bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombinant Mouse-Human Chimeric Antibodies as Calibrators in Immunoassays That Measure Antibodies toToxoplasma gondii
Authors: Hackett John , Hoff-Velk Jane , Golden Alan , Brashear Jeff , Robinson John , Rapp Margaret , Klass Michael , Ostrow David H. , Mandecki Wlodek
Published: December 2019

In the present study, we examined the feasibility of using recombinant antibodies containing murine variable regions and human constant regions as calibrators or controls in immunoassays. As a model system, we chose the Abbott IMx Toxo immunoglobulin M (IgM) and Toxo IgG assays designed to detect antibodies toToxoplasma gondii. Two mouse monoclonal antibodies were selected based on their reactivity to theT. gondiiantigens P30 and P66. Heavy- and light-chain variable-region genes were cloned from both hybridomas and transferred into immunoglobulin expression vectors containing human kappa and IgG1 or IgM constant regions. The constructs were stably transfected into Sp2/0-Ag14 cells. In the IMx Toxo IgG assay, immunoreactivity of the anti-P30 chimeric IgG1 antibody paralleled that of the positive human plasma-derived assay calibrators. Signal generated with the anti-P66 chimeric IgG1 antibody was observed to plateau below the maximal reactivity observed for the assay calibrator. Examination of the IgM chimeric antibodies in the IMx Toxo IgM assay revealed that both the anti-P30 and anti-P66 antibodies matched the assay index calibrator manufactured with human Toxo IgM-positive plasma. When evaluated with patient samples, the correlation between results obtained with the chimeric antibody calibrators and the positive human plasma calibrators was ≥0.985. These data demonstrate that chimeric mouse-human antibodies are a viable alternative to high-titer positive human plasma for the manufacture of calibrators and controls for diagnostic assays.
Phylogenetic Placement of Rickettsiae from the TicksAmblyomma americanumandIxodes scapularis
Authors: Weller Susan J. , Baldridge Gerald D. , Munderloh Ulrike G. , Noda Hiroaki , Simser Jason , Kurtti Timothy J.
Published: December 2019

A rickettsial isolate (isolate MOAa) belonging to the spotted fever group (SFG) was obtained from the lone star tickAmblyomma americanum. We used PCR to characterize the genes for the rickettsial outer membrane proteins rOmpA and rOmpB. We sequenced the PCR products (domains I of both therompAgene and therompBgene) of MOAa and WB-8-2, another rickettsial isolate fromA. americanum. To place MOAa and WB-8-2 and two other nonpathogenic isolates (Rickettsia rickettsiiHlp2 andRickettsia montanaM5/6) with respect to their putative sister species, we included them in a phylogenetic analysis of 9Rickettsiaspecies and 10Rickettsiastrains. Our phylogenetic results implied three evolutionary lineages of SFG rickettsiae and that WB-8-2 and MOAa were most closely related toR. montana. New World isolates were not the most closely related to each other (they did not form a clade). Rather, our results supported four independent origins (introductions) of rickettsiae into North America from different Old World regions. The results of our phylogenetic analysis did not support the hypothesis of a stable coevolution of rickettsiae and their tick hosts. Finally, we examined therompAgene of a nonpathogenic rickettsial symbiont isolated from the tickIxodes scapularis. In a phylogenetic analysis, the symbiont was placed as the sister toR. montanaand its isolates. The relationship of this symbiont toR. montanaraised questions as to the potential origin of pathogenic SFG rickettsiae from nonpathogenic tick symbionts, or vice versa.
Computerized Analysis of Restriction Fragment Length Polymorphism Patterns: Comparative Evaluation of Two Commercial Software Packages
Authors: Gerner-Smidt P. , Graves L. M. , Hunter Susan , Swaminathan B.
Published: December 2019

Two computerized restriction fragment length polymorphism pattern analysis systems, the BioImage system and the GelCompar system (Molecular Analyst Fingerprinting Plus in the United States), were compared. The two systems use different approaches to compare patterns from different gels. In GelCompar, a standard reference pattern in one gel is used to normalize subsequent gels containing lanes with the same reference pattern. In BioImage, the molecular sizes of the fragments are calculated from size standards present in each gel. The molecular size estimates obtained with the two systems for 12 restriction fragments of phage λ were between 97 and 101% of their actual sizes, with a standard deviation of less than 1% of the average estimated size for most fragments. At the window sizes used for analysis, the GelCompar system performed somewhat better than BioImage in identifying visually identical patterns generated by electrophoretic separation of
Hha
I-restricted DNA of
Listeria monocytogenes
. Both systems require the user to make critical decisions in the analysis. It is very important to visually verify that the systems are finding all bands in each lane and that no artifacts are being detected; both systems allow manual editing. It is also important to verify results obtained in the pattern matching or clustering portions of the analysis.
Effects of Following National Committee for Clinical Laboratory Standards and Deutsche Industrie Norm-Medizinische Mikrobiologie Guidelines, Country of Isolate Origin, and Site of Infection on Susceptibility of
Escherichia coli
to Amoxicillin-Clavulanate (Augmentin)
Authors: Simpson I. , Durodie J. , Knott S. , Shea B. , Wilson J. , Machka K.
Published: December 2019

Amoxicillin-clavulanate (Augmentin), as a combination of two active agents, poses extra challenges over single agents in establishing clinically relevant breakpoints for in vitro susceptibility tests. Hence, reported differences in amoxicillin-clavulanate percent susceptibilities among
Escherichia coli
isolates may reflect localized resistance problems and/or methodological differences in susceptibility testing and breakpoint criteria. The objectives of the present study were to determine the effects of (i) methodology, e.g., those of the National Committee for Clinical Laboratory Standards (NCCLS) and the Deutsche Industrie Norm-Medizinische Mikrobiologie (DIN), (ii) country of origin (Spain, France, and Germany), and (iii) site of infection (urinary tract, intra-abdominal sepsis, or other site[s]) upon the incidence of susceptibility to amoxicillin-clavulanate in 185 clinical isolates of
E. coli
. Cefuroxime and cefotaxime were included for comparison. The use of NCCLS methodology resulted in different distribution of amoxicillin-clavulanate MICs than that obtained with the DIN methodology, a difference highlighted by the 10% more strains found to be within the 8- to 32-μg/ml MIC range. This difference reflects the differing amounts of clavulanic acid present. NCCLS and DIN methodologies also produce different MIC distributions for cefotaxime but not for cefuroxime. Implementation of NCCLS and DIN breakpoints produced markedly different incidences of strains that were found to be susceptible, intermediate or resistant to amoxicillin-clavulanate. A total of 86.5% strains were found to be susceptible to amoxicillin-clavulanate by the NCCLS methodology, whereas only 43.8% were found to be susceptible by the DIN methodology. Similarly, 4.3% of the strains were found to be resistant by NCCLS guidelines compared to 21.1% by the DIN guidelines. The use of DIN breakpoints resulted in a fivefold-higher incidence of strains categorized as resistant to cefuroxime. There were no marked differences due to country of origin upon the MIC distributions for amoxicillin-clavulanate, cefuroxime, or cefotaxime, as determined with the NCCLS guidelines. Isolates from urinary tract and intra-abdominal infections were generally more resistant to amoxicillin-clavulanate than were isolates from other sites of infection.
Buffered Plate Antigen Test as a Screening Test for Diagnosis of Human Brucellosis
Authors: Lucero Nidia E. , Bolpe Jorge E.
Published: December 2019

Brucellosis in Argentina is currently investigated in bank donor blood by the standard plate agglutination test (PAT). This study evaluated the buffered plate antigen test (BPA), now used to screen for bovine brucellosis, as a screen for human disease. Of 57 sera from patients with culture-confirmed brucellosis, 100% were detected with the BPA. Of 142 sera positive by rose bengal (RB) and complement fixation (CF), from patients with clinical evidence of brucellosis, the BPA detected 100%. Of 307 sera from a nonsymptomatic population that were RB and CF negative, the BPA detected 99.67% of the negative sera. The data indicate that the BPA is satisfactory compared to the other agglutination tests employed. It is an inexpensive and practical screening test and reduces the nonspecific reactions detected by the PAT.
Evaluation of a Membrane Filter Assay System, Ortho HCV Ab Quik Pack, for Detection of Anti-Hepatitis C Virus Antibody
Authors: Kodama Takanari , Ichiyama Satoshi , Sato Kumiko , Nada Toshi , Nakashima Nobuo
Published: December 2019

A simple membrane immunoassay assay system, Quik Pack, for the detection of hepatitis C virus antibody was compared with two enzyme-linked immunosorbent assays (ELISAs) in a study of 600 serum samples. Quik Pack exhibited excellent sensitivity and specificity: 96.0 and 99.7%, respectively, versus the ELISA-2 and 99.7 and 99.4%, respectively, versus the ELISA-3.
Molecular Typing of Environmental and Patient Isolates of
Aspergillus fumigatus
from Various Hospital Settings
Authors: Chazalet Valérie , Debeaupuis Jean-Paul , Sarfati Jacqueline , Lortholary Jacques , Ribaud Patricia , Shah Pramod , Cornet Muriel , Thien Hoang Vu , Gluckman Eliane , Brücker Gilles , Latgé Jean-Paul
Published: December 2019

Fingerprinting of more than 700 clinical and environmental isolates of
Aspergillus fumigatus
from four differential hospital settings was undertaken with a dispersed repeated DNA sequence. The analysis of the environmental isolates showed that the airborne
A. fumigatus
population is extremely diverse, with 85% of the strains being represented as a single genotype isolated once. The remaining 15% of the strains were isolated several times and were able to persist for several months in the same hospital environment. No strains were found to be associated with a specific location inside the hospital, and identical strains were isolated from different buildings of the hospital and outdoors. Isolation of the same strain both from patients and from the environment of the same hospital is highly suggestive of a nosocomial infection. The characteristics of the environmental fungal population explains the two main results obtained from the typing of the clinical isolates: (i) the absence of a common strain responsible for an invasive aspergillosis outbreak results from the extreme diversity of the environmental population of
A. fumigatus
in contact with the patients, and (ii) patients hospitalized in different wards of the same hospital can be infected with the same strain since every patient might inhale the same spore population.
Production and Characterization ofEhrlichia risticii, the Agent of Potomac Horse Fever, from Snails (Pleuroceridae:Jugaspp.) in Aquarium Culture and Genetic Comparison to Equine Strains
Authors: Reubel Gerhard H. , Barlough Jeffrey E. , Madigan John E.
Published: December 2019

We report on the production and characterization ofEhrlichia risticii, the agent of Potomac horse fever (PHF), from snails (Pleuroceridae:Jugaspp.) maintained in aquarium culture and compare it genetically to equine strains. Snails were collected from stream waters on a pasture in Siskiyou County, Calif., where PHF is enzootic and were maintained for several weeks in freshwater aquaria in the laboratory. Upon exposure to temperatures above 22°C the snails released trematode cercariae tentatively identified as virgulate cercariae. Fragments of three different genes (genes for 16S rRNA, thegroESLheat shock operon, and the 51-kDa major antigen) were amplified from cercaria lysates by PCR and sequenced. Genetic information was also obtained fromE. risticiistrains from horses with PHF. The PCR positivity of snail secretions was associated with the presence of trematode cercariae. Sequence analysis of the three genes indicated that the source organism closely resembledE. risticii, and the sequences of all three genes were virtually identical to those of the genes of an equineE. risticiistrain from a property near the snail collection site. Phylogenetic analyses of the three genes indicated the presence of geographicalE. risticiistrain clusters.
Use of Tests for Acidification of Methyl-α-
d
-Glucopyranoside and Susceptibility to Efrotomycin for Differentiation of Strains of
Enterococcus
and Some Related Genera
Authors: Carvalho Maria Da Glória S. , Teixeira Lúcia M. , Facklam Richard R.
Published: December 2019

A total of 107
Enterococcus
strains, 10
Vagococcus fluvialis
strains, and 8
Lactococcus garvieae
strains were tested for acidification of methyl-α-
d
-glucopyranoside (MGP) and susceptibility to 100-μg efrotomycin (EFRO) disks. All 26 strains of
Enterococcus casseliflavus
, including 3 nonmotile and 2 nonpigmented strains, acidified MGP and were resistant to EFRO. All 22 strains of
Enterococcus gallinarum
, including 5 nonmotile strains, also acidified MGP and were resistant to EFRO. None of the 26 strains of
Enterococcus faecium
acidified MGP, and all were susceptible to EFRO. Although all 12
Enterococcus faecalis
strains were also negative in the MGP test, they were resistant to EFRO. Other enterococcal strains gave variable results. All 10 strains of
V. fluvialis
and all 8 strains of
L. garvieae
gave positive and negative results, respectively, in the MGP test and were, respectively, resistant and susceptible to EFRO. These results indicate that tests of the production of acid from MGP and susceptibility to EFRO can be used as adjunct tests in the identification of typical and atypical strains of enterococci in the clinical microbiology laboratory.
Rapid Extraction of Genomic DNA from Medically Important Yeasts and Filamentous Fungi by High-Speed Cell Disruption
Authors: Müller Frank-Michael C. , Werner Katherine E. , Kasai Miki , Francesconi Andrea , Chanock Stephen J. , Walsh Thomas J.
Published: December 2019

Current methods of DNA extraction from different fungal pathogens are often time-consuming and require the use of toxic chemicals. DNA isolation from some fungal organisms is difficult due to cell walls or capsules that are not readily susceptible to lysis. We therefore investigated a new and rapid DNA isolation method using high-speed cell disruption (HSCD) incorporating chaotropic reagents and lysing matrices in comparison to standard phenol-chloroform (PC) extraction protocols for isolation of DNA from three medically important yeasts (
Candida albicans
,
Cryptococcus neoformans
, and
Trichosporon beigelii
) and two filamentous fungi (
Aspergillus fumigatus
and
Fusarium solani
). Additional extractions by HSCD were performed on
Saccharomyces cerevisiae
,
Pseudallescheria boydii
, and
Rhizopus arrhizus
. Two different inocula (10
8
and 10
7
CFU) were compared for optimization of obtained yields. The entire extraction procedure was performed on as many as 12 samples within 1 h compared to 6 h for PC extraction. In comparison to the PC procedure, HSCD DNA extraction demonstrated significantly greater yields for 10
8
CFU of
C. albicans
,
T. beigelii
,
A. fumigatus
, and
F. solani
(
P
≤ 0.005), 10
7
CFU of
C. neoformans
(
P
≤ 0.05), and 10
7
CFU of
A. fumigatus
(
P
≤ 0.01). Yields were within the same range for 10
8
CFU of
C. neoformans
and 10
7
CFU of
C. albicans
for both HSCD extraction and PC extraction. For 10
7
CFU of
T. beigelii
, PC extraction resulted in a greater yield than did HSCD (
P
≤ 0.05). Yields obtained from 10
8
and 10
7
CFU were significantly greater for filamentous fungi than for yeasts by the HSCD extraction procedure (
P
< 0.0001). By the PC extraction procedure, differences were not significant. For all eight organisms, the rapid extraction procedure resulted in good yield, integrity, and quality of DNA as demonstrated by restriction fragment length polymorphism, PCR, and random amplified polymorphic DNA. We conclude that mechanical disruption of fungal cells by HSCD is a safe, rapid, and efficient procedure for extracting genomic DNA from medically important yeasts and especially from filamentous fungi.
Comparison of Culture and PCR for Detection of
Burkholderia cepacia
in Sputum Samples of Patients with Cystic Fibrosis
Authors: Whitby Paul W. , Dick Hani L. N. , Campbell Preston W. , Tullis D. Elizabeth , Matlow Anne , Stull Terrence L.
Published: December 2019

We investigated the utility of PCR to detect
Burkholderia cepacia
directly in sputum samples at two cystic fibrosis (CF) centers serving children and adults. Following liquefaction of the sputa by using
N
-acetyl-
l
-cysteine, DNA was isolated and analyzed by PCRs with three different primer pairs directed toward bacterial rRNA loci. Two primer pairs were putatively specific for
B. cepacia
. The other pair, which universally amplifies a band from all bacteria, served as a control. Sputum samples were obtained from 219 patients and analyzed independently by culture and by PCR to detect
B. cepacia
. The analyses were performed blinded with respect to each other. The results of the PCR with sputa demonstrated that the primers directed to the 16S loci demonstrated approximately 95% concordance with culture results and were more specific than those amplifying the 16S to 23S spacer region. In addition, the 16S primer pair putatively identified
B. cepacia
in seven patients whose sputa were culture negative at this time. Of these culture-negative patients, five had sputum samples that were culture positive for
B. cepacia
either prior or subsequent to this study. The results of this study indicate the utility of PCR as a diagnostic method for the rapid identification of
B. cepacia
in sputum samples of CF patients. We anticipate that improvements in our taxonomic understanding may allow the design of more specific primers for detection of each species of the
B. cepacia
complex in sputum samples.
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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Application of Competitive PCR to Cerebrospinal Fluid Samples from Patients with Herpes Simplex Encephalitis
Authors: Domingues R. B. , Lakeman F. D. , Mayo M. S. , Whitley R. J.
Published: December 2019

The purpose of the present study was to determine if the quantity of herpes simplex virus (HSV) DNA in the cerebrospinal fluid (CSF) of patients with herpes encephalitis would be useful in establishing the prognosis of the disease and to determine the effect of antiviral therapy on the clearance of viral DNA from the CSF. Quantitation of HSV DNA was done by constructing an internal standard (IS) from the glycoprotein B amplicon which had a 25-bp deletion between primer annealing sites. Each CSF specimen was coamplified with the IS and the ratio of the amount of HSV/amount of IS was compared to the ratios on a standard curve constructed with the same IS plus known amounts of HSV DNA. CSF specimens were available from 16 patients who were treated with intravenous acyclovir, and the amount of HSV DNA ranged from <25 to 18,000 copies per μl in CSF obtained before or within 4 days of the initiation of acyclovir therapy. Patients with >100 copies of HSV DNA per μl were older, were found by computed tomography to have lesions, and had poorer outcomes than patients with <100 copies. Follow-up CSF specimens were available from seven patients. In six of these seven patients, the HSV DNA levels decreased during therapy. One patient had a twofold increase in HSV DNA levels after 1 week of therapy and died on day 8. The application of this assay may be helpful in establishing the prognosis and in the monitoring of patients with herpes simplex encephalitis.
5′ Nuclease PCR Assay To Detect
Yersinia pestis
Authors: Higgins James A. , Ezzell John , Hinnebusch B. Joseph , Shipley Michelle , Henchal Erik A. , Ibrahim M. Sofi
Published: December 2019

The 5′ nuclease PCR assay uses a fluorescently labeled oligonucleotide probe (TaqMan) to rapidly detect and quantitate DNA templates in clinical samples. We developed a 5′ nuclease PCR assay targeting the plasminogen activator gene (
pla
) of
Yersinia pestis
. The assay is species specific, with a detection threshold of 2.1 × 10
5
copies of the
pla
target or 1.6 pg of total cell DNA. The assay detected
Y. pestis
in experimentally infected
Xenopsylla cheopis
fleas and in experimentally infected monkey blood and oropharyngeal swabs. The TaqMan assay is simple to perform and rapid and shows promise as a future field-adaptable technique.
Comparison of NNA Agar Culture and Selective Broth Culture for Detection of Group B Streptococcal Colonization in Women
Authors: Dunne W. Michael , Holland-Staley Carol A.
Published: December 2019

In 1996, the Centers for Disease Control and Prevention recommended the use of a selective broth culture for the improved detection of genital tract or anorectal carriage of group B streptococci (GBS) in pregnant women. In order to verify this recommendation in our laboratory, we compared the sensitivity of Todd-Hewitt medium with gentamicin and nalidixic acid (SBM) with our current method of direct plating on blood agar medium containing neomycin and nalidixic acid (NNA). Five hundred consecutive cervicovaginal and anorectal specimens submitted for GBS culture were included in the study. Swabs were plated onto NNA and the swabs were immersed in SBM, followed by overnight incubation at 35°C. On the following day, the NNA plates were examined for colonies typical of GBS and the organisms were identified by the CAMP test or by latex agglutination. SBM cultures were subcultured onto blood agar and CNA agar plates, and the plates were reincubated for 24 h. Negative specimens from either medium were incubated for an additional 24 h and were examined again before finalization of the results. GBS were recovered from 78 specimens by both methods; from SBM only for 17 specimens (sensitivity, 86%) and from NNA only for 16 specimens (sensitivity, 85%). A moderate to heavy growth of
Enterococcus faecalis
was observed on plates containing NNA-positive, SBM-negative specimens. Competitive growth studies suggested that
E. faecalis
suppressed the growth potential of GBS in SBM. Our study suggests that direct plating on NNA, as a single method, is equivalent in sensitivity to SBM for the recovery of GBS, and the results are often available 24 h sooner. However, it appears that both direct plating and selective broth amplification techniques are required for the maximum level of identification of colonization with GBS in pregnant women.
Evaluation of PCR, Culture, and Serology for Diagnosis of
Chlamydia pneumoniae
Respiratory Infections
Authors: Verkooyen R. P. , Willemse D. , Hiep-van Casteren S. C. A. M. , Mousavi Joulandan S. A. , Snijder R. J. , van den Bosch J. M. M. , van Helden H. P. T. , Peeters M. F. , Verbrugh H. A.
Published: December 2019

We prospectively studied 156 patients with a diagnosis of community-acquired pneumonia requiring admission. Several respiratory specimens were obtained for the detection of
Chlamydia pneumoniae
by cell culture and PCR. Three serum samples were obtained from each patient. Serological diagnosis of a
C. pneumoniae
infection was determined by the microimmunofluorescence (MIF) test, the complement fixation (CF) test, and recombinant lipopolysaccharide (LPS) enzyme-linked immunosorbent assay (ELISA; referred to as the rDNA LPS ELISA). Twenty-three patients (15%) had serological results compatible with acute
C. pneumoniae
infection; nine (39%) of these subjects were
C. pneumoniae
PCR positive. Twenty-two patients (14%) had positive PCR results without serological evidence of an acute
C. pneumoniae
infection. An attempt was made to calculate the sensitivities and specificities of the MIF test, rDNA LPS ELISA, and PCR for the diagnosis of chlamydial community-acquired pneumonia. Several “gold standards” were defined. Generally, the sensitivities of the rDNA LPS ELISA and MIF were comparable, while the sensitivity of the CF test was shown to be very low. Independent of the gold standard used, the best PCR results were obtained with nasopharyngeal specimens. However, the predictive value of a positive
C. pneumoniae
PCR result for patients with community-acquired pneumonia remains unknown and may be low. Although a widely accepted gold standard is still lacking, the rDNA LPS ELISA may currently be the preferred tool for diagnosing acute respiratory
Chlamydia
infections in routine clinical practice. However, the MIF test remains the method of choice for determining the prevalence of
C. pneumoniae
infections in a given community.