bluuebunny/crossref_metadata_2025 huggingface dataset:
Reverse Transcriptase PCR Amplification of
Rickettsia typhi
from Infected Mammalian Cells and Insect Vectors
Authors: Higgins James A. , Radulovic Suzana , Noden Bruce H. , Troyer Jill M. , Azad Abdu F.
Published: December 2019

We developed a reverse transcriptase PCR assay to detect expression of 120- and 17-kDa antigen genes in
Rickettsia typhi
. Infected Vero cell and flea RNAs were reverse transcribed by using random hexamers. The cDNA was amplified by using high concentrations of primer and template in an inexpensive, nonradioactive assay.
Characterization of IS
1245
for Strain Typing of
Mycobacterium avium
Authors: Pestel-Caron Martine , Arbeit Robert D.
Published: December 2019

IS
1245
is an insertion element widely prevalent among isolates of
Mycobacterium avium
. We used
Pvu
II Southern blots to analyze IS
1245
polymorphisms among 159
M. avium
isolates (141 clinical isolates from 40 human immunodeficiency virus-infected patients plus 18 epidemiologically related environmental isolates) that represented 40 distinct
M. avium
strains, as resolved by previous studies by pulsed-field gel electrophoresis (PFGE). All 40 strains carried DNA homologous to IS
1245
and thus were typeable. Twenty-five (63%) strains had ≥10 copies of the element, 6 (15%) had 4 to 9 copies, and 9 (23%) had only 1 to 3 copies. Among the last group of nine strains (each of which was distinct by PFGE analysis), IS
1245
typing resolved only four patterns and thus provided poor discriminatory power. To evaluate the in vivo stability of IS
1245
, we analyzed 32 strains for which sets of 2 to 19 epidemiologically related isolates were available. For 19 (59%) of these sets, all isolates representing the same strain had indistinguishable IS
1245
patterns. Within eight (25%) sets, one or more isolates had IS
1245
patterns that differed by one or two fragments from the modal pattern for the isolates of that strain. Five (16%) sets included isolates whose patterns differed by three or more fragments; on the basis of IS
1245
typing those isolates would have been designated distinct strains. IS
1245
was stable during in vitro passage, suggesting that the variations observed represented natural translocations of the element. IS
1245
provides a useful tool for molecular strain typing of
M. avium
but may have limitations for analyzing strains with low copy numbers or for resolving extended epidemiologic relationships.
Use of Multiple Competitors for Quantification of Human Immunodeficiency Virus Type 1 RNA in Plasma
Authors: Vener Tanya , Nygren Malin , Andersson AnnaLena , Uhlén Mathias , Albert Jan , Lundeberg Joakim
Published: December 2019

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma has rapidly become an important tool in basic HIV research and in the clinical care of infected individuals. Here, a quantitative HIV assay based on competitive reverse transcription-PCR with multiple competitors was developed. Four RNA competitors containing identical PCR primer binding sequences as the viral HIV-1 RNA target were constructed. One of the PCR primers was fluorescently labeled, which facilitated discrimination between the viral RNA and competitor amplicons by fragment analysis with conventional automated sequencers. The coamplification of known amounts of the RNA competitors provided the means to establish internal calibration curves for the individual reactions resulting in exclusion of tube-to-tube variations. Calibration curves were created from the peak areas, which were proportional to the starting amount of each competitor. The fluorescence detection format was expanded to provide a dynamic range of more than 5 log units. This quantitative assay allowed for reproducible analysis of samples containing as few as 40 viral copies of HIV-1 RNA per reaction. The within- and between-run coefficients of variation were <24% (range, 10 to 24) and <36% (range, 27 to 36), respectively. The high reproducibility (standard deviation, <0.13 log) of the overall procedure for quantification of HIV-1 RNA in plasma, including sample preparation, amplification, and detection variations, allowed reliable detection of a 0.5-log change in RNA viral load. The assay could be a useful tool for monitoring HIV-1 disease progression and antiviral treatment and can easily be adapted to the quantification of other pathogens.
Algorithmic Approach to High-Throughput Molecular Screening for Alpha Interferon-Resistant Genotypes in Hepatitis C Patients
Authors: Sreevatsan Srinand , Bookout Jack B. , Ringpis Fidel M. , Pottathil Mridula R. , Marshall David J. , De Arruda Monika , Murvine Christopher , Fors Lance , Pottathil Raveendran M. , Barathur Raj R.
Published: December 2019

This study was designed to analyze the feasibility and validity of using Cleavase Fragment Length Polymorphism (CFLP) analysis as an alternative to DNA sequencing for high-throughput screening of hepatitis C virus (HCV) genotypes in a high-volume molecular pathology laboratory setting. By using a 244-bp amplicon from the 5′ untranslated region of the HCV genome, 61 clinical samples received for HCV reverse transcription-PCR (RT-PCR) were genotyped by this method. The genotype frequencies assigned by the CFLP method were 44.3% for type 1a, 26.2% for 1b, 13.1% for type 2b, and 5% type 3a. The results obtained by nucleotide sequence analysis provided 100% concordance with those obtained by CFLP analysis at the major genotype level, with resolvable differences as to subtype designations for five samples. CFLP analysis-derived HCV genotype frequencies also concurred with the national estimates (N. N. Zein et al., Ann. Intern. Med. 125:634–639, 1996). Reanalysis of 42 of these samples in parallel in a different research laboratory reproduced the CFLP fingerprints for 100% of the samples. Similarly, the major subtype designations for 19 samples subjected to different incubation temperature-time conditions were also 100% reproducible. Comparative cost analysis for genotyping of HCV by line probe assay, CFLP analysis, and automated DNA sequencing indicated that the average cost per amplicon was lowest for CFLP analysis, at $20 (direct costs). On the basis of these findings we propose that CFLP analysis is a robust, sensitive, specific, and an economical method for large-scale screening of HCV-infected patients for alpha interferon-resistant HCV genotypes. The paper describes an algorithm that uses as a reflex test the RT-PCR-based qualitative screening of samples for HCV detection and also addresses genotypes that are ambiguous.
Effects of Different Test Conditions on MICs of Food Animal Growth-Promoting Antibacterial Agents for Enterococci
Authors: Butaye Patrick , Devriese Luc A. , Haesebrouck Freddy
Published: December 2019

The influence of the addition of sheep blood to Mueller-Hinton II agar and the effects of aerobic incubation with or without CO
2
and of anaerobic incubation were tested with bacitracin, tylosin, avoparcin, virginiamycin, avilamycin, narasin, and flavomycin on enterococci. The antibacterial activity of bambermycin (Flavomycin) was strongly inhibited by the addition of blood, except with the species
Enterococcus faecium
,
Enterococcus mundtii
,
Enterococcus hirae
,
Enterococcus casseliflavus
, and
Enterococcus gallinarum
, which were not susceptible to this antibiotic on blood-free medium. With all other antimicrobials except avoparcin and tylosin, the presence of blood resulted in MIC increases of 1 to 3 log
2
differences. Incubation in aerobic or anaerobic atmospheres enriched with CO
2
lowered the susceptibility of enterococci to tylosin and increased their susceptibility to avilamycin, narasin, and avoparcin. This effect was most pronounced in tests on blood-free media. Results of susceptibility tests incubated under anaerobiosis and in a CO
2
-enriched atmosphere did not differ. For all enterococcal species, the preferred conditions for testing the susceptibility are Mueller-Hinton II medium supplemented with blood and incubation in a CO
2
-enriched atmosphere. However, when only
E. faecium
and
Enterococcus faecalis
are being tested, Mueller-Hinton II medium without blood incubated aerobically gives satisfactory results.
Molecular Characterization of Vancomycin-Resistant Enterococci from Hospitalized Patients and Poultry Products in The Netherlands
Authors: van den Braak Nicole , van Belkum Alex , van Keulen Marrit , Vliegenthart John , Verbrugh Henri A. , Endtz Hubert P.
Published: December 2019

Vancomycin-resistant enterococci (VRE) pose an emerging health risk, but little is known about the precise epidemiology of the genes coding for vancomycin resistance. To determine whether the bacterial flora of consumer poultry serves as a gene reservoir, the level of contamination of poultry products with VRE was determined. VRE were genotyped by pulsed-field gel electrophoresis (PFGE), and transposon structure mapping was done by PCR. The
vanX-vanY
intergenic regions of several strains were further analyzed by sequencing. A total of 242 of 305 (79%) poultry products were found to be contaminated with VRE. Of these VRE, 142 (59%) were high-level-vancomycin-resistant
Enterococcus faecium
strains (VREF). PFGE revealed extensive VREF heterogeneity. Two genotypes were found nationwide on multiple occasions: type A (22 of 142 VREF [15%]) and type B (14 of 142 VREF [10%]). No PFGE-deduced genetic overlap was found when VREF from humans were compared with VREF from poultry. Two
vanA
transposon types were identified among poultry strains. In 59 of 142 (42%) of the poultry VREF, the size of the intergenic region between
vanX
and
vanY
was ∼1,300 bp. This transposon type was not found in human VREF. In contrast, all human strains and 83 of 142 (58%) of the poultry VREF contained an intergenic region 543 bp in size. Sequencing of this 543-bp intergenic
vanX-vanY
region demonstrated full sequence conservation. Though preliminary, these data suggest that dissemination of the resistance genes carried on transposable elements may be of greater importance than clonal dissemination of resistant strains. This observation is important for developing strategies to control the spread of glycopeptide resistance.
Survey of Incidence of
Clostridium difficile
Infection in Canadian Hospitals and Diagnostic Approaches
Authors: Alfa Michelle J. , Du Tim , Beda Gabi
Published: December 2019

A questionnaire relating to
Clostridium difficile
disease incidence and diagnostic practices was sent to 380 Canadian hospitals (all with >50 beds). The national questionnaire response rate was 63%. In-house testing was performed in 17.6, 61.5, and 74.2% of the hospitals with <300, 300 to 500, and >500 beds, respectively. The average test positivity rates were 17.2, 15.3, and 13.2% for hospitals with <300, 300 to 500, and >500 beds, respectively. The average disease incidences were 23.5, 30.8, and 40.3 cases per 100,000 patient days in the hospitals with <300, 300 to 500, and >500 beds, respectively. In the 81 hospitals where in-house testing was performed, cytotoxin testing utilizing tissue culture was most common (44.4%), followed by enzyme-linked immunosorbent assay (38.3%), culture for toxigenic
C. difficile
(32.1%), and latex agglutination (13.6%). The clinical criteria for
C. difficile
testing were variable, with 85% of hospitals indicating that a test was done automatically if ordered by a doctor. Our results show that
C. difficile
-associated diarrhea is a major problem in hospitals with ≥200 beds. Despite a lower disease incidence in smaller hospitals, there was a higher diagnostic test positivity rate. This may reflect the preference of smaller hospitals for culture and latex agglutination tests.
Coryneform Bacteria in Throat Cultures of Healthy Individuals
Authors: von Graevenitz Alexander , Pünter-Streit Verena , Riegel Philippe , Funke Guido
Published: December 2019

Throat swabs from 113 healthy individuals from Hamburg, Germany, and Zurich, Switzerland, were investigated for coryneform bacteria with nonselective and selective media. Ninety specimens contained 123 strains. Surprisingly, 76% of them were strains of
Corynebacterium durum
(47%) and
Rothia dentocariosa
(29%). Only two were strains of
Corynebacterium pseudodiphtheriticum
, and none were strains of
C. striatum
,
C. amycolatum
, or
C. diphtheriae.
Clonal Nature of Enterotoxigenic
Escherichia coli
Serotype O6:H16 Revealed by Randomly Amplified Polymorphic DNA Analysis
Authors: Pacheco Ana B. F. , Soares Kelen C. , de Almeida Darcy F. , Viboud Gloria I. , Binsztein Norma , Ferreira Luis C. S.
Published: December 2019

The genetic relatedness among 29 enterotoxigenic
Escherichia coli
strains of serotype O6:H16 was investigated by randomly amplified polymorphic DNA (RAPD) analysis. The strains were isolated in different parts of the world, displayed CS1-CS3 or CS2-CS3 profiles, and expressed heat-labile toxin (LT) and heat-stable toxin; a single strain expressed only LT. Ten RAPD types were distinguished and showed significant similarity, having on average 82% of the amplified bands in common. These results indicated that, irrespective of the different geographical origin or virulence factors, these strains belonged to a widespread clonal group.
Identification of Toxin A-Negative, Toxin B-Positive
Clostridium difficile
by PCR
Authors: Kato Haru , Kato Naoki , Watanabe Kunitomo , Iwai Naoichi , Nakamura Haruhi , Yamamoto Toshinobu , Suzuki Kanzo , Kim Shin-Moo , Chong Yunsop , Wasito Eddy Bagus
Published: December 2019

Toxigenic strains of
Clostridium difficile
have been reported to produce both toxins A and B nearly always, and nontoxigenic strains have been reported to produce neither of these toxins. Recent studies indicate that it is not always true. We established a PCR assay to differentiate toxin A-negative, toxin B-positive (toxin A−, toxin B+) strains from both toxin-positive (toxin A+, toxin B+) strains and both toxin-negative (toxin A−, toxin B−) strains as an alternative to cell culture assay and enzyme-linked immunosorbent assay (ELISA). By using the PCR primer set NK11 and NK9 derived from the repeating sequences of the toxin A gene, a shorter segment (ca. 700 bp) was amplified from toxin A−, toxin B+ strains compared to the size of the segment amplified from toxin A+, toxin B+ strains (ca. 1,200 bp), and no product was amplified from toxin A−, toxin B− strains. We examined a total of 421
C. difficile
isolates by PCR. Of these, 48 strains showed a shorter segment by the PCR, were negative by ELISAs for the detection of toxin A, and were positive by cell culture assay. Although the cytotoxin produced by the toxin A−, toxin B+ strains was neutralized by anti-toxin B serum, the appearance of the cytotoxic effects on Vero cell monolayers was distinguishable from that of toxin A+, toxin B+ strains. By immunoblotting, the 44 toxin A−, toxin B+ strains were typed to serogroup F and the remaining four strains were serogroup X. Pulsed-field gel electrophoresis separated the 48 strains into 19 types. The PCR assay for the detection of the repeating sequences combined with PCR amplification of the nonrepeating sequences of either the toxin A or the toxin B gene is indicated to be useful for differentiating toxin A−, toxin B+ strains from toxin A+, toxin B+ and toxin A−, toxin B− strains and will contribute to elucidation of the precise role of toxin A−, toxin B+ strains in intestinal diseases.
A Novel Toxinotyping Scheme and Correlation of Toxinotypes with Serogroups of
Clostridium difficile
Isolates
Authors: Rupnik Maja , Avesani Véronique , Janc Miha , von Eichel-Streiber Christoph , Delmée Michel
Published: December 2019

Two hundred nineteen
Clostridium difficile
isolates from 22 serogroups were screened for changes in the genes coding for toxin B (
tcdB
) and toxin A (
tcdA
). Parts of the toxin genes were amplified, and the PCR fragments were checked for length polymorphisms and cut with several restriction enzymes to monitor restriction fragment length polymorphisms (RFLPs). For 47 strains (21%), differences in the toxin genes were found compared to the toxin genes of reference strain VPI 10463. Polymorphisms were usually observed in both toxin genes. RFLPs were more commonly found in the
tcdB
gene, in which a single restriction enzyme could give up to five different patterns. Restriction sites seemed to be less heterogeneous in the
tcdA
gene, in which for most enzymes only two different RFLPs were recognized. However, deletions were observed in
tcdA
, and four new types of shortened
tcdA
genes are described. According to the changes in their toxin genes, variant strains could be divided into 10 groups (toxinotypes I to X). A toxinotype was characterized by similar patterns of changes in the toxin genes and in other regions of the pathogenicity locus and also similar pulsed-field gel electrophoresis patterns. Variant toxinotypes were found in 9 of the 22 serogroups studied, and some toxinotypes were clearly associated with specific serogroups. Toxinotype VIII is characteristic for all strains of serogroup F. Other serogroups in which variant toxinotypes were commonly found are A1, A15, E, and X. Testing of variability in
C. difficile
toxin genes not only might be useful as a molecular typing system but also could have implications in diagnostics and pathogenesis.
Evaluation of Mannitol Salt Agar for Detection of Oxacillin Resistance in
Staphylococcus aureus
by Disk Diffusion and Agar Screening
Authors: Kampf Günter , Lecke Christoph , Cimbal Ann-Katrin , Weist Klaus , Rüden Henning
Published: December 2019

Mannitol salt agar was evaluated for detection of oxacillin resistance in 136
Staphylococcus aureus
isolates. All
mecA
-positive isolates (
n
= 54) were correctly categorized as oxacillin resistant by the disk diffusion test (1-μg disk; zone diameter, <16 mm); the specificity was 97.6%. Agar screening (2 μg of oxacillin per ml) revealed a sensitivity of 98.1% and a specificity of 95.1%.
Microsatellite Markers for Typing
Aspergillus fumigatus
Isolates
Authors: Bart-Delabesse Emmanuelle , Humbert Jean-François , Delabesse Éric , Bretagne Stéphane
Published: December 2019

The use of microsatellites as highly polymorphic DNA markers for the typing of isolates of
Aspergillus fumigatus
was investigated. Four CA repeats were selected by screening an
A. fumigatus
DNA library with a (CA)
10
oligonucleotide. Primers flanking these CA repeats were designed to amplify each locus. One primer of each pair was labeled with a fluorophore, and the PCR products were analyzed with an automatic sequencer and the GeneScan software. For each primer set and for a given isolate, one band was detected and was assigned to an allele because
A. fumigatus
is haploid. With 50 clinical isolates, 50 environmental isolates, and 2 reference strains we obtained 12, 11, 10, and 23 different alleles for the four CA microsatellites, respectively (discriminatory power, 0.994). The results were identical by whatever DNA extraction technique was used. Interestingly, no clustering between environmental and clinical isolates was observed, suggesting that every isolate is potentially pathogenic. Microsatellite markers appear suitable for use in large epidemiological studies of invasive aspergillosis.
Rapid Identification of
Candida dubliniensis
by Indirect Immunofluorescence Based on Differential Localization of Antigens on
C. dubliniensis
Blastospores and
Candida albicans
Germ Tubes
Authors: Bikandi Joseba , Millán Rosario San , Moragues María D. , Cebas Gontzal , Clarke Mary , Coleman David C. , Sullivan Derek J. , Quindós Guillermo , Pontón José
Published: December 2019

There is a clear need for the development of a rapid and reliable test for the identification of
Candida dubliniensis
and for the discrimination of this species from
Candida albicans
. In the present study we have investigated the potential use of
C. dubliniensis
-specific antigens as a basis for its identification. We produced an anti-
C. dubliniensis
serum which, after adsorption with
C. albicans
blastospores, was found to differentially label
C. dubliniensis
isolates in an indirect immunofluorescence test. In this test, the antiserum reacted with blastospores and germ tubes of
C. dubliniensis
and with blastospores of
Candida krusei
and
Rhodotorula rubra
but did not react with blastospores of several other
Candida
species including
C. albicans
. The antiserum also reacted with
C. albicans
germ tubes. The anti-
C. dubliniensis
adsorbed serum reacted with specific components of 25, 28, 37, 40, 52, and 62 kDa in the
C. dubliniensis
extract and with a variety of antigens from other yeast species. The antigens from non-
C. dubliniensis
yeasts showing reactivity with the anti-
C. dubliniensis
adsorbed serum are mostly expressed within the cell walls of these yeast species, and this reactivity does not interfere with the use of the anti-
C. dubliniensis
adsorbed serum in an indirect immunofluorescence test for the rapid identification of
C. dubliniensis
.
Comparison of Amplified Ribosomal DNA Restriction Analysis, Random Amplified Polymorphic DNA Analysis, and Amplified Fragment Length Polymorphism Fingerprinting for Identification ofAcinetobacterGenomic Species and Typing ofAcinetobacter baumannii
Authors: Koeleman Johannes G. M. , Stoof Jeroen , Biesmans Dennis J. , Savelkoul Paul H. M. , Vandenbroucke-Grauls Christina M. J. E.
Published: December 2019

Thirty-one strains ofAcinetobacterspecies, including type strains of the 18 genomic species and 13 clinical isolates, were compared by amplified ribosomal DNA restriction analysis (ARDRA), random amplified polymorphic DNA analysis (RAPD), and amplified fragment length polymorphism (AFLP) fingerprinting. ARDRA, performed with five different enzymes, showed low discriminatory power for differentiatingAcinetobacterat the species and strain level. The standardized commercially available RAPD kit clearly enabled the discrimination of allAcinetobactergenomic species but showed great polymorphism between isolates ofAcinetobacter baumannii. AFLP fingerprinting with radioactively as well as fluorescently labelled primers showed high discriminatory power for the identification of 18Acinetobactergenomic species and typing of 13 clinicalAcinetobacterisolates. Compared to radioactive AFLP, fluorescent AFLP was technically fast and simple to perform, and it permitted analysis with an automated DNA sequencer. Fluorescent AFLP seems particularly well suited for studying the epidemiology of nosocomial infections and outbreaks caused byAcinetobacterspecies.
Development of rRNA-Targeted PCR and In Situ Hybridization with Fluorescently Labelled Oligonucleotides for Detection of
Yersinia
Species
Authors: Trebesius Karlheinz , Harmsen Dag , Rakin Alexander , Schmelz Jochen , Heesemann Jürgen
Published: December 2019

In this report, we present details of two rapid molecular detection techniques based on 16S and 23S rRNA sequence data to identify and differentiate
Yersinia
species from clinical and environmental sources. Near-full-length 16S rRNA gene (rDNA) sequences for three different
Yersinia
species and partial 23S rDNA sequences for three
Y. pestis
and three
Y. pseudotuberculosis
strains were determined. While 16S rDNA sequences of
Y. pestis
and
Y. pseudotuberculosis
were found to be identical, one base difference was identified within a highly variable region of 23S rDNA. The rDNA sequences were used to develop primers and fluorescently tagged oligonucleotide probes suitable for differential detection of
Yersinia
species by PCR and in situ hybridization, respectively. As few as 10
2
Yersinia
cells per ml could be detected by PCR with a seminested approach. Amplification with a subgenus-specific primer pair followed by a second PCR allowed differentiation of
Y. enterocolitica
biogroup 1B from biogroups 2 to 5 or from other pathogenic
Yersinia
species. Moreover, a set of oligonucleotide probes suitable for rapid (3-h) in situ detection and differentiation of the three pathogenic
Yersinia
species (in particular
Y. pestis
and
Y. pseudotuberculosis
) was developed. The applicability of this technique was demonstrated by detection of
Y. pestis
and
Y. pseudotuberculosis
in spiked throat and stool samples, respectively. These probes were also capable of identifying
Y. enterocolitica
within cryosections of experimentally infected mouse tissue by the use of confocal laser scanning microscopy.
Stable Phenotypic Resistance of
Candida
Species to Amphotericin B Conferred by Preexposure to Subinhibitory Levels of Azoles
Authors: Vazquez Jose A. , Arganoza Maria T. , Boikov Dina , Yoon Stephanie , Sobel Jack D. , Akins Robert A.
Published: December 2019

The fungicidal activity of amphotericin B (AmB) was quantitated for several
Candida
species.
Candida albicans
and
C. tropicalis
were consistently susceptible to AmB, with less than 1% survivors after 6 h of exposure to AmB.
C. parapsilosis
and variants of
C. lusitaniae
and
C. guilliermondii
were the most resistant, demonstrating 50 to 90% survivors in this time period and as high as 1% survival after a 24-h exposure time. All
Candida
species were killed (<1% survivors) after 24 h of exposure to AmB. In contrast, overnight exposure to either fluconazole or itraconazole resulted in pronounced increases in resistance to subsequent exposures to AmB. Most dramatically,
C. albicans
was able to grow in AmB cultures after azole preexposure. Several other
Candida
species did not grow in AmB but showed little or no reduction in viability after up to 24 h in AmB. Depending on the growth conditions,
Candida
cells preexposed to azoles may retain AmB resistance for days after the azoles have been removed. If this in vitro antagonism applies to the clinical setting, treatment of patients with certain antifungal combinations may not be beneficial. The ability of some
Candida
isolates to survive transient exposures to AmB was not reflected in the in vitro susceptibility changes as measured by standard MIC assays. This finding should be considered in studies attempting to correlate patient outcome with in vitro susceptibilities of clinical fungal isolates. Patients who fail to respond to AmB may be infected with isolates that are classified as susceptible by standard in vitro assays but that may be resistant to transient antifungal exposures which may be more relevant in the clinical setting.
Low False-Negative Rate of PCR Analysis for Detecting Human Papillomavirus-Related Cervical Lesions
Authors: Zazove Philip , Reed Barbara D. , Gregoire Lucie , Ferenczy Alex , Gorenflo Daniel W. , Lancaster Wayne D.
Published: December 2019

Although PCR analysis is a sensitive test for detection of human papillomavirus (HPV) in the cervix, the proportion of cases of cervical dysplasia missed, or the false-negative rate, has been unknown. We determined the accuracy of PCR analysis for HPV DNA as a predictor of HPV-related cervical lesions in a cross-sectional study of sexually active women, aged 18 to 50 years, from the University of Michigan Family Medicine HPV study. Of 133 eligible participants, 41 underwent colposcopy because of a positive result for HPV of the cervix by the PCR method and 92 underwent screening colposcopy with biopsy prior to knowing the HPV PCR results. Twenty-four of those screened were subsequently found to also be HPV DNA positive. In those found to be HPV positive, histological studies revealed the presence of condyloma or cervical intraepithelial neoplasia in 16 women (24.6%) and changes suggestive of condyloma in 5 (7.6%). No HPV-negative woman had an abnormal biopsy or cytology report (
P
= 0.000001). The false-negative rate (1 − sensitivity) for HPV PCR analysis for detection of the presence of a cervical HPV-related lesion was 0% (95% confidence interval, 0 to 0.047), and the specificity was 60.7%. In summary, PCR analysis for HPV DNA had a very low false-negative rate for predicting HPV-related lesions of the cervix in a community-based population. This supports the validity of using the absence of HPV at the cervix, as determined by PCR testing, as an inclusion criterion for patients in control groups in studies dealing with low-grade cervical lesions.
Detection of Rotavirus Types G8 and G10 among Brazilian Children with Diarrhea
Authors: Santos Norma , Lima Rita C. C. , Pereira Cláudio F. A. , Gouvea Vera
Published: December 2019

Characterization of 49 rotavirus-positive stool specimens from children with diarrhea in the state of Rio de Janeiro, Brazil, in 1996 and 1997 revealed a great diversity of rotavirus G types. Conventional types G1 and G3 accounted for 27 and 12% of the infections, respectively, whereas 60% of the infections were caused by unconventional types G5 (25%), G10 (16%), and G8 (4%) and mixed G types (16%).
Phenotypic and Genotypic Characterization of Virulent
Yersinia enterocolitica
Strains Unable To Ferment Sucrose
Authors: Guiyoule Annie , Guinet Françoise , Martin Liliane , Benoit Catherine , Desplaces Nicole , Carniel Elisabeth
Published: December 2019

Several atypical sucrose-negative
Yersinia
strains, isolated from clinical samples and sometimes associated with symptoms, proved to have full virulence potential in in vitro and in vivo testings. DNA-relatedness studies revealed that they were authentic
Yersinia enterocolitica
strains. Therefore, atypical sucrose-negative
Yersinia
isolates should be analyzed for their virulence potential.
Rapid Identification of Nine Microorganisms Causing Acute Respiratory Tract Infections by Single-Tube Multiplex Reverse Transcription-PCR: Feasibility Study
Authors: Gröndahl Britta , Puppe Wolfram , Hoppe Andrea , Kühne Inka , Weigl Josef A. I. , Schmitt Heinz-Josef
Published: December 2019

Acute respiratory tract infections (ARIs) are leading causes of morbidity and, in developing countries, mortality in children. A multiplex reverse transcription-PCR (RT-PCR) assay was developed to allow in one test the detection of nine different microorganisms (enterovirus, influenza A and B viruses, respiratory syncytial virus [RSV], parainfluenzaviruses type 1 and type 3, adenovirus,
Mycoplasma pneumoniae
, and
Chlamydia pneumoniae
) that do not usually colonize the respiratory tracts of humans but, if present, must be assumed to be the cause of respiratory disease. Clinical samples from 1,118 children admitted to the Department of Pediatrics because of an ARI between November 1995 and April 1998 were used for a first clinical evaluation. Detection of one of the microorganisms included in the assay was achieved for 395 of 1,118 (35%) clinical samples, of which 37.5% were RSV, 20% were influenza A virus, 12.9% were adenovirus, 10.6% were enterovirus, 8.1% were
M. pneumoniae
, 4.3% were parainfluenzavirus type 3, 3.5% were parainfluenzavirus type 1, 2.8% were influenza B virus, and 0.2% were
C. pneumoniae
. Seasonal variations in the rates of detection of the different organisms were observed, as was expected from the literature. The levels of concordance with the data obtained by commercially available enzyme immunoassays were 95% for RSV and 98% for influenza A. The results show that the multiplex RT-PCR–enzyme-linked immunosorbent assay is a useful and rapid diagnostic tool for the management of children with ARI. Studies of the overall benefit of this method with regard to the use of antibiotics, the use of diagnostic procedures including additional microbiological tests, and hospitalization rate and duration are warranted.
Comparison of PCR, Culture, and Serological Tests for Diagnosis of
Mycoplasma pneumoniae
Respiratory Tract Infection in Children
Authors: Dorigo-Zetsma J. W. , Zaat S. A. J. , Wertheim-van Dillen P. M. E. , Spanjaard L. , Rijntjes J. , van Waveren G. , Jensen J. S. , Angulo A. F. , Dankert J.
Published: December 2019

For diagnosis of
Mycoplasma pneumoniae
infection we compared two rapid tests, PCR and the immunoglobulin M immunofluorescence assay (IgM IFA), with culture and the complement fixation test (CFT), in a prospective study among 92 children with respiratory tract infection and 74 controls. Based on positivity of culture and/or CFT as the diagnostic criterion, nine patients (10%) were diagnosed with
M. pneumoniae
infection. All patients positive by culture were also positive by PCR. In all controls cultures, PCRs, and serological assays were negative, except in one with a positive IgM IFA. The IgM IFA had a low positive predictive value of 50%. Only a combination of PCR (seven patients) and CFT (seven patients) allowed diagnosis of all cases.
Detection of Penicillin Susceptibility in
Streptococcus pneumoniae
by
pbp2b
PCR-Restriction Fragment Length Polymorphism Analysis
Authors: O’Neill A. M. , Gillespie S. H. , Whiting G. C.
Published: December 2019

A PCR-restriction fragment length polymorphism strategy directed against the
pbp2b
gene was evaluated for identification of penicillin susceptibility. A total of 106 United Kingdom (U.K.), 30 Danish, and 11 Papua New Guinean strains were tested. Of the U.K. strains, all the susceptible and all but one of the resistant isolates were correctly assigned. By using conventional definitions of “not resistant” and “not susceptible,” the sensitivities were 97.5 and 94.4%, the specificities were 100 and 98.9%, the positive predictive values were 100 and 94.4%, and the negative predictive values were 93.1 and 98.9%, respectively. This technique may allow susceptible (MIC, <0.1 mg/liter) and resistant (MIC, >1 mg/liter) isolates to be distinguished in a single PCR.
Proficiencies of Three Anaerobic Culture Systems for Recovering Periodontal Pathogenic Bacteria
Authors: Doan Nguyen , Contreras Adolfo , Flynn Jane , Morrison John , Slots Jørgen
Published: December 2019

Anaerobic culture is employed routinely in the primary isolation of periodontal pathogenic bacteria. However, little or no data exist on the relative abilities of the Coy anaerobic chamber (Coy Laboratory Products, Grass Lake, Mich.), the GasPak (Becton Dickinson Microbiology Systems, Cockeysville, Md.), and the AnaeroPack (Mitsubishi Gas Chemical America, Inc., New York, N.Y.) systems to grow important periodontal species, including
Porphyromonas gingivalis
,
Prevotella intermedia/nigrescens
,
Bacteroides forsythus
,
Eubacterium
species,
Campylobacter
species,
Fusobacterium
species, and
Peptostreptococcus micros
. A total of 78 specimens from advanced periodontitis lesions were collected anaerobically, plated on enriched blood agar medium, and incubated at 35°C for 5 to 7 days in each anaerobic culture system. The three culture systems were equally efficient in isolating
Porphyromonas gingivalis
and
Prevotella intermedia/nigrescens
. The Coy anaerobic chamber yielded the highest proportional recoveries of
Campylobacter
(
P
= 0.0001; nonparametric analysis of variance) and
Eubacterium
(
P
= 0.009). The Coy anaerobic chamber and the GasPak system demonstrated higher proportional recoveries of
Bacteroides forsythus
(
P
= 0.0006) and
Peptostreptococcus micros
(
P
= 0.0001) than the AnaeroPack system. The AnaeroPack system was most efficient in growing
Fusobacterium
species (
P
= 0.0001). Overall, the Coy anaerobic chamber and the GasPak system showed the highest proportional recoveries of putative periodontal pathogens, but the recoveries by the various anaerobic test systems varied considerably from sample to sample.
Molecular Fine-Specificity Analysis of Antibody Responses to Human Cytomegalovirus and Design of Novel Synthetic-Peptide-Based Serodiagnostic Assays
Authors: Greijer Astrid E. , van de Crommert Jos M. G. , Stevens Servi J. C. , Middeldorp Jaap M.
Published: December 2019

To identify single immunodominant marker proteins which can replace complex virion antigen in serodiagnostic assays, we investigated in detail the molecular fine specificity of antibody responses in different individuals with latent or active human cytomegalovirus (HCMV) infection. An overview of the HCMV proteins recognized by human antibodies was obtained by immunoblotting. For selected immunodominant proteins the epitope fine specificity of the antibody response was determined by a peptide-scanning enzyme-linked immunosorbent assay (ELISA). Epitope clusters were synthesized as combination peptides and were used for further serologic analysis of immunoglobulin M (IgM) and IgG reactivities with panels of sera from different groups of patients in comparison to those with cytomegalovirus (CMV) virion antigen. Several serum samples had significantly higher reactivities with peptides than with the CMV virion antigen. However, individual serum samples occasionally recognized diverse peptide epitopes, stressing the importance of using combinations of peptides in serologic assays. From these studies we were able to define a specific combination of peptides derived from pp52 (UL44) and pp150 (UL32) for the specific and highly sensitive early detection of HCMV IgM, whereas a combination of peptides from pp150 (UL32), gB (UL55), and pp28 (UL99) was selected to give optimal and specific reactivity with HCMV IgG. On the basis of the results obtained with these peptide combinations, new, highly specific serodiagnostic assays were constructed. These assays had sensitivities of 98.9 and 96.4% for IgG and IgM, respectively, in comparison with the results obtained with the “gold standard,” the virion antigen-based ELISA. From the results of this study we conclude that specific combinations of highly defined synthetic peptides can replace complex HCMV virion extracts used in current serodiagnostics and may add to further standardization of HCMV serology.
Identity of Ehrlichial DNA Sequences Derived from
Ixodes ricinus
Ticks with Those Obtained from Patients with Human Granulocytic Ehrlichiosis in Slovenia
Authors: Petrovec M. , Sumner J. W. , Nicholson W. L. , Childs J. E. , Strle F. , Barlič J. , Lotrič-Furlan S. , AvšičŽupanc T.
Published: December 2019

Adult
Ixodes ricinus
(Acari: Ixodidae) ticks collected near Ljubljana, Slovenia, were tested for the agent of human granulocytic ehrlichiosis (HGE) by using PCR assays based on the 16S rRNA gene. Three (3.2%) of 93 ticks were found to contain granulocytic ehrlichiae. Nucleotide sequences of portions of the bacterial
groESL
heat shock operon amplified from these ticks were identical or nearly (99.8%) identical to those previously determined for human patients with HGE from Slovenia, providing additional evidence that the ticks were infected with the HGE agent. This study identified
I. ricinus
as the likely vector for these ehrlichial pathogens of humans in this part of Europe.
Improvement of Immunoglobulin M Capture Immunoassay Specificity: Toxoplasma Antibody Detection Method as a Model
Authors: Tuuminen Tamara , Seppänen Helena , Pitkänen Eeva-Marja , Palomäki Pekka , Käpyaho Kirsti
Published: December 2019

In the
Toxoplasma gondii
immunoglobulin M (IgM) capture fluorometric enzyme immunoassay used as a model, nonspecific responses due to the binding of human IgM to horseradish peroxidase (HRP) conjugates were observed despite the removal of the Fc portion of the immunoglobulin. This interaction may be mediated through the binding of human IgM to the HRP moiety of the conjugate. Addition of polymerized HRP into the reaction mixture reduced nonspecific signals in the majority of low false-positive serum reactions. Other plausible sites of interaction are conserved epitopes of mouse immunoglobulins presenting antigenic similarities with the allotopes of other species. Fragmentation of mouse antimicrobial IgG to Fab′ and selection of proper conjugation procedure improved assay specificity.
Multicenter Evaluation of the Abbott LCx
Mycobacterium tuberculosis
Ligase Chain Reaction Assay
Authors: Lumb Richard , Davies Kirsten , Dawson David , Gibb Robert , Gottlieb Thomas , Kershaw Clair , Kociuba Katherine , Nimmo Graeme , Sangster Norma , Worthington Michele , Bastian Ivan
Published: December 2019

Four Australian hospital laboratories evaluated the performance of the Abbott LCx
Mycobacterium tuberculosis
assay with 2,347 specimens (2,083 respiratory and 264 nonrespiratory specimens) obtained from 1,411 patients. A total of 152 specimens (6.5%) were culture positive for
Mycobacterium tuberculosis
complex (MTBC); of these, 79 (52%) were smear positive. After resolution of discrepant data, the overall sensitivity, specificity, and positive and negative predictive values for the LCx assay were 69.7, 99.9, 99.1, and 97.7% respectively. For smear-positive respiratory specimens that were culture positive for MTBC, the values were 98.5, 100, 100, and 98.4%, respectively, while the values for smear-negative respiratory specimens were 41.5, 99.9, 96.4, and 98%, respectively. Relative operating characteristic curves were constructed to demonstrate the relationship between sensitivity and specificity for a range of possible cutoff values in the LCx assay. These graphs suggested that the assay sensitivity for respiratory samples could be increased from 70.2 to 78.6%, while the specificity would be reduced from 99.9 to 99.4% by inclusion of a grey zone (i.e., LCx assay values of between 0.2 and 0.99). An algorithm is presented for the handling of specimens with LCx assay values within this grey zone.
Application of Infrequent-Restriction-Site PCR to Clinical Isolates of
Acinetobacter baumannii
and
Serratia marcescens
Authors: Yoo Jin-Hong , Choi Jung-Hyun , Shin Wan-Shik , Huh Dong-Ho , Cho Yoon-Kyung , Kim Kyung-Mi , Kim Mi-Young , Kang Moon-Won
Published: December 2019

We applied infrequent-restriction-site PCR (IRS-PCR) to the investigation of an outbreak caused by 23 isolates of
Acinetobacter baumannii
in an intensive care unit from November 1996 to May 1997 and a pseudoepidemic caused by 16 isolates of
Serratia marcescens
in a delivery room from May to September 1996. In the epidemiologic investigation of the outbreak caused by
A. baumannii
, environmental sampling and screening of all health care workers revealed the same species from the Y piece of a mechanical ventilator and the hands of two health care personnel. IRS-PCR showed that all outbreak-related strains were genotypically identical and that three strains from surveillance cultures were also identical to the outbreak-related strains. In a pseudoepidemic caused by
S. marcescens
, IRS-PCR identified two different genotypes, and among them one genotype was predominant (15 of 16 [93.8%] isolates). Extensive surveillance failed to find any source of
S. marcescens
. Validation of the result of IRS-PCR by comparison with that of field inversion gel electrophoresis (FIGE) showed that they were completely concordant. These results suggest that IRS-PCR is comparable to FIGE for molecular epidemiologic studies. In addition, IRS-PCR was less laborious and less time-consuming than FIGE. To our knowledge, this is the first report of the application of IRS-PCR to
A. baumannii
and
S. marcescens
.
Genetic Characterization of
Cryptosporidium
Strains from 218 Patients with Diarrhea Diagnosed as Having Sporadic Cryptosporidiosis
Authors: McLauchlin J. , Pedraza-Díaz S. , Amar-Hoetzeneder C. , Nichols G. L.
Published: December 2019

Samples of whole feces in which
Cryptosporidium
oocysts were recognized by hospital laboratories were collected from 218 patients with diarrhea. All samples were reexamined by light microscopy, and oocysts were detected in 211 samples. A simple and rapid procedure for the extraction of DNA from whole feces was developed, and this was used to amplify fragments of the
Cryptosporidium
outer wall protein (COWP), the thrombospondin-related adhesive protein C1 (TRAP-C1), and the 18S rRNA genes by PCR. For seven samples oocysts were not detected by microscopy and DNA failed to be amplified by the three PCR procedures. Among the 211 samples “positive” by microscopy, the sensitivities of PCRs for the 18S rRNA, COWP, and TRAP-C1 gene fragments were 97, 91, and 66%, respectively. The sensitivities of all three PCR procedures increased with increasing numbers of oocysts as observed by microscopy. Two genotypes of the COWP and TRAP-C1 genes can be detected by PCR-restriction fragment length polymorphism analysis. With this series of samples, the same genotypes of the COWP and TRAP-C1 genes always segregated together. A combined genotyping data set was produced for isolates from 194 samples: 74 (38%) were genotype 1 and 120 (62%) were genotype 2. Genotype 2 was detected in a significantly greater proportion of the samples with small numbers of oocysts, and genotype 1 was detected in a significantly greater proportion of the samples with larger numbers of oocysts. There were no significant differences in the distribution of the genotypes by patient sex and age. The distribution of the genotypes was significantly different both in patients with a history of foreign travel and in those from different regions in England.
Multicenter Laboratory Validation of Susceptibility Testing of
Mycobacterium tuberculosis
against Classical Second-Line and Newer Antimicrobial Drugs by Using the Radiometric BACTEC 460 Technique and the Proportion Method with Solid Media
Authors: Pfyffer Gaby E. , Bonato Donald A. , Ebrahimzadeh Adeleh , Gross Wendy , Hotaling Jacqueline , Kornblum John , Laszlo Adalbert , Roberts Glenn , Salfinger Max , Wittwer Franziska , Siddiqi Salman
Published: December 2019

In a large multicenter study involving six major study sites in the United States, Canada, and Europe, the susceptibilities of 272
Mycobacterium tuberculosis
strains to classical second-line antituberculosis (anti-TB) drugs (capreomycin, cycloserine, ethionamide, and kanamycin) and newer compounds (amikacin, clofazimine, ofloxacin, and rifabutin) were determined by the radiometric BACTEC 460 procedure and the conventional proportion method on Middlebrook 7H10 agar. Previously established critical concentrations for classical second-line anti-TB drugs were compared with several concentrations in liquid medium to establish equivalence. MICs of newer compounds determined in liquid medium were either the same or up to four times lower than those determined in agar medium. After establishing critical concentrations (breakpoints) in the extended testing of clinical isolates, we obtained an excellent overall correlation between the two systems, with no errors with amikacin, kanamycin, and ofloxacin and very few major or very major errors with the other drugs; however, for cycloserine, no breakpoint concentration could be recommended due to repeatedly inconsistent results by both methods. Based on these data we conclude that the BACTEC 460 procedure is a simple and rapid method requiring 4 to 8 days on average to generate accurate antimicrobial susceptibility testing (AST) results for eight anti-TB drugs other than those considered primary ones. These data not only fill a major gap of knowledge regarding the critical test concentrations of secondary anti-TB drugs but also provide a baseline for future evaluations of
M. tuberculosis
AST with the more recently developed, nonradiometric broth-based culture systems.