 13 pus specimens, 7 blood specimens, 4 urinary specimens, 1 catheter specimen, and 1 heparin vial. The strains were analyzed to determine their epidemiological relatedness and were characterized by their antibiotic resistance pattern determination, plasmid profiling, and genomic fingerprinting by macrorestriction analysis with pulsed-field gel electrophoresis (PFGE). The majority of the strains (82%) were multiply resistant to different commonly used antibiotics. Two major plasmid profiles were found: most strains (64%) harbored two plasmids of different sizes, whereas the others (20%) contained a single plasmid. PFGE with
Spe
I and/or
Xba
I restriction enzymes revealed that a single clone (80%) was responsible for causing infections or colonizations throughout the year, and this result was concordant with those obtained by plasmid profiling, with slight variations. By comparing the results of these three methods, PFGE and plasmid profiling were found to be the techniques best suited for investigating the epidemiological relatedness of
E. aerogenes
strains, and they are therefore proposed as useful tools for the investigation of nosocomial outbreaks caused by this organism.
Use of Lambda Phage DNA as a Hybrid Internal Control in a PCR-Enzyme Immunoassay To Detect
Chlamydia pneumoniae
Authors: Pham Dien G. , Madico Guillermo E. , Quinn Thomas C. , Enzler Mark J. , Smith Thomas F. , Gaydos Charlotte A. 
Published: December 2019

ABSTRACT

An inherent problem in the diagnostic PCR assay is the presence of ill-defined inhibitors of amplification which may cause false-negative results. Addition of an amplifiable fragment of foreign DNA in the PCR to serve as a hybrid internal control (HIC) would allow for a simple way to identify specimens containing inhibitors. Two oligonucleotide hybrid primers were synthesized to contain nucleic acid sequences of the
Chlamydia pneumoniae
16S rRNA primers in a position flanking two primers that target the sequences of a 650-bp lambda phage DNA segment. By using the hybrid primers, hybrid DNA comprising a large sequence of lambda phage DNA flanked by short pieces of chlamydia DNA was subsequently generated by PCR, cloned into a plasmid vector, and purified. Plasmids containing the hybrid DNA were diluted and used as a HIC by adding them to each
C. pneumoniae
PCR test. Consequently,
C. pneumoniae
primers were able to amplify both chlamydia DNA and the HIC DNA. The production of a 689-bp HIC DNA band on an acrylamide gel indicated that the specimen contained no inhibitors and that internal conditions were compatible with PCR. Subsequently, a biotinylated RNA probe for the HIC was transcribed from a nested sequence of the HIC and was used for its hybridization. Detection of the HIC DNA-RNA hybrid was achieved by enzyme immunoassay (EIA). This PCR-EIA system with a HIC was initially tested with 12 previously PCR-positive and 14 previously PCR-negative specimens. Of the 12 PCR-positive specimens, 11 were reconfirmed as positive; 1 had a negative HIC value, indicating inhibition. Of the 14 previously PCR-negative specimens, 13 were confirmed as true negative; 1 had a negative HIC value, indicating inhibition. The assay was then used with 237 nasopharyngeal specimens from patients with pneumonia. Twenty-one of 237 (8.9%) were positive for
C. pneumoniae
, and 42 (17.7%) were found to inhibit the PCR. Specimens showing inhibitory activity were diluted 1:10 and were retested. Ten specimens were still inhibitory to the PCR and required further DNA purification. No additional positive samples were detected and 3 nasopharyngeal specimens remained inhibitory to PCR. Coamplification of a HIC DNA can help confirm true-negative PCR results by ruling out the presence of inhibitors of DNA amplification.
Cytoplasmic, Nuclear, and Platelet Autoantibodies in Human Granulocytic Ehrlichiosis Patients
Authors: Wong Susan J. , Thomas Josephine A. 
Published: December 2019

ABSTRACT

Serum samples from patients with confirmed human granulocytic ehrlichiosis (HGE) were tested for cytoplasmic, nuclear, and platelet autoantibodies and rheumatoid factor. The indirect fluorescence antinuclear antibody test on Hep-2 cells demonstrated antinuclear titers of ≥40 and ≥160 in 44 and 10%, respectively, of serum samples from HGE patients. Two patients (4%) had anticytoplasmic (mitochondrial and spindle apparatus) antibodies with a titer of 80 and two patients (4%) had anticytoplasmic (mitochondrial) antibodies with a titer of 160 or greater. Flow cytometry was used to demonstrate antiplatelet antibodies in 80% of first serum samples from HGE patients. Rheumatoid factor was not detected. Nuclear and cytoplasmic autoantibodies are a major cause of interference when the indirect fluorescence antibody test is used to detect fluorescence of morulae in
Ehrlichia
-infected equine neutrophils or HL-60 promyelocytes. Antiplatelet antibodies may contribute to the profound thrombocytopenia which is a characteristic laboratory feature during the acute phase of HGE infection. Whether autoantibodies precede infection or are caused by immune activation of HGE deserves further study.
Comparison of the ABI 7700 System (TaqMan) and Competitive PCR for Quantification of IS
6110
DNA in Sputum during Treatment of Tuberculosis
Authors: Desjardin L. e. , Chen Y. , Perkins M. D. , Teixeira L. , Cave M. D. , Eisenach K. D. 
Published: December 2019

ABSTRACT

Mycobacterium tuberculosis
can persist in sputum for long periods of time after the initiation of antituberculosis chemotherapy. The purpose of this study was to determine whether quantitative estimates of
M. tuberculosis
DNA in sputum correlate with the numbers of viable bacilli and thus measure the therapeutic response of patients during treatment. Two methods of
M. tuberculosis
DNA quantification were examined by using DNA isolated from sputum specimens serially collected during the course of chemotherapy. A competitive PCR assay was compared to an automated system of real-time quantification with the ABI Prism 7700 Sequence Detection System (TaqMan). The ABI 7700 system uses standard PCR in conjunction with a fluorogenic probe in which the intensity of fluorescence is proportional to the amount of target DNA present. The results showed that both PCR systems are reproducible and accurate. The amounts of
M. tuberculosis
DNA quantified in sputum corresponded well with the numbers of acid-fast bacilli (AFB) counted by microscopy. Before initiation of antituberculosis therapy, measures of AFB,
M. tuberculosis
DNA, and cultivable bacilli were similar, suggesting that quantification of DNA is a good method for measuring the initial bacillary load. However, the rate of disappearance of both AFB and
M. tuberculosis
DNA did not correlate with the decline in cultivable bacilli in the specimen; therefore, these tests are not appropriate for monitoring treatment efficacy.
Comparison of Three Molecular Assays for Rapid Detection of Rifampin Resistance in
Mycobacterium tuberculosis
Authors: Watterson Simon A. , Wilson Stuart M. , Yates Malcolm D. , Drobniewski Francis A. 
Published: December 2019

ABSTRACT

Multidrug-resistant
Mycobacterium tuberculosis
(MDR-TB) is an emerging problem of great importance to public health, with higher mortality rates than drug-sensitive TB, particularly in immunocompromised patients. MDR-TB patients require treatment with more-toxic second-line drugs and remain infectious for longer than patients infected with drug-sensitive strains, incurring higher costs due to prolonged hospitalization. It is estimated that 90% of United Kingdom rifampin-resistant isolates are also resistant to isoniazid, making rifampin resistance a useful surrogate marker for multidrug resistance and indicating that second- and third-line drugs to which these isolates are susceptible are urgently required. Resistance in approximately 95% of rifampin-resistant isolates is due to mutations in a 69-bp region of the
rpoB
gene, making this a good target for molecular genotypic diagnostic methods. Two molecular assays, INNO-LiPA Rif.TB (Innogenetics, Zwijndrecht, Belgium) and MisMatch Detect II (Ambion, Austin, Tex.), were performed on primary specimens and cultures to predict rifampin resistance, and these methods were compared with the resistance ratio method. A third method, the phenotypic PhaB assay, was also evaluated in comparison to cultures in parallel with the genotypic assays. In an initial evaluation 16 of 16, 15 of 16, and 16 of 16 rifampin-resistant cultures (100, 93.8, and 100%, respectively), were correctly identified by line probe assay (LiPA), mismatch assay, and PhaB assay, respectively. Subsequently 38 sputa and bronchealveolar lavage specimens and 21 isolates were received from clinicians for molecular analysis. For the 38 primary specimens the LiPA and mismatch assay correlated with culture and subsequent identification and susceptibility tests in 36 and 38 specimens (94.7 and 100%), respectively. For the 21 isolates submitted by clinicians, both assays correlated 100% with routine testing.
Genetic Diversity among Strains of
Moraxella catarrhalis
: Analysis Using Multiple DNA Probes and a Single-Locus PCR-Restriction Fragment Length Polymorphism Method
Authors: Walker Elaine S. , Preston Robert A. , Post J. Christopher , Ehrlich Garth D. , Kalbfleisch John H. , Klingman Karin L. 
Published: December 2019

ABSTRACT

Moraxella
(
Branhamella
)
catarrhalis
, a causative agent of otitis media, sinusitis, and exacerbation of bronchitis, has acquired widespread ability to produce β-lactamase and can be nosocomially transmitted. The typing methods used in epidemiological analyses of
M. catarrhalis
are not optimal for genetic analyses. Two methods, a multiple-locus Southern blot (SB) method and a single-locus PCR-restriction fragment length polymorphism (RFLP) method, were developed and used to assess genetic diversity and potential clinical and geographic relationships in
M. catarrhalis
. Nine randomly cloned
M. catarrhalis
DNA fragments were used as probes of SBs containing DNA from 54 geographically and clinically diverse strains. For comparison, a PCR-RFLP method was developed as a quick, inexpensive, and discriminating alternative. A highly variable 3.7-kb genomic region (M46) was cloned and sequenced, and 3.5 kb of the cloned DNA was targeted for PCR amplification. DNAs from the 54 strains were subjected to PCR-RFLP. SB analysis distinguished all strains that had no apparent epidemiological linkage (40 of 54), and PCR-RFLP distinguished fewer strains (21 of 54). Epidemiologically linked strains appeared genetically identical by both methods. PCR-RFLP was compared to pulsed-field gel electrophoresis (PFGE) for 8 of the 54 strains and 23 additional strains. PCR-RFLP distinguished fewer strains than PFGE typing (16 of 31 versus 20 of 31 strains), but PCR-RFLP was more useful for inferring interstrain relatedness. Separate cluster analyses of multilocus SB and single locus PCR-RFLP data showed high genetic diversity within and across geographic locations and clinical presentations. The resultant dendrograms were not entirely concordant, but both methods often gave similar strain clusters at the terminal branches. High genetic diversity, nonconcordance of cluster analyses from different genetic loci, and shared genotypes among epidemiologically linked strains support a hypothesis of high recombination relative to spread of clones. Single-locus PCR-RFLP may be suitable for short-term epidemiological studies, but the SB data demonstrate that greater strain discrimination may be obtained by sampling variation at multiple genomic sites.
Utility of Random Amplified Polymorphic DNA PCR and TaqMan Automated Detection in Molecular Identification of
Aspergillus fumigatus
Authors: Brandt Mary E. , Padhye Arvind A. , Mayer Leonard W. , Holloway Brian P. 
Published: December 2019

ABSTRACT

We developed a method for the identification of
Aspergillus fumigatus
fungal isolates by using random amplified polymorphic DNA (RAPD) PCR (RAPD-PCR) cloning and the TaqMan LS50B fluorogenic detection system (Perkin-Elmer Corp., Applied Biosystems, Foster City, Calif.). DNA from seven clinically important
Aspergillus
species was screened by RAPD-PCR to identify section- or species-specific amplicons. With the OPZ19 RAPD primer a 1,264-bp product was amplified from all
A. fumigatus
strains initially examined but not from other species. A partial DNA sequence of this product was used to design a specific primer pair, which generated a single 864-bp fragment with DNA from 90 of 100
A. fumigatus
isolates when a “touchdown” (65→55°C) annealing protocol was used. The TaqMan system, a fluorogenic assay which uses the 5′→3′ endonuclease activity of
Taq
DNA polymerase, detected this 864-bp product with DNA from 89 of these 90
A. fumigatus
strains; 1 DNA sample generated an indeterminate result. With DNA from three morphologically typical
A. fumigatus
isolates, six white (“albino”)
A. fumigatus
isolates, and five of six
Neosartorya
species (non-
A. fumigatus
members of the section
Fumigati
), the 864-bp product was amplified differentially at an annealing temperature of 56°C but not with the touchdown annealing format. No amplicon was detected with DNA from 56 isolates of heterologous
Aspergillus
,
Penicillium
, and
Paecilomyces
species or from
Neosartorya fennelliae
; TaqMan assay results were either negative (51 isolates) or indeterminate (5 isolates) for all isolates. This RAPD-PCR and TaqMan assay offers promise as a nucleic acid-based system that can be used for the identification of filamentous fungal isolates and that requires no postamplification sample manipulations.
Quantification of Human Immunodeficiency Virus Type 1 RNA Levels in Plasma by Using Small-Volume-Format Branched-DNA Assays
Authors: Yeghiazarian Torange , Zhao Yuqi , Read Stanley E. , Kabat William , Li Xiaoyi , Hamren Sarah J. , Sheridan Patrick J. , Wilber Judith C. , Chernoff David N. , Yogev Ram 
Published: December 2019

ABSTRACT
We have developed small-volume (50 or 250 μl)-format branched-DNA assays for human immunodeficiency virus type 1 (HIV-1) RNA for use with specimens in which the volume is limited and/or a high viral load is anticipated. These formats exhibited good correlation with the standard 1-ml format; high specificity, reproducibility, and linearity; and no significant difference in the quantification of HIV-1 subtypes.
Genetic Analysis of Multiple Vancomycin-Resistant
Enterococcus
Isolates Obtained Serially from Two Long-Term-Care Patients
Authors: Schoonmaker Dianna J. , Bopp Lawrence H. , Baltch Aldona L. , Smith Raymond P. , Rafferty Mary Ellen , George Mary 
Published: December 2019

ABSTRACT

Fifty-eight vancomycin-resistant enterococcal isolates were obtained from two patients over 9 weeks. Numerous pulsed-field gel electrophoresis fingerprinting types were isolated from each patient. By PCR, all isolates were
vanA
+
. However, many isolates from patient B were found to lack
vanA
by hybridization. Our results demonstrate the importance of examining multiple isolates, especially from patients who are at high risk of infection.
Bacillus thuringiensis
subsp.
konkukian
(Serotype H34) Superinfection: Case Report and Experimental Evidence of Pathogenicity in Immunosuppressed Mice
Authors: Hernandez Eric , Ramisse Francoise , Ducoureau Jean-Pierre , Cruel Thierry , Cavallo Jean-Didier 
Published: December 2019

ABSTRACT

We present a case of severe war wounds infected by
Bacillus thuringiensis
serotype H34 and describe the experimental protocol used to demonstrate its ability to infect mice after cutaneous inoculation. This case is interesting because
B. thuringiensis
is considered to be a contaminant in laboratories and receives inadequate attention.
Line Probe Assay for Detection of Human Immunodeficiency Virus Type 1 Mutations Conferring Resistance to Nucleoside Inhibitors of Reverse Transcriptase: Comparison with Sequence Analysis
Authors: Descamps Diane , Calvez Vincent , Collin Gilles , Cécille Agnès , Apetrei Cristian , Damond Florence , Katlama Christine , Matheron Sophie , Huraux Jean-Marie , Brun-Vézinet Françoise 
Published: December 2019

ABSTRACT
We compared the line probe assay (LiPA) to sequence analysis for the detection of mutations conferring resistance to nucleoside inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Plasma samples from 40 patients who had received zidovudine, dideoxyinosine, and dideoxycytosine, alone or in combination, and who were enrolled in the ALTIS 2 clinical trial (lamivudine [3TC] plus stavudine) were tested at enrollment and at week 24. RT PCR products from plasma were used for LiPA, and DNA was used for sequence analysis. LiPA gave uninterpretable results for 8.5% of the analyzed codons corresponding to 63 samples, mainly for codons 41, 69, and 70. Several minor discrepancies between the two methods occurred, mainly due to the ability of LiPA to detect mixed populations while sequence analyses detect a single homogeneous population. LiPA is suitable for detecting mixed populations and easy to implement in clinical laboratories and might be useful for epidemiological surveys of primary HIV-1 resistance.
Genetic Relationship between
Cryptococcus neoformans
var.
neoformans
Strains of Serotypes A and D
Authors: Franzot Sarah P. , Fries Bettina C. , Cleare Wendy , Casadevall Arturo 
Published: December 2019

ABSTRACT

Cryptococcus neoformans
serotypes A and D are responsible for the overwhelming majority of infections in patients with AIDS. The genetic relationship between the serotypes is poorly understood, but there are significant differences in the epidemiology and clinical presentation of serotype A and D infections. We evaluated the genetic relationship between reference
C. neoformans
strains belonging to serotypes A and D by analyzing their
URA5
sequences and restriction fragment length polymorphisms (RFLPs) with the
C. neoformans
repetitive element 1 (CNRE-1) probe. The results were compared to those previously obtained for isolates from Brazil and New York City by the same typing methods, and dendrograms were generated. Serotype A and D strains produced distinct RFLP patterns consistent with their separation into two major clusters in the dendrogram generated on the basis of RFLP data. Similarly, serotype A and D strains clustered independently on the basis of the nucleotide sequences of their
URA5
genes. Pairwise comparisons revealed average numbers of nucleotide differences within serotypes A and D of 3.0 ± 1.7 and 7.2 ± 3.4, respectively (
P
< 0.0001), and between serotypes A and D of 41.9 ± 2.7. In summary, our results indicate phylogenetic differences between the two serotypes of
C. neoformans
var.
neoformans
and suggest that these serotypes could probably be considered different varieties of
C. neoformans
.
Outbreak of
Staphylococcus schleiferi
Wound Infections: Strain Characterization by Randomly Amplified Polymorphic DNA Analysis, PCR Ribotyping, Conventional Ribotyping, and Pulsed-Field Gel Electrophoresis
Authors: Kluytmans Jan , Berg Hans , Steegh Paul , Vandenesch François , Etienne Jerome , van Belkum Alex 
Published: December 2019

ABSTRACT

Within a 1-year period, six surgical-site infections (SSI) caused by
Staphylococcus schleiferi
were observed in the department of cardiac surgery of Ignatius Hospital, Breda, The Netherlands. Since outbreaks caused by this species of coagulase-negative staphylococci have not been described before, an extensive environmental survey and a case control study were performed in combination with molecular typing of the causative microorganism in order to identify potential sources of infection. Variability, as detected by four different genotyping methods (random amplification of polymorphic DNA [RAPD], conventional and PCR-mediated ribotyping, and pulsed-field gel electrophoresis [PFGE] of DNA macro restriction fragments), appeared to be limited both among the clinical isolates and among several control strains obtained from various unrelated sources. Among unrelated strains, RAPD and PCR-mediated ribotyping identified two types only, whereas seven different types were identified in a relatively concordant manner by conventional ribotyping and PFGE. The latter two procedures proved to be the most useful tools for tracking the epidemiology of
S. schleiferi
. Four of the outbreak-related strains were identical by both methods, and two isolates showed limited differences. In the search for a potential source of
S. schleiferi
infection, two slightly different PFGE types were encountered on several occasions in the nose of a single surgeon. These strains were, however, clearly different from the outbreak type. In contrast,
S. schleiferi
cultures remained negative for two persons identified on the basis of case control analysis. It was demonstrated that SSI caused by
S. schleiferi
had a clinical impact for patients comparable to that of a wound infection caused by
Staphylococcus aureus
. This report describes the first well-documented outbreak of
S. schleiferi
infection. A source of the outbreak was not detected.