bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of restriction endonuclease analysis, ribotyping, and pulsed-field gel electrophoresis for molecular differentiation of Clostridium difficile strains
Authors: Kristjánsson M , Samore M H , Gerding D N , DeGirolami P C , Bettin K M , Karchmer A W , Arbeit R D
Published: January 2020

A combined clinical and molecular epidemiologic analysis of 46 strains of Clostridium difficile, including 16 nosocomial isolates from one ward (outbreak ward) plus 17 other nosocomial isolates and 13 community-acquired isolates, was performed. HindIII digests of total cellular DNA were analyzed by restriction enzyme analysis (REA) and ribotyping; SmaI digests were analyzed by pulsed-field gel electrophoresis (PFGE). Isolates were assigned to typing groups on the basis of the profiles detected; isolates with closely related profiles were assigned to subgroups. The 16 isolates from the outbreak ward were resolved by both REA and PFGE into five distinct groups; 13 isolates represented two REA groups and three PFGE groups and two isolates were resolved as distinct groups by both techniques. DNA obtained from one isolate was persistently partially degraded, precluding analysis by PFGE. Seventeen sporadic nosocomial isolates were resolved by REA and PFGE into comparable numbers of groups (i.e., nine groups) and subgroups (i.e., 15 and 14 subgroups, respectively), with two isolates not evaluable by PFGE. The 13 epidemiologically unrelated community-acquired isolates were assigned to 11 groups by REA and to 12 groups by PFGE. Overall, ribotyping identified only nine groups among the 46 isolates. We conclude that REA and PFGE have comparable discriminatory powers for epidemiologic typing of C. difficile isolates and that ribotyping is appreciably less discriminatory. For a few isolates, partial DNA degradation prevented analysis by PFGE but not by REA or ribotyping; the cause of the degradation is unknown.
Persistent and transient antibody responses to hepatitis E virus detected by western immunoblot using open reading frame 2 and 3 and glutathione S-transferase fusion proteins
Authors: Li F , Zhuang H , Kolivas S , Locarnini S A , Anderson D A
Published: January 2020

Recombinant proteins containing amino acid sequences from open reading frame (ORF) 2 and ORF3 of a Chinese strain of hepatitis E virus (HEV) were constructed as fusions with glutathione S-transferase (GST). Stable fusion proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were transferred to nitrocellulose membranes, and the immobilized proteins were probed with sera from hepatitis E patients from various regions or from rhesus monkeys (Macaca mulatta) experimentally infected with the Chinese strain of HEV. Immunoglobulin G-class antibodies were detected with chemiluminescence and anti-human immunoglobulin G conjugated to horseradish peroxidase. Anti-ORF3 antibodies were detected in most patients and monkeys within 17 days of exposure, but this humoral response declined with time and was usually undetectable by approximately 100 days. Anti-ORF2.1 antibody was usually detected as early as anti-ORF3 but persisted in all animals and many patients, whereas reactivity to the larger GST-ORF2.2 fusion protein was more transient, even though all sequences present in GST-ORF2.1 are present in GST-ORF2.2. Rechallenge of these monkeys with HEV suggested that immunity to reinfection was incomplete, as levels of anti-ORF2.1 (but not anti-ORF2.2) were boosted after each rechallenge. The results demonstrate that the carboxy-terminal region of HEV ORF2 contains epitopes which are recognized by convalescent-phase antibody and are likely to be associated with limited immunity to infection, but these epitopes may be masked when larger portions of ORF2 are expressed as recombinant proteins.
Controlled evaluation of 5 versus 10 milliliters of blood cultured in aerobic BacT/Alert blood culture bottles
Authors: Weinstein M P , Mirrett S , Wilson M L , Reimer L G , Reller L B
Published: January 2020

Bottles developed for use in the BacT/Alert automated blood culture system (Organon Teknika Corp., Durham, N.C.) can accept up to 10 ml of blood without falling below a 1:5 ratio of blood to broth. We compared the yield and speed of detection of microorganisms in 13,128 adequately filled, paired, aerobic bottles inoculated with 5 versus 10 ml of blood at three university hospitals. A total of 798 microorganisms causing sepsis grew in one or both bottles. The overall recovery of microorganisms from 10-ml samples exceeded that from 5-ml samples (P < 0.001); the increased yield attributed to the additional 5 ml in the samples was 7.2%. The increased yield from 10-ml inocula was most marked for Escherichia coli (P < 0.01) and other members of the family Enterobacteriaceae (P < 0.001). Ten-milliliter samples did not yield more gram-positive bacteria, nonfermentative gram-negative rods, or yeasts. When both bottles were positive, the bottles inoculated with 10 ml of blood showed growth sooner (P < 0.001). Earlier detection with 10-ml inocula was especially notable for coagulase-negative staphylococci (P < 0.001), streptococci (P < 0.001), E. coli (P < 0.025), and other members of the family Enterobacteriaceae (P < 0.025). We conclude that an increase in the volume of blood inoculated into BacT/Alert aerobic blood culture bottles from 5 to 10 ml will increase the overall yield and the speed of detection of clinically important blood pathogens.
Western immunoblot analysis of Ehrlichia chaffeensis, E. canis, or E. ewingii infections in dogs and humans
Authors: Rikihisa Y , Ewing S A , Fox J C
Published: January 2020

Ehrlichia chaffeensis, E. canis, and E. ewingii are genetically closely related, as determined by 16S rRNA gene base sequence comparison, but they exhibit biologic differences. E. chaffeensis is the etiologic agent of human ehrlichiosis. E. canis and E. ewingii cause two distinctly different forms of canine ehrlichiosis and infect different types of leukocytes, monocytes and granulocytes, respectively. E. chaffeensis can also infect dogs. In the study, Western immunoblot analysis of sera from dogs inoculated with E. chaffeensis, E. canis, or E. ewingii was performed to determine antigenic specificity and the intensities of the reactions to purified E. chaffeensis and E. canis antigens. At 2 to 3 weeks postexposure, antisera from four dogs inoculated with E. chaffeensis reacted with 64-, 47-, 31-, and 29-kDa proteins of E. chaffeensis but reacted poorly with E. canis antigen. In contrast, at 2 to 3 weeks postexposure, antisera from four E. canis-inoculated dogs reacted strongly with the 30-kDa major antigen of E. canis but reacted poorly with proteins from E. chaffeensis. At 4 weeks postexposure, the sera from three E. ewingii-inoculated dogs showed weak binding to 64- and 47-kDa proteins of both E. chaffeensis and E. canis. Convalescent-phase sera from human ehrlichiosis patients and sera from dogs chronically infected with E. ewingii strongly reacted with similar sets of proteins of E. chaffeensis and E. canis with similar intensities. However, sera from dogs chronically infected with E. canis reacted more strongly with a greater number of E. canis proteins than with E. chaffeensis proteins. The protein specificity described in the report suggests that dogs with E. canis infections can be distinguished from E. chaffeensis-infected animals by Western immunoblot analysis with both E. canis and E. chaffeensis antigens.
Comparison of sensititre broth microdilution and agar dilution susceptibility testing techniques for meropenem to determine accuracy, reproducibility, and predictive values
Authors: Dowzicky M J , Nadler H L , Sheikh W
Published: January 2020

The stability, accuracy, reproducibility, and predictive value of Sensititre MIC panels containing meropenem (Merrem) were evaluated by using National Committee for Clinical Laboratory Standards (NCCLS)-recommended American Type Culture Collection (ATCC) strains and 110 selected strains of rapidly growing and fastidious aerobes and anaerobes with various degrees of susceptibility to meropenem. The NCCLS-recommended agar dilution method was used as a standard reference method. Meropenem-containing Sensititre MIC panels were monitored for their stabilities at room temperature and reproducibilities over 24 months by using six ATCC strains. Ninety-nine percent of the MICs of both meropenem and imipenem obtained for NCCLS-recommended ATCC strains were within the established ranges after 2 years. The overall agreement (+/- 1 twofold dilution) between the Sensititre and the agar dilution meropenem MICs was greater than 93%. The predictive value of meropenem MICs for indicating suspeptibility or resistance obtained by the Sensititre method was greater than 90%. No major or very major interpretive errors were observed, and only 5% of meropenem MICs were associated with minor interpretive errors. Problematic organisms were not observed. The Sensititre MIC panels containing meropenem offer a convenient and valid alternative to the NCCLS reference method for the susceptibility testing of potential pathogens likely to be recovered from mixed infections.
Stabilities of quantitative plasma culture for human immunodeficiency virus, RNA, and p24 antigen from samples collected in VACUTAINER CPT and standard VACUTAINER tubes
Authors: Mole L , Margolis D , Carroll R , Todd J , Holodniy M
Published: January 2020

We evaluated the stability of human immunodeficiency virus (HIV) load markers from blood samples collected in VACUTAINER CPT or standard VACUTAINER brand tubes using sodium heparin or sodium citrate as anticoagulants. Quantitative plasma culture and p24 antigen concentrations were determined, and HIV RNA levels in plasma were measured by both reverse transcription-PCR-enzyme-linked immunosorbent assay (RT-PCR-ELISA) and branched DNA methods. All tubes were stored at room temperature for analysis at 2, 24, 48, and 72 h after the blood samples were drawn. No difference was seen between tube types with respect to the HIV titer in plasma or the positivity rate for all samples that demonstrated a fall in titer over time. Unbound p24 antigen levels in plasma decreased during the initial 48-h period in both tube types. Immune complex-dissociated p24 antigen levels decreased in CPT tubes but not in standard VACUTAINER tubes. The HIV RNA copy number in plasma measured by RT-PCR-ELISA was stable in most subjects and was significantly higher in CPT tubes than in standard VACUTAINER tubes at 24 and 72 h after the blood samples were drawn. The branched DNA probe assay detected a significant decline in HIV RNA equivalent in plasma over 72 h in both collection tubes, the decline being more dramatic in the standard VACUTAINER tube than the CPT tube. Overall, interday variability suggests that samples collected for a particular assay should be processed at the same time after blood is drawn and that a particular tube type be used throughout a given study.
Strain relatedness of Candida albicans strains isolated from children with leukemia and their bedside parents
Authors: Doi M , Homma M , Iwaguchi S , Horibe K , Tanaka K
Published: January 2020

Candida yeasts are occasionally recovered from patients with leukemia in spite of antifungal therapy used during chemotherapy. It is not yet known whether yeasts in these patients are of endogenous or exogenous origin. We examined the strain relatedness of Candida albicans isolated from three patients with leukemia (A, B, and C) and their bedside parents using pulsed-field gel electrophoresis (PFGE), restriction fragment length polymorphism (RFLP) by SmaI digestion, and the Southern hybridization patterns of the RFLPs by the C. albicans-specific probe RPS1. SmaI digestion and Southern hybridization by RPS1 showed identical or similar patterns among Candida isolates in patients A and his mother, although their karyotypes were different. Isolates from patients B and both parents showed identical electrophoretic karyotypes, SmaI digestion patterns, and hybridization patterns. Since electrophoretic karyotypes are more variable than RFLPs and their hybridization patterns, the identity of the last two suggests a close relatedness between strains. Our results also suggest that transmission of yeast strains may have occurred between patient A and his mother and between patient B and her parents. Isolates from patient C and her mother are thought to have originated from different strains, since different patterns were obtained in electrophoretic karyotypes, SmaI digestion patterns, and Southern hybridization patterns.
Case report and review of septicemia due to Serratia ficaria
Authors: Darbas H , Jean-Pierre H , Paillisson J
Published: January 2020

Serratia ficaria was first described in 1979 as part of the fig tree ecosystem (P.A.D. Grimont, F. Grimont, and M. P. Starr, Curr. Microbiol. 2:277-282, 1979). Since then, it has been isolated from clinical specimens from a few human patients (C. Bollet, J. Freney, P. de Micco, F. Grimont, and P.A.D. Grimont, Méd. Mal. Infect. 20:97-100, 1990; J.A. Brouillard, W. Hansen, and A. Compere, J. Clin. Microbiol. 19:902-904, 1984; H. Darbas, H. Jean-Pierre, G. Boyer, and M. Riviere, Méd. Mal. Infect. 23:269-270, 1993; V.J. Gill, J.J. Farmer, III, P.A.D. Grimont, M.A. Asbury, and C.L. McIntosh, J. Clin. Microbiol. 14:234-236, 1981; F.D. Pien and J.J. Farmer III, South. Med. J. 76:1591-1592, 1983; C. Richard, J. de Coquet, and C. Suc, Méd. Mal. Infect. 19:45-47, 1989), but the pathogenicity of S. ficaria was always questionable. We are reporting the case of an aged cancer patient who developed S. ficaria septicemia. The habitat of this organism and its potential role as a pathogen are discussed.
Genome analysis of South American adenovirus strains of serotype 7 collected over a 7-year period
Authors: Kajon A , Wadell G
Published: January 2020

DNA restriction analysis was carried out on a sample of 212 epidemiologically unrelated adenovirus (Ad) strains of serotype 7 isolated in Chile, Uruguay, and Argentina from nasopharyngeal aspirates of children with acute lower respiratory disease between 1984 and 1990. The identified genome types were Ad7b (n = 12), Ad7c (n = 21), and Ad7h (n = 179). The analysis of the occurrence of the three genome types throughout the study period revealed that Ad7c was the predominant virulent variant in 1984 and indicated that a shift to genome type 7h took place in 1986. Ad7b cocirculated with the other genomic variants at a relatively low frequence.
Usefulness of PCR for detection of Pneumocystis carinii DNA
Authors: Roux P , Lavrard I , Poirot J L , Chouaid C , Denis M , Olivier J L , Nigou M , Miltgen M
Published: January 2020

Diagnosis of Pneumocystis carinii pneumonia is based on the identification of the various stages of the parasite in lung samples by standard staining techniques. We therefore assessed the value of the PCR for detection of P. carinii in bronchoalveolar lavage, induced sputum, and blood samples relative to that of standard staining techniques.
Phenotypic analysis of outer surface protein C (OspC) of Borrelia burgdorferi sensu lato by monoclonal antibodies: relationship to genospecies and OspA serotype
Authors: Wilske B , Jauris-Heipke S , Lobentanzer R , Pradel I , Preac-Mursic V , Rössler D , Soutschek E , Johnson R C
Published: January 2020

Molecular analyses of the genes encoding OspC, a major immunodominant protein of Borrelia burgdorferi sensu lato, revealed a considerable degree of heterogeneity. In the present study, we investigated whether a similar heterogeneity of the OspC phenotype can be shown by analysis with monoclonal antibodies (MAbs). Thirteen OspC-specific MAbs (L22 MAbs) were produced by immunizing mice with either different combinations of whole-cell antigens or recombinantly expressed OspCs cloned from strains belonging to different Borrelia spp. Ten of them differed in their reactivities with various strains. Western blot (immunoblot) analyses of 38 B. burgdorferi sensu lato strains resulted in 13 different reactivity patterns. These 13 different patterns were observed among only six different OspA serotypes, indicating that OspC is more heterogeneous than OspA. Patterns 1 to 4 were present only in B. burgdorferi sensu stricto, patterns 5 to 7 were present only in Borrelia afzelii, and patterns 9 to 13 were present only in Borrelia garinii. Pattern 8 was observed among B. afzelii and B. garinii strains but not among B. burgdorferi sensu stricto strains. One L22 MAb (2B8) recognized a common OspC-specific epitope of all 38 B. burgdorferi sensu lato strains analyzed, and another one (22C11) recognized a common epitope of OspC from both B. afzelii and B. garinii and was not reactive with OspC from B. burgdorferi sensu stricto. Western blot and sequence analysis of truncated OspCs located the 22C11 epitope as well as a species-specific sequence motif between amino acids 20 and 35. Other broadly reactive L22 MAbs were 10D3, 1F8, and 7G5. Some L22 MAbs (1C3, 1C3, 12E5, 1B11, 1F10, and 6C8) bound to epitopes present only in a few strains. Relapsing fever borreliae (Borrelia hermsii, Borrelia turicatae, and Borrelia duttoni) were nonreactive, with the following exception: three L22 MAbs (2B8, 6C4, and 10C5) recognized an abundantly expressed 20-kDa-range protein of B. turicatae. Because OspC is an immunodominant protein during the early immune response in Lyme borreliosis and has been shown to be effective as a vaccine in an animal model, our findings have important implications for the development of diagnostic reagents as well as vaccine research.
New variant of Vibrio cholerae O1 from clinical isolates in Amazonia
Authors: Coelho A , Andrade J R , Vicente A C , Salles C A
Published: January 2020

A survey of pathogenic Vibrio cholerae O1 strains from the north of Brazil by using arbitrarily primed PCR fingerprints revealed a group of strains with similar fingerprint patterns that are distinct from those of the current El Tor epidemic strain. These strains have been analyzed by in vivo and in vitro techniques and the group has been denominated the Amazonian variant of V. cholerae O1.
Clinical impact of bacteria and fungi recovered only from broth cultures
Authors: Morris A J , Wilson S J , Marx C E , Wilson M L , Mirrett S , Reller L B
Published: January 2020

We prospectively evaluated 356 bacteria and fungi recovered from broth enrichment tubes from cultures with sterile direct plates to determine the clinical impact of isolates recovered only from broth cultures. These "broth only" isolates (BOI) were classified as contaminants or true on the basis of review of patient charts. True isolates were considered clinically relevant only if they altered or should have altered patient management. Of 356 BOI, 259 (73%) were considered contaminants (mostly coagulase-negative staphylococci and Propionibacterium spp.) and 97 (27%) were considered true. For individual microorganisms, 9 of 9 (100%) Staphylococcus aureus isolates, 13 of 13 (100%) members of the family Enterobacteriaceae, 10 of 12 (83%) fungi, 7 of 10 (70%) enterococci, 7 of 11 (64%) other gram-negative bacilli, 13 of 31 (45%) anaerobic bacteria, 10 of 24 (42%) streptococci, 22 of 140 (16%) coagulase-negative staphylococci, 6 of 92 (7%) Propionibacterium spp., and 0 of 14 (0%) diphtheroids and Bacillus spp. were classified as true. Eleven of 97 (11%) patients with true BOI had clinically relevant isolates. Fifty-nine of the 97 (61%) patients with true isolates already were on therapy, and no change was made because of the BOI. Six (6%) patients with contaminants received therapy for their BOI. We conclude that broth inoculated as an adjunct to direct plating seldom yields results that favorably alter patient management and could be omitted for most specimens without compromising patient care.
Phylogeny and PCR identification of the human pathogenic fungus Penicillium marneffei
Authors: LoBuglio K F , Taylor J W
Published: January 2020

The phylogenetic position of the human pathogenic fungus Penicillium marneffei was assessed from the nucleotide sequences of the nuclear and mitochondrial ribosomal DNA regions. Phylogenetic analysis determined that P. marneffei is closely related to species of Penicillium subgenus Biverticillium and sexual Talaromyces species with asexual biverticillate Penicillium states. Knowledge of the phylogenetic position of P. marneffei facilitated the design of unique oligonucleotide primers, from the nuclear ribosomal DNA internal transcribed spacer region, for the specific amplification of P. marneffei DNA. These primers were successful at selectively amplifying DNA from six isolates of P. marneffei and excluding the other species tested, which included Penicillium subgenus Biverticillium and Talaromyces species and several well-known fungal pathogens, namely, Aspergillus fumigatus, Coccidioides immitis, Histoplasma capsulatum, and Pneumocystis carinii. The primers that we have developed for the specific amplification of P. marneffei have the potential to be incorporated in a PCR identification system which could be used for the identification of this pathogenic agent from clinical material.
Clinical evaluation of a Mycobacterium tuberculosis PCR assay
Authors: Beige J , Lokies J , Schaberg T , Finckh U , Fischer M , Mauch H , Lode H , Köhler B , Rolfs A
Published: January 2020

On the basis of previously published PCR primer sequences, we have designed a sensitive system for detecting DNA of the Mycobacterium tuberculosis complex (MTB) in patient sputum samples which employs a fast and simplified sample preparation method appropriate for routine diagnostic testing. In order to evaluate the accuracy of the PCR assay, we performed a prospective study with 103 patients, comparing PCR results with culture results of samples obtained from a parallel culture assay as well as with subsequent culture results. Using two MTB-specific PCR primer systems, we found 48 of 49 tuberculosis (Tb) patients to be PCR positive (PCR sensitivity, 0.98). Sixteen of 54 presumably non-Tb patients showed amplifiable MTB DNA (specificity, 0.7). The study demonstrates that for diagnostic applications of MTB PCR two MTB-specific primer pairs should be used. MTB infection is extremely unlikely in cases of MTB PCR-negative samples: with our method for the exclusion of active Tb, the validity of one PCR assay seems to be equivalent to those of at least three culturing procedures. Positive PCR results do not necessarily reflect active MTB infection. It remains to be shown whether positive PCR results in Tb-negative patients mean false-positivity, an early laboratory finding which predicts a subsequent reactivation of a prior Tb infection, or whether asymptomatic patients may carry PCR-amplifiable MTB DNA without any clinical relevance. It is important to point out that the validity of PCR results in clinical studies depends on the use of contamination controls parallel to all PCR steps and the simplicity of the DNA extraction method as well as on the specificity of the PCR results.
Molecular identification of bacteria by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene
Authors: Widjojoatmodjo M N , Fluit A C , Verhoef J
Published: January 2020

PCR-single-strand conformation polymorphism (PCR-SSCP) analysis is a rapid and convenient technique for the detection of mutations and allelic variants. We have adapted this technique for the identification of bacteria by PCR with fluorescein-labeled primers chosen from the conserved regions of the 16S rRNA gene flanking a variable region. The PCR product was denatured, separated on a nondenaturing gel, and detected by an automated DNA sequencer. The mobility of the single-stranded DNA is sequence dependent and allows the identification of a broad panel of bacteria. A single nucleotide difference in the amplified region was sufficient to obtain different PCR-SSCP patterns. The simultaneous amplification of multiple polymorphic regions by multiplex PCR with subsequent multiplex SSCP increased the discriminatory power of PCR-SSCP. A broad range of gram-negative and gram-positive bacteria were tested by PCR-SSCP, including, e.g., Escherichia coli, Enterobacter spp., Klebsiella spp., Haemophilus spp., Neisseria spp., Staphylococcus spp, Streptococcus spp., Enterococcus spp., and Bacillus spp. In total, a panel of 178 strains of bacteria representing 51 species in 21 genera was examined. Although a limited number of strains from each species were tested, the strains tested gave species-specific patterns, with only one exception: Shigella species were indistinguishable from E. coli. PCR is a sensitive technique; as few as 10 CFU of E. coli was sufficient to produce PCR-SSCP patterns suitable for identification. The whole fluorescence PCR-SSCP procedure takes approximately 8 h for the detection and identification of low numbers of bacteria.2+ fluorescence PCR-SSCP seems to be a promising method for the differentiation of a broad range of pathogens found in usually sterile clinical sites, such as blood and cerebrospinal fluid.
Characterization of Newcastle disease virus isolates by reverse transcription PCR coupled to direct nucleotide sequencing and development of sequence database for pathotype prediction and molecular epidemiological analysis
Authors: Seal B S , King D J , Bennett J D
Published: January 2020

Degenerate oligonucleotide primers were synthesized to amplify nucleotide sequences from portions of the fusion protein and matrix protein genes of Newcastle disease virus (NDV) genomic RNA that could be used diagnostically. These primers were used in a single-tube reverse transcription PCR of NDV genomic RNA coupled to direct nucleotide sequencing of the amplified product to characterize more than 30 NDV isolates. In agreement with previous reports, differences in the fusion protein cleavage sequence that correlated genotypically with virulence among various NDV pathotypes were detected. By using sequences generated from the matrix protein gene coding for the nuclear localization signal, lentogenic viruses were again grouped phylogenetically separate from other pathotypes. These techniques were applied to compare neurotropic velogenic viruses isolated from an outbreak of Newcastle disease in cormorants and turkeys. Cormorant NDV isolates and an NDV isolate from an infected turkey flock in North Dakota had the fusion protein cleavage sequence 109SRGRRQKRFVG119. The R-for-G substitution at position 110 may be unique for the cormorant-type isolates. Although the amino acid sequences from the fusion protein cleavage site were identical, nucleotide sequence data correlate the outbreak in turkeys to a cormorant virus isolate from Minnesota and not to a cormorant virus isolate from Michigan. On the basis of sequence information, the cormorant isolates are virulent viruses related to isolates of psittacine origin, possibly genotypically distinct from other velogenic NDV isolates. These techniques can be used reliably for Newcastle disease epidemiology and for prediction of pathotypes of NDV isolates without traditional live-bird inoculations.
Intermethod variation in detection of human papillomavirus DNA in cervical smears
Authors: Smits H L , Bollen L J , Tjong-A-Hung S P , Vonk J , Van Der Velden J , Ten Kate F J , Kaan J A , Mol B W , Ter Schegget J
Published: January 2020

In order to investigate the reliability of detection of human papillomavirus (HPV) DNA in cervical smears, we have compared the performance of two HPV PCR systems, the CPI/IIG and MY09/11 primer-mediated PCRs and the Hybrid Capture System HPV DNA detection test (hybrid capture assay), in detecting HPV DNA in cervical smears. We also included in our study the MY09/11B PCR plus SHARP (solution hybridization assay for PCR products) Signal System. This SHARP Signal System was recently developed to detect MY09/11B-generated biotinylated PCR products. The detection rate of the hybrid capture assay was lower than those of the CPI/IIG and MY09/11 PCRs and the MY09/11B PCR plus SHARP Signal System. The detection rates of the CPI/IIG PCR and the MY09/11B PCR plus SHARP Signal System were similar and higher than that of the conventional MY09/11 PCR system. The agreement beyond chance of the PCR methods was nearly perfect (kappa value between 0.82 and 0.84). The agreement beyond chance of the hybrid capture assay and the PCR methods was fair to good (kappa value between 0.64 and 0.70). The systems detected HPV DNA in different but overlapping sets of smears. Our results indicate that each of the detection methods alone underestimates the prevalence of HPV.
Diagnosis of Helicobacter pylori infection by PCR: comparison with other invasive techniques and detection of cagA gene in gastric biopsy specimens
Authors: Lage A P , Godfroid E , Fauconnier A , Burette A , Butzler J P , Bollen A , Glupczynski Y
Published: January 2020

A PCR assay for the detection of Helicobacter pylori in gastric biopsy specimens with specific primers for ureC gene amplification (herein referred to as ureC PCR) was compared with other routine invasive methods (culture, the rapid-urease test, and Giemsa staining of histological sections) with samples from a group of 104 consecutive dyspeptic patients. Bacteria were found in 40 (38.5%), 38 (36.5%), 36 (34.6%), and 35 (33.7%) of the patients by ureC PCR, culture, the rapid-urease test, and Giemsa stain, respectively. Sixty-three patients had negative cultures, negative histological examinations, and negative rapid-urease test results, and 61 of these patients were also negative by ureC PCR. ureC PCR detected H. pylori in two culture-negative patients. In parallel, a PCR-based assay to detect the H. pylori cytotoxin-associated antigen (cagA) gene, a putative virulence gene, was also developed. To assess the likelihood of detection of H. pylori genes directly from gastric biopsy samples and from the corresponding H. pylori isolates, specimens from 31 patients were subjected to PCR with ureC- and cagA-targeting primers. All 31 biopsy specimens and the corresponding H. pylori isolates were positive in the ureC PCR. H. pylori strains that were cagA positive also gave positive cagA PCR fragments with biopsy specimens from the same patients. All ureC PCR-positive patients were examined; biopsy specimens from 10 of 11 (91.7%) duodenal ulcer patients harbored H. pylori cagA-positive strains, whereas 19 of26 (73%) of those from patients with chronic gastritis only were found to be cagA positive. These findings indicate first that ureC PCR is at least as sensitive as culture for diagnosing H. pylori infection and second that the presence of the H. pylori cagA gene can also be detected directly in biopsy specimens by PCR amplification.
Distribution of serotypes of Streptococcus pneumoniae isolated from invasive infections over a 16-year period in the greater São Paulo area, Brazil
Authors: Brandileone M C , Vieira V S , Zanella R C , Landgraf I M , Melles C E , Taunay A , de Moraes J C , Austrian R
Published: January 2020

Capsular types of pneumococci from normally sterile body sites of 1,622 patients in Brazil were analyzed. Of 1,477 isolates from cerebrospinal fluid, 76.1% were of types represented in the currently available pneumococcal vaccine. The importance of age, time, and place in determining the optimal formulation of pneumococcal vaccine is considered.
Typing of Pneumocystis carinii strains with type-specific oligonucleotide probes derived from nucleotide sequences of internal transcribed spacers of rRNA genes
Authors: Lu J J , Bartlett M S , Smith J W , Lee C H
Published: January 2020

We have recently developed a method for typing Pneumocystis carinii strains that infect humans. The method takes advantage of nucleotide sequence variations in internal transcribed spacers (ITSs) of the rRNA genes of P. carinii. To date, two types of nucleotide sequences (designated types A and B) have been found in the ITS1 region, and three types of nucleotide sequences (designated types a, b and c) have been found in the ITS2 region. Of the six potential combination types, we have detected four, designated types Ac, Bb, Ba, and Bc. To simplify typing, we have designed five oligonucleotide probes, probes 1-A, 1-B, 2-a, 2-b, and 2-c, which are specific to ITS1 type A and type B and ITS2 type a, type b, and type c, respectively, of P. carinii strains that infect humans. We also have designed an oligonucleotide which reacts specifically with P. carinii strains that infect rats. The ITS region were amplified by PCR, and the PCR products were then probed with these type-specific oligonucleotide probes. Typing with the type-specific oligonucleotide probes was found to be effective with specimens containing only one type of P. carinii. These methods are rapid and simple to perform and will be useful for studying the epidemiology of P. carinii infections.
Multilaboratory evaluation of screening methods for detection of high-level aminoglycoside resistance in enterococci. National Committee for Clinical Laboratory Standards Study Group on Enterococci
Authors: Swenson J M , Ferraro M J , Sahm D F , Clark N C , Culver D H , Tenover F C
Published: January 2020

Since the early 1970s, the synergistic activity of an aminoglycoside with a cell wall-active agent has been predicted by determining the ability of an enterococcus to grow in the presence of high levels of the aminoglycoside (usually > or = 2,000 micrograms/ml). However, a variety of media and concentrations of aminoglycosides has been used for this screening procedure. In the present study, we sought to optimize the agar dilution, broth microdilution, and disk diffusion tests used to detect high-level gentamicin and streptomycin resistance in enterococci. For dilution tests, brain heart infusion agar or broth gave the best growth and performance. For agar dilution, 500 micrograms of gentamicin per ml, 2,000 micrograms of streptomycin per ml, and an inoculum of 1 x 10(6) CFU/ml were optimal, while for broth microdilution, 500 micrograms of gentamicin per ml, 1,000 micrograms of streptomycin per ml, and an inoculum of 5 x 10(5) CFU/ml were best. Growth of more than one colony in the agar dilution test was determined to be the best indicator of high-level resistance. For disk diffusion, Mueller-Hinton agar, 120-micrograms gentamicin disks, and 300-micrograms streptomycin disks with breakpoints of no zone for resistance and > or = 10 mm for susceptibility gave the best sensitivity and specificity if results for strains with zones of 7 to 9 mm are considered inconclusive, indicating that a broth or agar test should be performed to determine susceptibility or resistance.
Comparison of Gram stain with DNA probe for detection of Neisseria gonorrhoeae in urethras of symptomatic males
Authors: Juchau S V , Nackman R , Ruppart D
Published: January 2020

The comparison of Gram-stained urethral smears with Gen-Probe for the detection of Neisseria Gonorrhoeae in the urethras of males with symptomatic urethritis revealed a 99.6% correlation between the two methods. A simple Gram stain would appear to be the method of choice for the detection of gonorrhea in symptomatic males, because it is much less expensive and much more rapid than the Gen-Probe method.
Plasma (1-->3)-beta-D-glucan and fungal antigenemia in patients with candidemia, aspergillosis, and cryptococcosis
Authors: Miyazaki T , Kohno S , Mitsutake K , Maesaki S , Tanaka K , Ishikawa N , Hara K
Published: January 2020

(1-->3)-beta-D-Glucan is one of the major structural components of fungi, and it seems that it can be detected by the fractionated (1-->3)-beta-D-glucan-sensitive component from a Limulus lysate, factor G. We evaluated the concentration of (1-->3)-beta-D-glucan by using factor G and other fungal antigens in 24 patients with clinical evidence of mycosis and 36 healthy subjects. The mean concentration of (1-->3)-beta-D-glucan in the plasma of the healthy subjects was found to be 2.7 +/- 1.9 pg/ml (range, < 6.9 pg/ml), and it was found to be substantially higher in all 11 patients with candidemia (mean, 2,207.4 pg/ml; range, 325.4 to 8,449.0 pg/ml). Eight of those 11 patients with candidemia (73%) were positive for the Cand-Tec heat-labile candida antigen and only 3 patients (27%) were positive for mannan antigen. Three patients with invasive pulmonary aspergillosis were positive for galactomannan and had, in addition, high concentrations of (1-->3)-beta-D-glucan (mean, 323.3 pg/ml; range, 27.0 to 894.0 pg/ml). All 10 patients with cryptococcosis (including 2 patients with probable cryptococcosis) were positive for cryptococcal antigen by the Eiken latex test; however, (1-->3)-beta-D-glucan levels were not elevated in these patients (mean, 7.0 pg/ml; range, < 16.5 pg/ml). Our results indicated that (1-->3)-beta-D-glucan levels are elevated in patients with candidiasis and aspergillosis but not in those with cryptococcosis.
Comparison of arbitrarily primed PCR with restriction endonuclease and immunoblot analyses for typing Clostridium difficile isolates
Authors: Tang Y J , Houston S T , Gumerlock P H , Mulligan M E , Gerding D N , Johnson S , Fekety F R , Silva J
Published: January 2020

Arbitrarily primed PCR (AP-PCR) was used to genotype 26 clinical isolates of Clostridium difficile previously analyzed by immunoblotting (IB) and 20 isolates typed by restriction endonuclease analysis (REA) with HindIII. Two levels of differentiation were achieved with the AP-PCR approach by use of two different arbitrary primers. With the 19-mer arbitrary primer T-7 (first level of differentiation), a good correlation was found between IB and AP-PCR typing. Twenty isolates grouped into six IB types were separated into seven major AP-PCR types. These seven AP-PCR groups were further discriminated into 12 subtypes after genotyping with the arbitrary primer PG-05 (second level of differentiation). The remaining six isolates, all of different IB types, showed a unique and distinct DNA banding pattern with both of the arbitrary primers, T-7 and PG-05. Twenty isolates representing 20 REA types from 15 REA groups were resolved into 13 AP-PCR DNA profiles with the arbitrary primer T-7. A good correlation was found at this level of differentiation between the major REA groups, Y and M, and AP-PCR typing. While AP-PCR with this primer failed to differentiate isolates in REA groups J, G, R, and B, AP-PCR with PG-05 resolved these four isolates into four distinct AP-PCR types. In addition, one of three M strains and one of four Y strains displayed a slightly different DNA banding pattern by AP-PCR (with PG-05) from that of the other strains in the group. We conclude that AP-PCR is a rapid and sensitive method which not only complements other typing schemes but also may be a substitute and prove to be especially suited for immediate epidemiological tracking of nosocomial infections due to C. difficile.
Evaluation of PCR and indirect enzyme-linked immunosorbent assay on milk samples for diagnosis of brucellosis in dairy cattle
Authors: Romero C , Pardo M , Grillo M J , Diaz R , Blasco J M , Lopez-Goñi I
Published: January 2020

A study was performed to evaluate the previously described PCR (C. Romero, C. Gamazo, M. Pardo, and I. López-Goñi, J. Clin. Microbiol. 33:615-617, 1995) for the diagnosis of brucellosis in dairy cattle. Milk samples from 56 Brucella milk culture-positive cattle and from 37 cattle from Brucella-free herds were examined for Brucella DNA by PCR and for specific antibodies by an indirect enzyme-linked immunosorbent assay (ELISA). The specificities of both tests were 100% when testing the milk samples from Brucella-free cattle. The milk samples from 49 infected cattle were positive by PCR (87.5% sensitivity), and 55 were positive by ELISA (98.2% sensitivity). A PCR-positive sample was negative by ELISA, and 7 ELISA-positive samples were PCR negative, yielding an observed proportion of agreement of 0.91 for the two tests. Although the results suggest that ELISA is a better screening test than PCR, the combined sensitivity of the two assays was 100%, and their simultaneous application could be more useful than one test alone for a rapid screening of brucellosis in dairy cattle.
Screening for Escherichia coli O157:H7--a nationwide survey of clinical laboratories
Authors: Boyce T G , Pemberton A G , Wells J G , Griffin P M
Published: January 2020

The Association of State and Territorial Public Health Laboratory Directors has recommended that clinical laboratories screen at least all bloody stools for Escherichia coli O157:H7. We contacted the microbiology supervisors of 230 randomly selected clinical laboratories in the United States and administered a standardized questionnaire regarding stool culture practices. Of the 129 laboratories that performed stool cultures, only 70 (54%) reported screening either all stools or all bloody stools submitted for culture for E. coli O157:H7.
Antigens from Taenia crassiceps cysticerci used in complement fixation, enzyme-linked immunosorbent assay, and western blot (immunoblot) for diagnosis of neurocysticercosis
Authors: Garcia E , Ordoñez G , Sotelo J
Published: January 2020

Antigens from Taenia solium cysticerci for the immunodiagnosis of cysticercosis are scarce and difficult to obtain. We studied the reliability of antigens from Taenia crassiceps cysticerci as a substitute for those from T. solium in three diagnostic tests: complement fixation, enzyme-linked immunosorbent assay, and Western blot (immunoblot). Precision rates of the three tests of 93, 99, and 96%, respectively, were found. Cysticerci from T. crassiceps can be easily obtained in large quantities and can be effectively used for the diagnosis of human neurocysticercosis.
Evaluation of API Campy in comparison with conventional methods for identification of thermophilic campylobacters
Authors: Huysmans M B , Turnidge J D , Williams J H
Published: January 2020

API Campy was compared with conventional biochemical methods for its ability to identify 100 thermophilic campylobacter isolates. When the results were read according to the manufacturer's instructions, API Campy showed 92% agreement with conventional methods. Extended incubation of the assimilation strip resulted in the correct identification of an additional two isolates. Discrepant results occurred for six isolates. Overall, API Campy offered no advantages over conventional methods.
Isolation and characterization of a beta-D-glucuronidase-producing strain of Escherichia coli serotype O157:H7 in the United States
Authors: Hayes P S , Blom K , Feng P , Lewis J , Strockbine N A , Swaminathan B
Published: January 2020

A phenotypic variant of Escherichia coli serotype O157:H7 (G5101) was isolated from a patient with bloody diarrhea. Strain G5101 does not ferment sorbitol but is beta-D-glucuronidase and urease positive. Serotyping and colony hybridization using a serotype-specific DNA probe confirmed that the isolate was O157:H7. G5101 produces Shiga-like toxins I and II and contains an eae gene that is highly conserved in the O157:H7 serotype. This strain would have been missed by laboratories that screen for the sorbitol-negative, beta-D-glucuronidase-negative phenotype in isolating E. coli O157:H7 from clinical and food specimens.
Geographical heterogeneity between Far East and Europe in prevalence of ypm gene encoding the novel superantigen among Yersinia pseudotuberculosis strains
Authors: Yoshino K , Ramamurthy T , Nair G B , Fukushima H , Ohtomo Y , Takeda N , Kaneko S , Takeda T
Published: January 2020

Yersinia pseudotuberculosis is known to produce a novel superantigen designated Y. pseudotuberculosis-derived mitogen. In the present study, we demonstrated that there was a distinct geographical heterogeneity between the Far East (higher prevalence) and Europe (lower prevalence) in the prevalence of the ypm gene encoding the superantigen among Y. pseudotuberculosis strains.
Evaluation of API An-IDENT and RapID ANA II systems for identification of Actinomyces species from clinical specimens
Authors: Miller P H , Wiggs L S , Miller J M
Published: January 2020

We compared the accuracy of the An-IDENT system (bioMerieux Vitek, Inc., Hazelwood, Mo.) and the RapID ANA II system (Innovative Diagnostic Systems, Norcross, Ga.) with that of conventional biochemical tests for the identification of 85 strains of Actinomyces species. In our hands, the overall accuracy of the An-IDENT was 59% and that of the RapID ANA II was 24%. The error rate for the An-IDENT was 18% and that for the RapID ANA II was 38%. The results of this study suggest that although the An-IDENT was more accurate than the RapID ANA II (P < 0.005), neither system, in our hands, was able to identify Actinomyces species with an acceptable degree of accuracy. It is recommended that suspected Actinomyces isolates be identified by conventional testing.
Enzyme-linked immunosorbent assay for detecting antibodies to Borna disease virus-specific proteins
Authors: Briese T , Hatalski C G , Kliche S , Park Y S , Lipkin W I
Published: January 2020

Borna disease virus is a unique neurotropic RNA virus that causes neurologic disease in a wide variety of animal hosts. We established an enzyme-linked immunosorbent assay for the detection of antibodies to Borna disease virus on the basis of the use of three recombinant viral proteins (recp40, recp23, and recp18). This assay system is more sensitive and rapid than the methods currently used for the serologic diagnosis of infection such as Western blotting (immunoblotting), indirect immunofluorescence test, or immunoprecipitation.
Evaluation of enzyme immunoassay for hepatitis B virus DNA based on anti-double-stranded DNA
Authors: Garcia F , Garcia F , Bernal M C , Leyva A , Piedrola G , Maroto M C
Published: January 2020

We have evaluated a new enzyme immunoassay technology to detect the products of PCR-based amplification that may be applicable to routine testing of hepatitis B virus (HBV) DNA. Two hundred eight serum samples were studied: 73 were basal samples and 135 were sequential serum samples from patients with chronic hepatitis, some of whom were being treated with alpha interferon. We compared the new detection method (PCR-DNA enzyme immunoassay [DEIA]) with dot blot hybridization performed without prior PCR amplification and with two other methods for detection of PCR products: agarose gel electrophoresis with ethidium bromide staining (PCR-EB) and dot blot (PCR-dot blot). For hepatitis B-antigen-positive basal samples, HBV DNA was detected in 70.4% by dot blot, 74.1% by PCR-EB, and 100% by PCR-DEIA and PCR-dot blot; for anti-hepatitis B e-antigen basal samples, HBV DNA was found in 10.5% by dot blot and PCR-EB and in 42.1% by PCR-DEIA and PCR-dot blot. Chi-square tests showed a strong association between dot blot and PCR-EB and between PCR-DEIA and PCR dot blot. Using PCR-dot blot as the reference, dot blot shows a 56.9% sensitivity and a 100% specificity, PCR-EB shows a 55.0% sensitivity and a 100% specificity, and PCR-DEIA shows a 95.4% sensitivity and a 97% specificity. We conclude that the technical advantages of the DEIA method and its high sensitivity and specificity may facilitate the use of PCR in routine testing for HBV DNA in clinical microbiology laboratories.
First reported case of Aspergillus granulosus infection in a cardiac transplant patient
Authors: Fakih M G , Barden G E , Oakes C A , Berenson C S
Published: January 2020

We report a case of disseminated infection with Aspergillus granulosus in a cardiac transplant recipient on immunosuppressive therapy. This is the first reported case in which this organism has been described as a pathogen. This organism bears morphological features different from those of more common Aspergillus species and should be considered a potential pathogen in immunocompromised patients.
Serologic evaluation of human immunodeficiency virus type 1-infected individuals from Argentina and the United States indicates a similar distribution of subgroup B isolates
Authors: Warren R Q , Wong M T , Melcher G P , Blatt S P , Cahn P , Perez H , Zapiola I , Bouzas M B , Muchinik G , Anderson S A
Published: January 2020

Utilizing peptides based on the V3 region of gp120, we undertook a serologic examination of human immunodeficiency virus type 1 (HIV-1)-infected individuals from Argentina to determine if prevalent HIV-1 isolates could be identified in this population. Our findings suggest that a similar pool of HIV-1 subgroup B isolates exists in both Argentina and the United States.