bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of Roche Amplicor PCR assay for Mycobacterium tuberculosis
Authors: Wobeser W L , Krajden M , Conly J , Simpson H , Yim B , D'costa M , Fuksa M , Hian-Cheong C , Patterson M , Phillips A , Bannatyne R , Haddad A , Brunton J L , Krajden S
Published: January 2020

The Roche Amplicor Mycobacterium tuberculosis PCR test (RMtb-PCR) was compared with mycobacterial culture, with the BACTEC 460 system and inoculation on Lowenstein-Jensen media. Results were interpreted with an adjusted "gold standard" incorporating clinical diagnosis. A total of 1,480 clinical specimens from 1,155 patients, including tissues and fluids, as well as 141 specimens which demonstrated a positive growth index on the BACTEC 460 system were assessed. The sensitivity, specificity, and positive and negative predictive values of RMtb-PCR compared with the adjusted gold standard for clinical specimens were 79, 99, 93, and 98%, respectively. In smear-positive specimens, the sensitivity of RMtb-PCR was 98% versus 53% for smear-negative specimens. When RMtb-PCR was performed two times per week, PCR results were available an average of 21 days before the culture results. For specimens demonstrating a positive growth index on the BACTEC 460 system, RMtb-PCR had a sensitivity and specificity of 98 and 100%, respectively. This study demonstrates the value of a commercial nucleic acid amplification kit for rapid diagnosis of M. tuberculosis, particularly in smear-positive specimens or BACTEC culture-positive specimens.
Diagnosis of viral respiratory tract infections in children by using a reverse transcription-PCR panel
Authors: Gilbert L L , Dakhama A , Bone B M , Thomas E E , Hegele R G
Published: January 2020

Reverse transcription-PCR (RT-PCR) is a sensitive method for detection of RNA virus nucleic acid sequences in clinical respiratory specimens. Previous studies have focused on RT-PCR for a single virus, but this approach is limited by the inability to establish a specific etiology when the RT-PCR result is negative and by the inability to document simultaneous infections involving more than one virus. The purpose of this study was to apply a panel of RT-PCR protocols for respiratory syncytial virus, parainfluenza virus, and picornaviruses to respiratory specimens from 80 children suspected to have acute viral respiratory tract infections and to correlate RT-PCR results with viral culture results and clinical diagnosis. In comparison with viral culture, the RT-PCR panel had a sensitivity of over 94% and showed evidence of simultaneous infections in a significantly greater proportion of specimens (20.0% versus 3.8%; P < 0.002). For specimens in which no viruses were detected by culture, the proportion of specimens with positive picornavirus RT-PCR results was significantly greater than the proportion of specimens with positive respiratory syncytial virus or parainfluenza virus RT-PCR results (P < 0.001). There were no statistically significant associations between RT-PCR results and clinical diagnosis. In summary, the RT-PCR panel provides an improved approach to obtain new insights into acute viral respiratory tract infections in children.
Comparison of ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis for molecular typing of Listeria monocytogenes
Authors: Louie M , Jayaratne P , Luchsinger I , Devenish J , Yao J , Schlech W , Simor A
Published: January 2020

Fifty-one clinical isolates of Listeria monocytogenes (15 isolates from two outbreaks and 36 epidemiologically unrelated isolates) were typed by conventional serotyping, ribotyping (RT), pulsed-field gel electrophoresis (PFGE), and arbitrarily primed PCR (AP-PCR). Serotyping was unable to distinguish between related and unrelated strains of L. monocytogenes. Each of the three molecular methods showed excellent typeability and reproducibility. Restriction with EcoRI and PvuII gave 16 and 23 RT patterns, respectively. Restriction with ApaI or SmaI generated 22 and 26 PFGE profiles, respectively. ApaI profiles were easier to interpret, with 10 to 15 bands each, while SmaI profiles had 15 to 20 bands each. AP-PCR with two different primers yielded 29 and 31 randomly amplified polymorphic DNA patterns, respectively. Strains from the same outbreak shared concordant patterns by each of the three methods. Of the three techniques evaluated, RT was the least discriminating and could not distinguish between strains from the two outbreaks. The abilities of AP-PCR and PFGE to differentiate between strains were comparable. However, AP-PCR was more rapid and easier to perform. We conclude that the DNA profiles generated by either AP-PCR or PFGE can be used to differentiate outbreak strains from epidemiologically unrelated strains and to clearly identify unrelated strains as being distinct from one another. We recommend that at least two independent primers be used for AP-PCR typing in order to improve its discriminatory power.
Comparison of spiral gradient endpoint and agar dilution methods for susceptibility testing of anaerobic bacteria: a multilaboratory collaborative evaluation
Authors: Wexler H M , Molitoris E , Murray P R , Washington J , Zabransky R J , Edelstein P H , Finegold S M
Published: January 2020

A multilaboratory collaborative study was carried out to assess the utility of the spiral gradient endpoint (SGE) method for the determination of the antimicrobial susceptibilities of anaerobes and to evaluate the equivalence of the MICs obtained by the SGE method with those obtained by the reference agar dilution method of the National Committee for Clinical Laboratory Standards. The standard deviation of the MIC obtained by the SGE method for the five participating laboratories was +/- 0.26 of a twofold dilution, whereas it was +/- 1 twofold dilution by the reference method. The interlaboratory reproducibility of the results for two control strains tested with imipenem, chloramphenicol, and metronidazole indicated that 96% of the measurements fell within +/- 1 twofold dilution of the mode. The equivalence of the SGE method with the agar dilution method was assessed with a wide variety of anaerobic organisms. The MICs by both methods were within 1 doubling dilution in 93% of the measurements (n = 1,074). Discrepancies generally occurred with those organism-drug combinations that resulted in tailing endpoints (Fusobacterium nucleatum, 86% agreement) or in cases of light growth (Peptostreptococcus spp., 86% agreement).
Natamycin as a selective antifungal agent in media for growth of Legionella spp
Authors: Edelstein P H , Edelstein M A
Published: January 2020

The growth of 18 different Legionella sp. strains and 76 different yeast isolates was tested on buffered charcoal yeast extract medium supplemented with alpha-ketoglutarate (BCYE alpha medium) and with natamycin, an antifungal agent. Bacterial growth was no different on BCYE alpha medium made with or without natamycin, whereas complete inhibition of yeasts occurred in BCYE alpha medium containing 200 to 500 micrograms of natamycin per ml. Selective BCYE alpha media made with natamycin rather than anisomycin had no (formulation with vancomycin, polymyxin B, and agar) or little (formulation with cefamandole, polymyxin B, and agar) inhibitory effect on the growth of 14 different Legionella sp. bacteria. Natamycin is an inexpensive alternative to anisomycin in the formulation of selective BCYE alpha media.
Comparison of ribotyping and genome fingerprinting of Pseudomonas aeruginosa isolates from cystic fibrosis patients
Authors: Bennekov T , Colding H , Ojeniyi B , Bentzon M W , Høiby N
Published: January 2020

Forty Pseudomonas aeruginosa strains, previously characterized by pulsed-field gel electrophoresis, were ribotyped with EcoRI, BamHI, ClaI, and PvuII. Ribotyping with PvuII proved to be as discriminatory as pulsed-field gel electrophoresis with XbaI or DraI while EcoRI and BamHI were not. ClaI contributed further ribotypes, some of which might be due to a transposable element.
Diagnosis of parvovirus B19 infection by detection of specific immunoglobulin M antibody in saliva
Authors: Cubel R C , Oliveira S A , Brown D W , Cohen B J , Nascimento J P
Published: January 2020

Serum and saliva samples were simultaneously collected from patients with B19 infection. Specimens were collected in a period of 1 to 18 days after the onset of symptoms. Saliva samples were collected with a commercial device, OraSure. The quality of these samples was evaluated by determining the concentration of total immunoglobulin G (IgG) by an enzyme immunoassay. The concentration of IgG in these samples ranged from 4.8 to > 250 mg/liter. B19 infection was confirmed for 20 patients by testing sera in a 1: 100 dilution by an IgM capture enzyme immunoassay (MACEIA) and an IgM capture hemadherence test (MACHAT). Saliva samples from these IgM-positive patients were tested neat by MACEIA and MACHAT. IgM could be detected in 11 of 20 (55%) samples by MACEIA and in 15 of 18 (83%) samples by MACHAT. Serum and saliva samples from a further 17 patients with rash were also tested. All of these specimens were unreactive by both assays. These results show that saliva may be a convenient alternative to serum for the diagnosis of recent B19 infection.
Molecular typing by random amplification of polymorphic DNA and M13 southern hybridization of related paired isolates of Aspergillus fumigatus
Authors: Anderson M J , Gull K , Denning D W
Published: January 2020

Three forms of DNA-based typing procedures for Aspergillus fumigatus isolates have been developed over the last five years. The procedures are random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) detection, and Southern hybridizations with various repetitive sequence-based probes. Using two of these procedures, we compared 16 selected isolates, grouped into eight pairs on the basis of epidemiology or previously assigned RFLP types. RAPD with four primers (R108, RC08, 2, and 4), including three previously used with A. fumigatus, showed that one primer, R108, gave the best discrimination (8 types). Southern hybridization of total genomic DNA digested with HindIII and probed with the total bacteriophage M13 genome resulted in the highest overall level of discrimination. Combination of the RAPD and Southern hybridization with the previously assigned RFLP types discriminated 10 isolates of 16. Isolates closely linked epidemiologically could not be distinguished from each other. In addition, three pairs of isolates previously unlinked by epidemiology had the same overall types. Two pairs were obtained from the same hospital within 2 years of each other, whereas the third pair were isolated from California and Germany. A full understanding of the epidemiology and ecology of A. fumigatus requires multiple discriminatory typing procedures.
DNA fingerprinting by infrequent-restriction-site amplification
Authors: Mazurek G H , Reddy V , Marston B J , Haas W H , Crawford J T
Published: January 2020

Identification of bacterial strains by DNA fingerprinting facilitates epidemiologic studies and improves disease control. For some species of organisms, no typing method is available; for others, typing methods are tedious. We developed a method of amplifying DNA sequences flanking infrequent restriction sites by PCR and used the method to produce strain-specific electrophoretic patterns from crude bacterial lysates. This method of fingerprinting is rapid, sensitive, and widely applicable. Identical enzymes, adaptors, primers, and PCR conditions were used to characterize 32 Mycobacterium avium-M. intracellulare isolates, 4 Pseudomonas aeruginosa isolates, and 4 Staphylococcus aureus isolates.
Comparison of fluorescent BACTEC 9000 MB system, Septi-Chek AFB system, and Lowenstein-Jensen medium for detection of mycobacteria
Authors: van Griethuysen A J , Jansz A R , Buiting A G
Published: January 2020

The newly developed fluorescent BACTEC 9000 MB system for automated culture of mycobacteria was compared with the Septi-Chek AFB system and Lowenstein-Jensen medium (LJ). A total of 2,005 clinical specimens were included in the study. Mycobacteria were isolated from 202 (10.1%) specimens, including 155 Mycobacterium tuberculosis complex isolates and 47 Mycobacteria other than M. tuberculosis isolates. Of 131 isolates detected by the BACTEC system, the Septi-Chek AFB system, or both, 120 (91.6%) were detected by the BACTEC system and 105 (80.2%) were detected by the Septi-Chek AFB system (P < 0.02). The recovery rate in the BACTEC system compared with that in the Septi-Chek AFB system was significantly higher for M. tuberculosis complex isolates (P < 0.005) and for isolates from acid-fast smear-negative specimens (P < 0.01). Of 148 isolates detected by the BACTEC system, LJ, or both, 142 (95.9%) were detected by the BACTEC system and 118 (79.9%) were detected by LJ (P < 0.001). The recovery rate in the BACTEC system compared with that on LJ was significantly higher for M. tuberculosis complex isolates (P < 0.001). The BACTEC system detected more mycobacteria from both smear-positive and smear-negative specimens than LJ. The mean times to detection of mycobacteria were 17.6 days for the BACTEC system, 26.0 days for the Septi-Chek AFB system, and 29.4 days for LJ. The BACTEC fluorescent 9000 MB system is a rapid, sensitive, and efficient method for the isolation of mycobacteria.
Acinetobacter species identification by using tRNA spacer fingerprinting
Authors: Ehrenstein B , Bernards A T , Dijkshoorn L , Gerner-Smidt P , Towner K J , Bouvet P J , Daschner F D , Grundmann H
Published: January 2020

Identification of Acinetobacter spp. to the DNA group level by phenotypic techniques is problematic, and there is a need for an alternative identification method for routine use. The present study validated the suitability of a rapid identification technique based on tRNA spacer (tDNA) fingerprinting in comparison with that of a commercially available assay involving carbon source utilization tests (Biolog MicroStation System) for identifying the 21 DNA-DNA hybridization groups belonging to the genus. For this purpose, 128 strains identified previously by DNA-DNA hybridization were analyzed by both techniques. tDNA fingerprinting was highly reproducible and classified all strains into 17 groups. Six DNA groups belonging to the A. calcoaceticus-A. baumannii complex were grouped into two distinct clusters, indicating the high degree of genetic similarity within this complex. Strains of the more recently described DNA groups BJ13 to BJ16 were ambiguously grouped and displayed three pattern types. The software used with the commercial carbon source utilization method grouped the 128 strains into 12 clusters, explaining the less discriminatory power of this system. We conclude that tDNA fingerprinting offers a quick and reliable method for the routine differentiation of most Acinetobacter spp. at the subgenus level.
Detection of Neisseria gonorrhoeae from air-dried genital samples by single-tube nested PCR
Authors: Herrmann B , Nyström T , Wessel H
Published: January 2020

A single-tube nested PCR method was developed for the detection of Neisseria gonorrhoeae. The optimized assay had a detection limit of less than 0.3 cell. Five different storage conditions for gonococcal specimens were compared with respect to the PCR detection of bacteria. For air-dried gonococcal slides containing three bacteria, DNA was detected after 8 weeks at ambient temperature, and for slides containing 300 bacteria, DNA could be detected after 24 weeks at ambient temperature. Air-dried storage combined with analysis by the single-tube nested PCR and a commercially available PCR (Amplicor) was used to test 350 cervical specimens from women in the West African island nation of Cape Verde. The in-house PCR detected 17 cases of N. gonorrhoeae infection, while the Amplicor system detected 14 cases of N. gonorrhoeae infection. No specimen was negative by the in-house PCR assay and positive by the Amplicor PCR. This sensitive nested PCR assay, combined with air-dried storage, allows for the detection of gonococci when specimen storage and transport times are extended and freezing conditions are not available.
Surrogate disks for predicting cefotaxime and ceftriaxone susceptibilities of Streptococcus pneumoniae
Authors: Barry A L , Fuchs P C
Published: January 2020

Cefotaxime- and ceftriaxone-resistant Streptococcus pneumoniae is now appearing in some medical centers, but 30-micrograms cefotaxime or 30-micrograms ceftriaxone disks are not reliable for detecting such strains. Studies were undertaken to select another cephalosporin disk that might be used as a screening test that could be used in conjunction with a 1-micrograms oxacillin disk. A 30-micrograms cefuroxime disk is proposed: strains with zones > or = 28 mm in diameter are predictably susceptible to cefotaxime and ceftriaxone, and those with smaller zones should be further studied to confirm resistance to either drug. A 30-micrograms ceftizoxime disk may also be used as a screening test with zones > or = 26 mm indicating susceptibility, but cefuroxime disks are preferred.
Reverse transcription-PCR detection of hepatitis G virus
Authors: Schlueter V , Schmolke S , Stark K , Hess G , Ofenloch-Haehnle B , Engel A M
Published: January 2020

Hepatitis G virus (HGV) was recently identified as a new member of the Flaviviridae, but its clinical significance is still unclear. Since no immunoassay for the diagnosis of HGV is available, we developed a sensitive reverse transcription-PCR (RT-PCR) assay to facilitate the detection of the viral genome by mass screening in the clinical laboratory. Sequences within the 5'-noncoding region and within the putative NS5a region are independently amplified in the presence of digoxigenin-11-dUTP and are detected by hybridization with biotinylated capture probes binding to a streptavidin-coated matrix. Semiquantitative Enzymun-Test DNA detection via chemiluminescence can be performed either in a microtiter plate format or on fully automated ES 300 machines. We were able to detect at least 8 x 10(2) genome equivalents per ml of serum using both primer pairs. HGV was shown to be present in 43 of 130 (33%) serum samples from intravenous drug abusers with a high risk of parenteral exposure. However, only two of the patients were positive when the NS5a primers only were used, and only one patient was positive when only the 5'-noncoding region primers were used, demonstrating the increased sensitivity of HGV detection with two sets of primers. Among these patients, there was no obvious correlation with other viral infections like hepatitis B virus, hepatitis C virus, or human immunodeficiency virus. Within a blood donor panel, 3 of 92 (3%) samples were found to be HGV positive, suggesting that donated blood may need to be screened for HGV.
Amplification and restriction endonuclease digestion of a large fragment of genes coding for rRNA as a rapid method for discrimination of closely related pathogenic bacteria
Authors: Ibrahim A , Gerner-Smidt P , Sjöstedt A
Published: January 2020

By use of primers specific to conserved regions of the rRNA gene cluster, a discrete amplicon of approximately 5 kb was amplified by PCR from all 21 bacterial genera investigated. Subsequent endonuclease digestion of the PCR product with HaeIII distinguished between the three species of the human pathogen Francisella spp. on the one hand and four clinically relevant genomic groups of Acinetobacter spp. on the other hand. The described technique has the potential as a rapid method for discriminating between closely related species that are of clinical importance.
Epidemiology of Burkholderia cepacia infection in patients with cystic fibrosis: analysis by randomly amplified polymorphic DNA fingerprinting
Authors: Mahenthiralingam E , Campbell M E , Henry D A , Speert D P
Published: January 2020

We fingerprinted a collection of 627 Burkholderia cepacia isolates from 255 patients with cystic fibrosis (CF) and 43 patients without CF and from the environment, by a PCR-based randomly amplified polymorphic DNA (RAPD) method with primers selected for their ability to produce discriminatory polymorphisms. The RAPD typing method was found to be reproducible and discriminatory, more sensitive than PCR ribotyping, and able to group epidemiologically related B. cepacia strains previously typed by both pulsed-field gel electrophoresis and conventional ribotyping. Seven strain types infecting multiple CF patients were found at several different CF treatment centers in Canada, the United States, the United Kingdom, France, and Australia, indicating the presence of epidemic strain types. Most CF patients were each colonized with a single strain type, and several patients harbored the same strain type for 5 or more years. B. cepacia isolates recovered from other clinical sources (44 isolates examined) and from the environment (58 isolates examined) possessed RAPD fingerprints that were generally distinct from CF-associated strain types (525 isolates examined). RAPD is a versatile fingerprinting method for studying the epidemiology of B. cepacia.
Enhanced isolation of Serpulina hyodysenteriae by using sliced agar media
Authors: Olson L D
Published: January 2020

A method has been developed for separating Serpulina hyodysenteriae, a large spirochete and the causative agent of swine dysentery (SD), from other fecal anaerobic bacteria in rectal and colonic swabs. This was done by cutting the blood agar in parallel cuts and streaking perpendicular to the cuts in the center of the petri dish. Migration of S. hyodysenteriae from the central streak was apparent by the presence of strong beta-hemolysis along the edges of the cuts. If only S. hyodysenteriae migrated in the cut, they migrated to the end of the cut. However, if both motile bacteria and S. hyodysenteriae migrated in the cut, the motile bacteria migrated to the end of the cut where they formed colonies and the S. hyodysenteriae located along the edges of the cut between the colonies of motile bacteria and the central streak. Although motile bacteria were present where S. hyodysenteriae located, the growth of the motile bacteria was partially inhibited since they rarely formed visible colonies and were low in number. The cut in the agar was thought to improve traction for the serpentine movement of the S. hyodysenteriae and for the flagellar movement of the motile bacteria. Use of sliced blood agar was superior to conventionally streaked blood agar in that (i) it was easier to see strong beta-hemolysis on sliced agar; (ii) frequently, a confirmatory diagnosis could be made using only one petri dish with sliced agar, thereby saving time and media; (iii) S. hyodysenteriae could sometimes be isolated free of other bacteria; and (iv) sliced agar was more effective in isolating S. hyodysenteriae from swine with chronic diarrhea and nondiarrhetic carriers of SD in which the shedding of S. hyodysenteriae was low.
Multicenter comparison of three commercial methods for quantification of human immunodeficiency virus type 1 RNA in plasma
Authors: Schuurman R , Descamps D , Weverling G J , Kaye S , Tijnagel J , Williams I , van Leeuwen R , Tedder R , Boucher C A , Brun-Vezinet F , Loveday C
Published: January 2020

Three procedures for the quantification of human immunodeficiency virus type 1 (HIV-1) RNA from plasma were compared at three laboratories. The comparison involved the Quantiplex branched DNA assay (version 1.0) by Chiron Diagnostics, the NASBA-QT assay by Organon Teknika, and the Amplicor Monitor assay by Roche Molecular Systems. The laboratories performed each of the three assays with the same sets of reconstructed HIV-1-infected human plasma samples, cross-sectionally collected clinical plasma samples and longitudinally collected plasma samples from patients starting zidovudine therapy. Analysis of the reconstruction panel results for interlaboratory variation demonstrated that no laboratory differences in results were detected for any of the assays. A comparison of the reproducibilities of duplicate samples analyzed by batch and in separate assay runs demonstrated that the reproducibilities of the test results were similar within one assay and appeared to be independent of the HIV-1 concentration. The best reproducibility was obtained with the Quantiplex assay, but all three assays demonstrated equal reliability, which was independent of batched or unbatched analysis of replicate samples. Differences in the absolute concentrations calculated were observed for the assays, in particular in the analysis of reconstructed samples. In all assays, similar changes in plasma HIV-1 RNA concentrations were determined for longitudinally collected clinical samples.
Genotyping of outbreak-related and sporadic isolates of Clostridium difficile belonging to serogroup C
Authors: van Dijck P , Avesani V , Delmée M
Published: January 2020

Serogroup C of Clostridium difficile is the serogroup most frequently related to outbreaks. Fifty-six toxigenic serogroup C isolates of C. difficile were genotyped by ribotyping PCR (ribo-PCR), random amplified polymorphic DNA (RAPD) assay, and pulsed-field gel electrophoresis (PFGE). Thirty-five of the 56 isolates were recovered from four unrelated outbreaks (Belgium, 1987, 1992, and 1995; France, 1992 to 1993) 7 derived from a spatiotemporal cluster in Cotonou, Benin (1992), and 14 were sporadic isolates. The serogroup C reference strain, also isolated during an outbreak (Belgium, 1983), was genotyped too. Ribo-PCR, the RAPD assay, and PFGE generated 2, 5, and 11 major genotypes, respectively. Combination of the three methods finally yielded 13 general types, although ribo-PCR did not play any role in enhancing resolution. Three general types were recovered from all the isolates from the five outbreaks and the cluster, with two types being predominant. The 14 sporadic serogroup C isolates were divided into 11 overall genotypes. These results indicate that genotyping methods, and more particularly the combination of the RAPD assay and PFGE, can resolve genetic diversity within toxigenic, serogroup C C. difficile strains. Also, this study suggests that outbreak-related serogroup C strains are limited to a few genetically stable and apparently very widely (internationally and intercontinentally) distributed genotypes.
Differentiation of bacterial 16S rRNA genes and intergenic regions and Mycobacterium tuberculosis katG genes by structure-specific endonuclease cleavage
Authors: Brow M A , Oldenburg M C , Lyamichev V , Heisler L M , Lyamicheva N , Hall J G , Eagan N J , Olive D M , Smith L M , Fors L , Dahlberg J E
Published: January 2020

We describe here a new approach for analyzing nucleic acid sequences using a structure-specific endonuclease, Cleavase I. We have applied this technique to the detection and localization of mutations associated with isoniazid resistance in Mycobacterium tuberculosis and for differentiating bacterial genera, species and strains. The technique described here is based on the observation that single strands of DNAs can assume defined conformations, which can be detected and cleaved by structure-specific endonucleases such as Cleavase I. The patterns of fragments produced are characteristic of the sequences responsible for the structure, so that each DNA has its own structural fingerprint. Amplicons, containing either a single 5'-fluorescein or 5'-tetramethyl rhodamine label were generated from a 620-bp segment of the katG gene of isoniazid-resistant and -sensitive M. tuberculosis, the 5' 350 bp of the 16S rRNA genes of Escherichia coli O157:H7, Salmonella typhimurium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, Shigella dysenteriae, Campylobacter jejuni, staphylococcus, hominis, Staphylococcus warneri, and Staphylococcus aureus and an approximately 550-bp DNA segment comprising the intergenic region between the 16S and 23S rRNA genes of Salmonella typhimurium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, and Shigella dysenteriae serotypes 1, 2, and 8. Changes in the structural fingerprints of DNA fragments derived from the katG genes of isoniazid-resistant M. tuberculosis isolates were clearly identified and could be mapped to the site of the actual mutation relative to the labeled end. Bland patterns which clearly differentiated bacteria to the level of genus and, in some cases, species were generated from the 16S genes. Cleavase I analysis of the intergenic regions of Salmonella and Shigella species differentiated genus, species, and serotypes. Structural fingerprinting by digestion with Cleavase I is a rapid, simple, and sensitive method for analyzing nucleic acid sequences and may find wide utility in microbial analysis.
Use of pulsed-field gel electrophoresis typing to study an outbreak of infection due to Serratia marcescens in a neonatal intensive care unit
Authors: Miranda G , Kelly C , Solorzano F , Leanos B , Coria R , Patterson J E
Published: January 2020

Serratia marcescens is a well-known cause of nosocomial infections and outbreaks, particularly in critically ill neonates and immunocompromised patients. Numerous methods have been proposed for typing. We used pulsed-field gel electrophoresis (PFGE) typing to analyze an outbreak in a neonatal intensive care unit (NICU). We included 23 patient isolates from an outbreak (March to July 1995), and 10 patient isolates from different wards during the same time period. PFGE of whole-cell DNA digested by SpeI was used as a marker of strain identity. The most common presentation of the infection was sepsis in 18 of 23 (78%) neonates. Only four different biotypes were identified; biotype A8d accounted for 84% of the strains. PFGE typing revealed two clones responsible for two different clonal strain dissemination outbreaks from March to July, with 24 patient isolates being pattern A and 4 patient isolates being pattern E. PFGE typing suggests cross transmission between patients in the NICU and other wards. The isolates from 5 other patients showed distinct PFGE patterns. Extensive investigation and cultures failed to identify any environmental or staff reservoir of S. marcescens. This is one of the first reports applying PFGE to the study of S. marcescens, and this method was a useful marker of strain identity. PFGE typing distinguished strains which appeared to be the same by biotyping.
Evaluations of enzyme-linked immunosorbent assay procedure for determining specific Epstein-Barr virus serology and of rapid test kits for diagnosis for infectious mononucleosis
Authors: Gerber M A , Shapiro E D , Ryan R W , Bell G L
Published: January 2020

Using the results of Epstein-Barr virus-specific immunofluorescence serology as the "gold standard," we found that the sensitivities of the five rapid test kits varied from 78 to 84% and specificities varied from 89 to 100%. Enzyme-linked immunosorbent assay-determined specific Epstein-Barr virus antibody profiles had a sensitivity and specificity of 98.6 and 95.5%, respectively.
Identification of enteroviruses in clinical specimens by competitive PCR followed by genetic typing using sequence analysis
Authors: Arola A , Santti J , Ruuskanen O , Halonen P , Hyypiä T
Published: January 2020

A sensitive method based on competitive PCR was developed to detect and quantitate enteroviral RNA in clinical specimens, with special emphasis on controlling contamination and the presence of potential inhibitory factors in the specimens. Oligonucleotide primers from the conserved parts of the 5' untranslated and VP2 capsid protein-coding regions were selected to differentiate between enteroviruses and rhinoviruses on the basis of the length of the cDNA amplicons. RNA transcribed from a truncated cDNA copy of the echovirus 11 genome was used as an internal control for the reverse transcription reaction and PCR. This allowed simple differentiation of the control and viral PCR products from each other by agarose gel electrophoresis and nonradioactive quantitation of the viral RNA in the clinical specimens. By direct sequencing of the PCR products and subsequent computer analysis of the data, potential laboratory contaminations could be monitored and enteroviruses from clinical samples could be grouped into four distinct clusters, thus enabling genetic typing of the viruses. The described method can be applied to the diagnosis and epidemiology of enteroviral infections.
Correlation between DNA restriction fragment length polymorphisms in Leptospira interrogans serovar pomona type kennewicki and host animal source
Authors: Bolin C A , Zuerner R L
Published: January 2020

Isolates (n = 147) of Leptospira interrogans serovar pomona type kennewicki from cattle, swine, horses, and wildlife were analyzed by DNA restriction endonuclease analysis. Restriction fragment length polymorphisms were identified in DNA digested with HpaII, and the restriction fragment length polymorphisms were correlated with the host animal source of the isolates. These results will be useful in understanding the epidemiology of serovar pomona infections in livestock.
Unusual diversity of human rotavirus G and P genotypes in India
Authors: Ramachandran M , Das B K , Vij A , Kumar R , Bhambal S S , Kesari N , Rawat H , Bahl L , Thakur S , Woods P A , Glass R I , Bhan M K , Gentsch J R
Published: January 2020

Between April and December 1993, we determined P and G genotypes of group A rotavirus strains obtained from children admitted to diarrhea treatment centers in five Indian cities. From a total of 63 rotavirus-positive specimens, we identified 10 different strains with five different G genotypes and four distinct P types by using reverse transcription-PCR. The common worldwide strains G1P8, G2P4, G3P8, and G4P8 were underrepresented among Indian children (33%), whereas strains of P type 6 (G1P6, G2P6, G3P6, G4P6, and G9P6), which primarily infect asymptomatic newborns but are rare in children with diarrhea were common in India (43%). Of these, G9P6, a strain not previously reported to be found in children with diarrhea, was the most prevalent (22%). Eleven percent of the strains were nontypeable, and another 11% of the specimens had mixed infections. Using digoxigenin-labeled, genotype-specific hybridization probes, we confirmed all G9 strains and mixed infections tested and identified three nontypeable strains (one G9 and two P8). The epidemiological significance of G9 rotavirus strains, if confirmed in other settings, may have important implications for vaccine development.
Epidemiological study of Mycoplasma pneumoniae infections in japan based on PCR-restriction fragment length polymorphism of the P1 cytadhesin gene
Authors: Sasaki T , Kenri T , Okazaki N , Iseki M , Yamashita R , Shintani M , Sasaki Y , Yayoshi M
Published: January 2020

Two hundred fifty strains of Mycoplasma pneumoniae isolated during the past 20 years in Japan were classified into two groups (I and II) based upon different PCR-restriction fragment length polymorphism patterns of their P1 cytadhesin genes. Clear shifts between the M. pneumoniae groups were observed but did not appear to be correlated with M. pneumoniae epidemic cycles. Patients' sera showed relatively higher levels of antiadhesin antibodies to M. pneumoniae strains homologous with the infecting strain.
Enterobacter cloacae producing a Shiga-like toxin II-related cytotoxin associated with a case of hemolytic-uremic syndrome
Authors: Paton A W , Paton J C
Published: January 2020

Two Shiga-like toxin-producing organisms were isolated from the feces of an infant with hemolytic-uremic syndrome by PCR followed by colony blot hybridization. One strain was identified as Escherichia coli OR:H9, while the other was identified as Enterobacter cloacae. Both isolates were highly cytotoxic for Vero cells, and Southern hybridization analysis of chromosomal DNA indicated that both contained a single slt-II-related gene and that these genes were located on similarly sized restriction fragments. Nucleotide sequence analysis indicated that the toxin encoded by the E. cloacae slt-II-related gene was very similar to Shiga-like toxin II variants from E. coli, differing from the most closely related toxin by 3 residues in the A subunit.
Serotyping of Cryptococcus neoformans by dot enzyme assay
Authors: Belay T , Cherniak R , O'Neill E B , Kozel T R
Published: January 2020

A method is described for the serotyping of Cryptococcus neoformans based on direct analysis of culture supernatants for the major type-specific capsular antigen, glucuronoxylomannan. Factor sera prepared by absorption of polyclonal rabbit antisera (Iatron Laboratories, Inc., Tokyo, Japan) or selected anti-C. neoformans monoclonal antibodies were used in a dot enzyme assay to detect the presence of antigen.
Rapid laboratory confirmation of human brucellosis by PCR analysis of a target sequence on the 31-kilodalton Brucella antigen DNA
Authors: Matar G M , Khneisser I A , Abdelnoor A M
Published: January 2020

We developed a PCR-based assay for the rapid and specific laboratory diagnosis of human brucellosis directly from whole blood. Specimens were collected in EDTA tubes from 17 patients with acute serologic brucellosis and 3 patients with chronic relapsing brucellosis as determined by serologic tests and the patient's clinical picture. DNA was extracted from peripheral mononuclear cells obtained from the blood of patients with brucellosis and control individuals. Specific primers for the PCR amplification of a 223-bp region on the sequence encoding the 31-kDa immunogenic Brucella abortus protein (BCSP 31) were used. All amplicons had the expected size of 223 bp. The specificity of amplification was determined by Southern hybridization and restriction endonuclease analysis. DNA extracted from blood taken from 30 healthy individuals as well as from 9 patients with typhoid fever did not show any amplification with the primers used. The test proved to be rapid and specific for the laboratory confirmation of acute human brucellosis. Further studies must be conducted to assess the utility of this test on additional patients with chronic relapsing brucellosis as well as patients under treatment.
Susceptibility testing of fungi: current status of correlation of in vitro data with clinical outcome
Authors: Ghannoum M A , Rex J H , Galgiani J N
Published: January 2020

In summary, it is clear that in vitro susceptibility testing can predict outcome in selected clinical situations. The clearest data are from the fluconazole-treated AIDS patients with oropharyngeal candidiasis. In this setting, the homogeneity of the underlying immune defect, combined with the ease of identification and monitoring of the infection, creates a near-perfect test situation. In more complex scenarios, such as the heterogeneous population of patients enrolled in a recent study of candidemia, no such clear-cut correlation was present. The importance of host factors in the correlation of the MIC with outcome cannot be overemphasized. Examples of these parameters include patient status (underlying disease, the presence of intravascular catheters, and CD4+ T-cell number), drug pharmacokinetics (absorption and distribution), patient compliance, and drug-drug interactions. Identification of relevant factors can substantially improve the degree of the MIC-outcome correlation and thus improve the clinical utility of in vitro testing. An important feature in this entire process is the role of standardized susceptibility testing procedures. While not without flaws, the proposed NCCLS reference method has been invaluable in allowing multiple investigators to contribute data that can be used to clarify the correlation between the fluconazole MIC and outcome. While the development of simplified second-generation methods is eagerly anticipated, the role of the reference method as a common touchstone is critical. Only by use of either the reference method itself or methods with a known relationship to the reference method can this broad collaborative process really proceed. Current work is focusing on defining interpretive breakpoints for fluconazole and Candida species, refinement of the in vitro procedures used to measure susceptibility to amphotericin B, ketoconazole, and itraconazole, and the acquisition of a broad base of data on the relationship between the MIC and outcome for these three drugs. Although considerable work remains to be done, the available data suggest that solutions to each of these problems are possible and that routine susceptibility testing of fungi will become meaningful for clinical decision making in the foreseeable future.
Comparison of broth microdilution and E-test for susceptibility testing of Neisseria meningitidis
Authors: Pascual A , Joyanes P , Martinez-Martinez L , Suarez A I , Perea E J
Published: January 2020

The susceptibilities of 54 clinical isolates of Neisseria meningitidis to penicillin, cefotaxime, ceftriaxone, cefepime, imipenem, ciprofloxacin, chloramphenicol, and rifampin were determined by the microdilution method in both cation-adjusted Mueller-Hinton broth (CAMHB) and Haemophilus test medium (HTM). Poor growth was observed in 16.6 and 9% of the strains in CAMHB and HTM, respectively. As a result, the growth of the 54 N. meningitidis strains was evaluated in three other commercially available batches of CAMHB and in one in-house batch of HTM. Poor growth was observed for 9.3 to 16.6% of the strains in all four batches. More important, three of the CAMHB batches failed to support growth for 3.7 to 33.3% of the strains; 3.7% of the strains did not grow in the in-house-prepared HTM. Ten (18.7%) strains were relatively resistant to penicillin (RRP; MIC, > 0.125 mu g/ml) in CAMHB and 13 (24%) strains were RRP in HTM. The percentages of agreement obtained by using CAMHB as the reference ranged from 78% for cefepime to 100% for ceftriaxone. Seven minor errors were observed for penicillin; five of them were for strains susceptible to penicillin in CAMHB and RRP in HTM. All strains were susceptible to the other antimicrobial agents evaluated. The growth of N. meningitidis was also evaluated in four batches of Mueller-Hinton agar (MHA). In two of them, 3.7 and 44.4% of the strains did not grow, and considering all four batches, 5.5 to 11.1% grew poorly. All strains grew adequately in MHA supplemented with blood (MHA-b). The activities of penicillin and cefotaxime were also evaluated by the E-test in MHA and MHA-b. The proportion of RRP strains were 24% in MHA and 59% in MHA-b. For penicillin, the percentages of agreement of the E-test with the microdilution method in CAMHB (reference) were 64.8 and 70.3% in MHA and MHA-b, respectively. For cefotaxime, the agreement was 98.1%. Minor errors for the penicillin MIC were detected for 38% of the strains tested. Further studies are needed to define adequate culture media for reference methods to evaluate the susceptibility of N. meningitidis to antimicrobial agents.
Characterization of clinical isolates of Escherichia coli showing high levels of fluoroquinolone resistance
Authors: Lehn N , Stower-Hoffmann J , Kott T , Strassner C , Wagner H , Kronke M , Schneider-Brachert W
Published: January 2020

During the years 1992 to 1994, an increase in fluoroquinolone-resistant Escherichia coli was observed at the Medical Center of the Technical University in Munich, Germany. Nineteen strains were collected and were thus available for further analysis. Pulsed-field gel electrophoresis showed clonal diversity in all but two strains. The majority of the patients from whom the strains were isolated had been previously treated with fluoroquinolones. Quinolone resistance was associated with mutations of the quinolone resistance-determining region of the gyrA gene in all cases. Direct sequencing of gyrA PCR amplification products revealed a mutation in codon 83 of the gyrA gene. In some instances the Ser-83-->Leu mutation was accompanied by an Asp-87-->Asn or Asp-87-->Gly mutation. Furthermore, the strains exhibited two different genotypes: in almost half of the fluoroquinolone-resistant E. coli strains as well as in the fluoroquinolone-susceptible E. coli reference strains ATCC 25922 and 35218, silent mutations were detected at bases 255, 273, 300, and 333. Although fluoroquinolones solved major problems in antimicrobial chemotherapy, in certain departments of our hospital the number of resistant E. coli isolates has become so high that susceptibility to fluoroquinolones can no longer be taken for granted.
Comparative evaluation of virological and serological methods in prenatal diagnosis of parvovirus B19 fetal hydrops
Authors: Zerbini M , Musiani M , Gentilomi G , Venturoli S , Gallinella G , Morandi R
Published: January 2020

Human parvovirus B19 infection in pregnancy represents a potential hazard to the fetus since fetal loss or fetal hydrops can occur. The risk of fetal loss due to transplacental B19 transmission has been evaluated in several studies using different diagnostic methods on maternal and fetal specimens. We analyzed the diagnostic value of virological and serological techniques on maternal serum, fetal cord blood, and amniotic fluid specimens obtained at the time of clinical diagnosis of fetal hydrops in 18 cases of B19 fetal hydrops. B19 DNA was detected by nested PCR, dot blot hybridization, and in situ hybridization assay. Anti-B19 immunoglobulin M and G antibodies were detected by immunoassays using recombinant B19 antigens. Our data suggest that for maternal sera, virological and serological methods have a complementary role in diagnosis, while for fetal specimens the in situ detection of B19 DNA in fetal cord blood is the most sensitive diagnostic system.
Rapid subgenus identification of human adenovirus isolates by a general PCR
Authors: Kidd A H , Jonsson M , Garwicz D , Kajon A E , Wermenbol A G , Verweij M W , De Jong J C
Published: January 2020

In most clinical situations involving adenovirus infection, subgenus (subgroup) identification of an adenovirus isolate is as informative as a finer identification by serotype. A PCR method which allows the identification of human adenovirus isolates as members of subgenera A, B:1, B:2, C, D, E, or F is described. It is based on a simple (nonnested) PCR using primers which bind to regions immediately flanking the VA RNA-encoding regions of human adenovirus genomes. The PCR allows amplification of DNA from all 49 human adenovirus prototype strains so far described. Since there are differences in the lengths of the VA RNA-encoding regions in adenoviruses of different subgenera, it is possible to differentiate some subgenera according to the size of the PCR product determined by electrophoresis. This forms the basis of an initial broad categorization of isolates as belonging to either (i) subgenus B:1, C, D, or E or (ii) subgenus A, B:2, or F. Subgenus identification is completed by a one-step restriction enzyme digestion and gel electrophoresis. The method was assessed by blind subgenus identification of 200 miscellaneous primate adenovirus isolates prepared by the reference laboratory at Bilthoven, The Netherlands. Identification at the subgenus level by PCR correlated 91.5% with the results of serotyping. A further 5.5% of isolates were correctly identified as belonging to one of two specified subgenera. Six of the 200 identifications (3%) were unsuccessful for various reasons, including weak PCR products, intermediate strains, and mistaken primate host. The method should serve as a rapid means of confirming adenovirus cytopathic effects in laboratories performing virus culture, with simultaneous subgenus identification of the isolate. It will also have relevance as an aid to conventional serotyping for epidemiological purposes, since for all adenoviruses except those belonging to subgenus D, neutralization tests need only involve a maximum of four type-specific antisera.