bluuebunny/crossref_metadata_2025 huggingface dataset:
Demonstration of circulating group B streptococcal immune complexes in neonates with meningitis
Authors: Vallejo J G , Baker C J , Edwards M S
Published: January 2020

Group B streptococci are the major cause of sepsis and fatal shock in neonates in the United States. Although a number of clinical features have been associated with enhanced severity of disease, the role of soluble immune complex formation in group B streptococcal infection has not been evaluated. We determined the frequency with which circulating immune complexes occurred in 16 infants with nonfatal type III, group B streptococcal meningitis, using an immunoglobulin-specific C1q enzyme immunoassay. Ten healthy, age-matched infants served as a control group. Elevated levels of immunoglobulin M (IgM)-containing immune complexes were present in the sera of four (25%) patients with group B streptococcal meningitis. Group B antigen was detected in precipitated IgM immune complexes from each of these four infants by competitive enzyme-linked immunosorbent assay. In addition, IgG-containing immune complexes were present in 56% of sick and 60% of control infants. Group B antigen was demonstrated in the serum of a sick neonate containing only IgG immune complexes but not in controls. Our findings indicate that a subset of infants with type III, group B streptococcal meningitis develop IgM immune complexes containing group B-specific antigen, and these may persist for up to 3 months in some patients.
Evaluation of third-generation assays for detection of anti-hepatitis C virus (HCV) antibodies and comparison with presence of HCV RNA in blood donors reactive to c100-3 antigen
Authors: Lavanchy D , Mayerat C , Morel B , Schneider P , Zufferey C , Gonvers J J , Pécoud A , Frei P C
Published: January 2020

We tested serum samples from blood donors by using first-, second-, and third-generation enzyme immunoassays or recombinant immunoblot assays. Second- and third-generation assays gave comparable results. Circulating hepatitis C virus RNA was found in a high proportion of reactive samples. A lack of reactivity or low-level reactivity predicted the absence of hepatitis C virus RNA in 100% of the cases.
High frequency of yeast carriage on hands of hospital personnel
Authors: Strausbaugh L J , Sewell D L , Ward T T , Pfaller M A , Heitzman T , Tjoelker R
Published: January 2020

The hands of 36 nurses and 21 nonnursing hospital employees were tested by culture with a modification of the broth wash technique. Seventy-five percent of the nurses and 81% of the nonnurses were found to harbor yeasts on their hands; 58% of nurses and 38% of nonnurses were carrying Candida spp.
Rotavirus serotypes causing severe acute diarrhea in young children in six Australian cities, 1989 to 1992
Authors: Masendycz P J , Unicomb L E , Kirkwood C D , Bishop R F
Published: January 2020

Rotavirus VP7 serotypes were identified in stools from 72.9% (1,302/1,784) of hospitalized Australian children in six cities (1989 to 1992) and comprised 1,088 (83.6%) serotype G1 isolates, 84 (6.4%) serotype G2 isolates, 64 (4.9%) serotype G3 isolates, 49 (3.8%) serotype G4 isolates, and 17 (1.3%) isolates of mixed serotypes. The most densely populated cities yielded the greatest diversity of serotypes.
Demonstration of persistence of Salmonella typhimurium in an AIDS patient by molecular methods
Authors: Fica A E , Horowitz H W , Lior H , Cabello F C
Published: January 2020

We document microbiological persistence of the same Salmonella typhimurium strain in an AIDS patient during 7 months of clinical observation despite prolonged quinolone therapy. Persistence was demonstrated by phage types that closely resembled one another, similar antibiotic resistance patterns, conserved restriction fragment length polymorphism of chromosomal DNA digested with different DNA restriction enzymes, identical ribotypes, and IS200 types in four characterized sequential isolates of S. typhimurium.
Genotypic and phenotypic analysis of Borrelia burgdorferi isolates from The Netherlands
Authors: Nohlmans L M , de Boer R , van den Bogaard A E , van Boven C P
Published: January 2020

Sixty-three Borrelia burgdorferi isolates recovered from Ixodes ricinus ticks collected in 17 locations in The Netherlands and three Dutch human skin isolates were characterized by rRNA gene restriction fragment length polymorphism, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting (immunoblotting). All three human isolates belonged to B. burgdorferi group VS461. Of the tick isolates, 29 (46%) were B. burgdorferi sensu stricto, 2 (3%) were group VS461, 19 (30%) were Borrelia garinii, and 13 (21%) were different from any previously described genomic species. On the basis of the criteria described, 12 isolates formed a distinct genomic group, designated M19. rRNA gene restriction patterns of the group M19 isolates resembled but were not identical to the B. garinii patterns. Hybridization of digested DNA with a flagellin probe confirmed the separation of group M19 from the B. garinii isolates. One isolate, M63, was different from all the others. In conclusion, the occurrence of B. burgdorferi sensu stricto, B. garinii, and B. burgdorferi group VS461 in ticks from The Netherlands corresponds with the occurrence of these genomic species among tick isolates from other European countries. However, our findings suggest that B. burgdorferi sensu lato probably contains more than three genomic species.
Detection and quantitation of the glucuronoxylomannan-like polysaccharide antigen from clinical and nonclinical isolates of Trichosporon beigelii and implications for pathogenicity
Authors: Lyman C A , Devi S J , Nathanson J , Frasch C E , Pizzo P A , Walsh T J
Published: January 2020

Sera from patients with systemic infections caused by the opportunistic fungus Trichosporon beigelii have been shown to cross-react with anticryptococcal antibodies. We quantitatively compared the amounts of antigen produced and examined the expression of O-acetyl epitopes from 35 strains of T. beigelii isolated from deep and superficial infections. By counterimmunoelectrophoresis, 10 of 10 isolates from deep infections were positive for polysaccharide, compared with 7 of 13 isolates from superficial infections (P = 0.02). All 23 strains tested were positive for polysaccharide when screened by immunodot. By enzyme immunoassay, the cross-reactive antigen produced by deep isolates (n = 9) had a mean titer of 1:5,500. In contrast, superficial isolates (n = 22) produced significantly less antigen than the deep isolates (P < 0.001), with a mean titer of 1:700. Isolates from environmental sources (n = 3) were similar to the superficial isolates, with a mean titer of 1:600. The mean concentrations +/- standard errors of cross-reactive polysaccharide released by deep isolates and superficial isolates were 3.09 +/- 0.44 and 1.74 +/- 0.30 micrograms/ml, respectively, when measured by rocket immunoelectrophoresis (P = 0.02). O-Acetyl epitopes were detected on polysaccharide from 8 of 9 strains of T. beigelii isolated from deep sources, while only 2 of 12 superficial isolates expressed detectable O-acetyl epitopes (P = 0.01). Thus, while all isolates of T. beigelii tested were capable of producing glucuronoxylomannan-like cross-reactive antigen, pathogenic isolates produced significantly more antigen than superficial or environmental isolates. Furthermore, significantly more pathogenic isolates than superficial or environmental isolates expressed antigen that was O acetylated.
Isolation of the newly described species Mycobacterium celatum from AIDS patients
Authors: Tortoli E , Piersimoni C , Bacosi D , Bartoloni A , Betti F , Bono L , Burrini C , De Sio G , Lacchini C , Mantella A
Published: January 2020

Mycobacterium celatum is a recently described species which, on the basis of conventional tests, may be misidentified as Mycobacterium xenopi or as belonging to the Mycobacterium avium complex. Only genomic sequencing or high-performance liquid chromatography of cell wall mycolic acids can presently allow a correct identification of this mycobacterium. Two cases of infection due to M. celatum, in AIDS patients, are described here. The quantitative susceptibility pattern of the isolates to a wide spectrum of drugs is also reported.
Suggested modifications for disk diffusion susceptibility testing criteria for levofloxacin and sparfloxacin following tests with a predictor panel of ciprofloxacin-resistant clinical isolates
Authors: Sutton L D , Jones R N
Published: January 2020

A predictor panel of 300 clinical bacterial isolates (200 resistant to ciprofloxacin) was used to compare 5-micrograms disk diffusion test results with the MICs of ofloxacin (control), levofloxacin, and sparfloxacin. Regression analysis demonstrated high correlations between the methods for all three fluoroquinolones (r > or = 0.95). In order to minimize disk diffusion testing errors among the fluoroquinolone-resistant strains, the following modifications to previously proposed or published interpretive criteria were suggested: for levofloxacin, susceptible at > or = 17 mm (< or = 2 micrograms/ml) and resistant at < or = 13 mm (> or = 8 micrograms/ml); for sparfloxacin, susceptible at > or = 20 mm (< or = 1 microgram/ml) and resistant at < or = 16 mm (> or = 4 micrograms/ml). The study control drug, ofloxacin, did not appear to possess a significant error rate (5% minor error) when fluoroquinolone-resistant strains were tested, and no modifications were proposed. Under these proposed interpretive criteria, the absolute categorical agreements between standardized susceptibility testing methods for levofloxacin and sparfloxacin results were 91.3 and 94.0%, respectively (< or = 0.3% major errors and nil very major errors).
Quantitative molecular monitoring of human immunodeficiency virus type 1 activity during therapy with specific antiretroviral compounds
Authors: Bagnarelli P , Menzo S , Valenza A , Paolucci S , Petroni S , Scalise G , Sampaolesi R , Manzin A , Varaldo P E , Clementi M
Published: January 2020

Methods for the absolute quantitation of nucleic acids present in small amounts in biological samples (competitive PCR and competitive reverse transcription PCR) were applied to the direct monitoring of specific anti-human immunodeficiency virus type 1 (HIV-1) therapy. With these techniques, different parameters of HIV-1 activity (including genomic RNA copy numbers in plasma, proviral and late transcript copy numbers in peripheral blood lymphocytes, and mean transcriptional activity per each HIV-1 provirus) were monitored during therapy with azidothymidine or ddI. In most of these treated patients, a direct response to the antiretroviral compounds employed was detected during the first few weeks of treatment, as documented by a fast decrease of all molecular indexes of HIV-1 activity. However, residual viral replication (albeit at minimal levels) was documented during therapy in all subjects monitored in this study. In a minority of the patients under study (3 of 12), the drug-dependent viral inhibition was maintained throughout the observation time (213 to 791 days), but in 9 patients a rebound in viremia level was detected during therapy with competitive reverse transcription PCR. Sequencing analysis of a portion of the HIV-1 gene pol from cell-free virions showed that circulating viral variants bearing at least two mutations compatible with azidothymidine or ddI resistance were detectable in the patients who exhibited a rebound in cell-free HIV-1 genomic RNA copy numbers in plasma but not in one patient who maintained (for 455 days) lowered levels of viral load during ddI treatment.
Low human immunodeficiency virus type 1 (HIV-1) DNA burden as a major cause for failure to detect HIV-1 DNA in clinical specimens by PCR
Authors: Zazzi M , Romano L , Catucci M , De Milito A , Almi P , Gonnelli A , Rubino M , Valensin P E
Published: January 2020

To determine the sensitivity of a nested PCR procedure for detecting human immunodeficiency virus type 1 DNA in clinical specimens, 553 peripheral blood mononuclear cell samples obtained from 268 human immunodeficiency virus type 1-seropositive subjects were assayed by use of two independent primer sets for each sample. Overall, 1,088 of 1,106 (98.37%) reactions were positive. Investigation of the negative reactions showed that a low viral burden in some infected subjects, rather than primer-template mismatches, was the primary cause for the false-negative PCR results.
Quantitative analysis of syncytium-inducing and non-syncytium-inducing virus in patients infected with human immunodeficiency virus type 1
Authors: Shafer R W , Aguiniga E , Merigan T C
Published: January 2020

Among 75 consecutive human immunodeficiency virus type 1 (HIV-1)-infected patients with moderate and advanced immunosuppression, those harboring syncytium-inducing (SI) HIV-1 had a lower CD(4+)-cell count (145 versus 278 cells per microliter, P < 0.001) and 10-fold-higher virus titers than patients with non-SI HIV-1 (398 versus 39 infectious units per 10(6) CD4+ lymphocytes; P < 0.001). In patients with SI virus, the mean titer of SI virus, determined with a quantitative MT-2 cell assay, was 135 SI infectious units per 10(6) CD4+ lymphocytes. Virus titer correlated inversely with CD(4+)-cell count in patients with SI (r = -0.67) but not non-SI (r = -0.29) virus.
Length of incubation time for human immunodeficiency virus cultures
Authors: Fiscus S A , Welles S L , Spector S A , Lathey J L
Published: January 2020

Qualitative human immunodeficiency virus culture is a slow, labor-intensive, and expensive procedure, yet critical for the diagnosis of infants born to human immunodeficiency virus-seropositive mothers. We report that the cultures can be terminated at day 21 with minimal false-negative results but with considerable savings in both time and money.
Subtyping of hepatitis C virus isolates by a line probe assay using hybridization
Authors: Andonov A , Chaudhary R K
Published: January 2020

A reverse hybridization test (Inno-LiPA HCV; Innogenetics, N.V., Zwijnaarde, Belgium) was used for typing hepatitis C virus. All 38 samples, typed by PCR with primers from core and NS5 genes, were also genotyped by this test. Of the samples, 33 (87%) had the same subtypes by both assays. The correlations between PCR and Inno-LiPA for individual types were 77% for type I (1a), 90% for type II (1b), 100% for type III (2a), 100% for type IV (2b), and 100% for type V (3a). One of the type III (2a) samples also reacted with type I (1a) probes in the Inno-LiPA test.
Detection of Helicobacter pylori gene expression in human gastric mucosa
Authors: Peek R M , Miller G G , Tham K T , Pérez-Pérez G I , Cover T L , Atherton J C , Dunn G D , Blaser M J
Published: January 2020

Mucosal and systemic immunologic recognition of cagA by Helicobacter pylori-infected individuals is associated with peptic ulcer disease; however, in the laboratory, expression of cagA is subject to artificial conditions which may not accurately reflect the conditions in host tissues. Gastric antral and body biopsy specimens and serum for anti-H. pylori immunoglobulin G serology were obtained from 42 patients. Biopsy specimens were studied by histology, culture, and reverse transcription PCR (RT-PCR). Oligonucleotide primers specific for H. pylori (16S rRNA, ureA, and cagA) were used to detect bacterial mRNA in gastric biopsy specimens. PCR was performed on DNA from corresponding H. pylori isolates to detect genomic 16S rRNA, ureA, and cagA. Of the 42 patients from whom clinical specimens were obtained, 25 were infected with H. pylori on the basis of both serology and histology or culture (i.e., tissue positive); 13 were negative by serology, histology, and culture; and 4 were positive by serology only. RT-PCR with 16S rRNA primers detected 24 of 25 tissue-positive and 0 of 17 tissue-negative patients (P < 0.001). RT-PCR with ureA primers detected 16 of 25 tissue-positive and 0 of 17 tissue-negative patients (P < 0.001). CagA mRNA was detected by RT-PCR in 14 of 25 gastric biopsy specimens in the tissue-positive group and in 0 of 17 gastric biopsy specimens in the tissue-negative group. PCR of genomic DNA for the presence of the cagA gene in the corresponding bacterial isolates correlated absolutely with cagA gene expression in gastric tissue. These results indicate that RT-PCR is a sensitive and specific method for the detection of the presence of H. pylori and the expression of H. pylori genes in human gastric tissue. Detection of H. pylori gene expression in vivo by this approach may contribute to improving the diagnosis and understanding the pathogenesis of H. pylori infections.
Comparative study of five methods for intratypic differentiation of polioviruses
Authors: van der Avoort H G , Hull B P , Hovi T , Pallansch M A , Kew O M , Crainic R , Wood D J , Mulders M N , van Loon A M
Published: January 2020

A coded panel of 90 poliovirus isolates, 30 of each of the three known serotypes, was used to evaluate five methods for the intratypic differentiation of polioviruses: (i) an enzyme-linked immunosorbent assay with polyclonal cross-absorbed antisera (PAb-E), (ii) a neutralization assay with type-specific monoclonal antibodies (MAb-N), (iii) a restriction fragment length polymorphism (RFLP) assay, (iv) a Sabin vaccine strain-specific PCR assay, and (v) a Sabin vaccine strain-specific cRNA probe hybridization (ProHyb) assay. Sequence analysis was used for the definitive characterization of the strains. The panel was distributed to five laboratories; each laboratory analyzed the strains by at least two methods. Each method was used by three or four laboratories. The total performance scores (percentage correct results per number of tests) of the five methods were 96.7% for PAb-E, 93.9% for MAb-N, 91.9% for RFLP assay, 93.3% for Sabin vaccine strain-specific PCR, and 97.4% for Sabin vaccine strain-specific ProHyb. Consistent results were obtained by each laboratory for 88 of 90 isolates (97.8%) examined by PAb-E, 81 of 90 isolates (90.0%) examined by MAb-N, 78 of 90 isolates (86.7%) examined by RFLP assay, 81 of 90 isolates (90.0%) examined by PCR, and 89 of 90 isolates (98.9%) examined by ProHyb assay. Six strains were classified differently by different methods. It is recommended that at least two methods be used for the intratypic differentiation of poliovirus isolates, and each method should be based on a different principle (i.e., antigenic properties and nucleotide sequence composition). If two assays yield discrepant results, further characterization, preferably by partial sequence determination, will be required for correct identification.
Use of PCR and direct immunofluorescence microscopy for confirmation of results obtained by Syva MicroTrak Chlamydia enzyme immunoassay
Authors: Ostergaard L , Møller J K
Published: January 2020

A procedure for use of the Amplicor Chlamydia PCR with the Syva MicroTrak enzyme immunoassay (EIA) medium was developed, and the performance of the Syva MicroTrak EIA was evaluated by use of PCR and the Syva MicroTrak direct immunofluorescence assay (DFA) as confirmatory methods. PCR detected Chlamydia organisms at a 10-fold greater dilution than did DFA. Of 366 specimens, 119 specimens were positive by both PCR and DFA, 6 specimens were positive only by PCR, and 241 specimens were negative by both PCR and DFA. Subsequently, DFA and the developed PCR procedure were used prospectively for confirmation of EIA results in a defined negative gray zone between the cutoff value and 30% of the cutoff value (70% below the cutoff value). All specimens with results above the EIA cutoff value were also subjected to confirmation with DFA and PCR. EIA was performed on 7,748 endocervical swab specimens, of which 494 (6.4%) were subjected to confirmation, and on 968 male urethral swab specimens, of which 185 (19.1%) were subjected to confirmation. A "gold standard" was based on the findings by DFA and PCR, and divergent results were resolved by a major outer membrane protein-based PCR. Forty-five of 160 female specimens (28.1%) and 11 of 93 male specimens (11.8%) within the defined negative gray zone were found to be positive. Of 334 female specimens having absorbance unit (AU) values above the EIA cutoff value, 258 could be confirmed, thereby giving a positive predictive value of 77% (258/334). Accordingly, the positive predictive value with male specimens was 95% (87/92). The prevalence of Chlamydia trachomatis-positive specimens was 3.9% (303/7,748) in females and 10.1% (98/968) in males. All male specimens having AU values above 1.0 in the EIA were confirmed positive. In contrast to this, 16 females with AU values above 1.0 in the EIA could not be confirmed positive with either DFA or PCR. The mean age of these females was higher than that of patients testing negative for C. trachomatis (P < 0.005). This might suggest an age-dependent change in vaginal colonization with an organism(s) crossreacting in the EIA. Thus, the PCR procedure developed can be used for confirmation of EIA results, testing specimens with AU values in the defined negative gray zone improves the sensitivity of EIA, and all specimens testing positive in EIA should be subjected to confirmation.
Detection of Theileria annulata in blood samples of carrier cattle by PCR
Authors: d'Oliveira C , van der Weide M , Habela M A , Jacquiet P , Jongejan F
Published: January 2020

We report the detection of Theileria annulata, the causative agent of tropical theileriosis, by PCR in blood samples obtained from carrier cattle. The assay employs primers specific for the gene encoding the 30-kDa major merozoite surface antigen of T. annulata. A 721-bp fragment was amplified from blood samples taken monthly from calves experimentally infected with one of four different stocks of T. annulata originating in either Mauritania, Portugal, Spain, or Turkey. At the end of the experiment, five animals carried the infection for 12 months and two animals remained infected for 15 months. DNAs from six other Theileria species, T. parva, T. mutans, T. sergenti, T. buffeli, T. velifera, and T. taurotragi, were not amplified. Moreover, DNAs from four other hemoparasites (Anaplasma centrale, Anaplasma marginale, Babesia bovis, and Babesia bigemina) were also not amplified. As a control, primers derived from the small subunit rRNA gene of Theileria spp. amplified a 1.1-kb DNA fragment from all Theileria species examined but not from the other four hemoparasites. As few as two to three parasites per microliter of infected blood in a 50-microliters sample volume were detected by Southern or microplate hybridization with a T. annulata-specific cDNA probe. In addition, 92 field samples obtained from cattle in Spain were tested; 22% were positive in blood smears, 40% were positive by immunofluorescent antibody test, and 75% were positive for T. annulata by PCR. The method provides a useful diagnostic tool for detecting T. annulata carrier cattle.
Cervical lymphadenitis caused by a fastidious mycobacterium closely related to Mycobacterium genavense in an apparently immunocompetent woman: diagnosis by culture-free microbiological methods
Authors: Bosquée L , Böttger E C , De Beenhouwer H , Fonteyne P A , Hirschel B , Larsson L , Meyers W M , Palomino J C , Realini L , Rigouts L
Published: January 2020

Fastidious mycobacteria usually infect immunocompromised hosts (human immunodeficiency virus-infected or otherwise immunosuppressed patients). We here describe severe lymphadenitis, caused by a fastidious mycobacterium closely related to Mycobacterium genavense, in an apparently immunocompetent woman, whose brother had died from an unidentified mycobacterial infection in 1969. A variety of techniques, including inoculation of nude mice, histopathology, electron microscopy, lipid analysis, ATP measurements, and molecular biology, were used to characterize this mycobacterium. All attempts to culture the etiological agent on many different media failed. The organism multiplied only in congenitally athymic nude mice. Although phenotypically similar to M. genavense, the mycobacterium differs from M. genavense by three nucleotides of the 16S rRNA gene sequence. Various antimycobacterial drugs were administered, including gamma interferon, but multiple relapses occurred. Finally, therapy with a combined regimen of clarithromycin, clofazimine, rifabutin, and ethambutol was curative. To our knowledge, this is the first report of lymphadenitis in an apparently immunocompetent patient, caused by a noncultivable Mycobacterium sp. closely related to M. genavense. This study emphasizes the importance of employing a variety of diagnostic approaches such as the inoculation of laboratory animals, histopathology, electron microscopy, lipid analysis, ATP measurements, and molecular biology to characterize novel microorganisms that cannot be cultured in vitro.
Identification of "Streptococcus milleri" group isolates to the species level with a commercially available rapid test system
Authors: Flynn C E , Ruoff K L
Published: January 2020

Clinical isolates of the "Streptococcus milleri" species group were examined by conventional methods and a rapid, commercially available method for the identification of these strains to the species level. The levels of agreement between the identifications obtained with the commercially available system (Fluo-Card Milleri; KEY Scientific, Round Rock, Tex.) and conventional methods were 98% for 50 Streptococcus anginosus strains, 97% for 31 Streptococcus constellatus strains, and 88% for 17 isolates identified as Streptococcus intermedius. Patient records were also studied in order to gain information on the frequency and sites of isolation of each of the three "S. milleri" group species.
Characterization of ear fluid isolates of Alloiococcus otitidis from patients with recurrent otitis media
Authors: Bosley G S , Whitney A M , Pruckler J M , Moss C W , Daneshvar M , Sih T , Talkington D F
Published: January 2020

Nineteen isolates of Alloiococcus otitidis from ear fluid samples collected by tympanostomy from patients at four geographic locations were identified by phenotypic characterization and genetic relatedness. Initial growth of A. otitidis isolates occurred after 3 days at 37 degrees C on brain heart infusion (BHI) agar with 5% rabbit blood. Heavy growth occurred in BHI broth supplemented with 0.07% lecithin and 0.5% Tween 80 after 4 days of incubation. The isolates were gram-positive cocci that divided on an irregular plane and produced metabolic lactic acid, pyrrolidonyl arylamidase, and leucine aminopeptidase. These cocci grew sparsely in 6.5% NaCl-BHI broth, were asaccharolytic on both fermentative and oxidative bases, and were cytochrome negative by the iron-porphyrin test. The cellular fatty acid profile of A. otitidis was distinguished from those of related genera and characterized by major amounts ( > or = 14%) of 16:0, 18:2, 18:1 omega 9c, and 18:0 and smaller amounts of 14:0, 16:1 omega 7c, 17:0, and 18:1 omega 7c. Fifteen isolates demonstrated > 69% relatedness by DNA-DNA hybridization. Four isolates plus the original 15 were confirmed as A. otitidis by dot blot hybridization with a digoxigenin-labeled nucleotide probe specific for this species. The intergenic space between the genes coding for the 16S and 23S rRNAs of alloiococci was amplified by PCR, analyzed by restriction fragment length polymorphism, and determined to consist of three different genetic types. Although beta-lactamase negative, A. otitidis demonstrated intermediate levels of resistance to beta-lactams, including expanded-spectrum cephalosporins, and were resistant to trimethoprim-sulfamethoxazole and erythromycin.
Case of recurrent Flavimonas oryzihabitans bacteremia associated with an implanted central venous catheter (Port-A-Cath): assessment of clonality by arbitrarily primed PCR
Authors: Verhasselt B , Claeys G , Elaichouni A , Verschraegen G , Laureys G , Vaneechoutte M
Published: January 2020

Flavimonas oryzihabitans bacteremias, which occurred immediately after the flushing or use of an implanted central venous catheter (Port-A-Cath) in two patients at the same pediatric ward, were studied by arbitrarily primed PCR. We conclude that the colonization of the Port-A-Cath with F. oryzihabitans described here lasted for several months.
Screening for antimicrobial resistance in fecal samples by the replica plating method
Authors: Osterblad M , Leistevuo T , Huovinen P
Published: January 2020

Replica plating can be used for the detection of antibiotic resistance in normal flora. We have evaluated this application of the replica plating method by comparing it with a five-colony method. The replica plating method uses a single plate for each antibiotic, with a concentration just above that for borderline resistance. By the five-colony method, five colonies per sample were picked, chosen to represent all different colony morphologies present, and MICs were determined by a standard agar dilution method. The gram-negative, aerobic floras of 131 fecal samples were screened for resistance to ampicillin, cefuroxime, nalidixic acid, trimethoprim, sulfamethoxazole, and tetracycline by both methods. The rate of resistance detection by the two methods did not differ statistically for any of the antibiotics tested. The breakpoint concentrations used for the replica plates in the study gave results similar to those produced by the agar dilution method and the breakpoint values of the National Committee for Clinical Laboratory Standards and can thus be recommended. As the only currently used resistance detection method, replica plating facilitates an exact determination of the percentage of resistant colonies/total number of colonies (between 1 and 100%) in a sample. This revealed an uneven distribution, with only 23% of the samples having resistance frequencies in the range of 10 to 85%; usually, the resistant flora either was a small minority or was very dominant in samples with resistance. This phenomenon was present for all of the antibiotics.
Typing of LaCrosse, snowshoe hare, and Tahyna viruses by analyses of single-strand conformation polymorphisms of the small RNA segments
Authors: Black W C , Vanlandingham D L , Sweeney W P , Wasieloski L P , Calisher C H , Beaty B J
Published: January 2020

Single-strand conformation polymorphism analysis was developed to differentiate the small RNA segments of three California serogroup bunyaviruses. The small RNA segments of La Crosse, snowshoe hare, and Tahyna viruses were reverse transcribed and PCR amplified. The cDNAs were then denatured, rapidly chilled to promote intrastrand reassociation, separated electrophoretically on a nondenaturing gel at room temperature, and silver stained. The resulting single-strand conformation polymorphism patterns were specific for the respective viruses. This molecular technique offers great potential for virus typing and taxonomic studies.
Prolonged Bartonella bacteremia in cats associated with cat-scratch disease patients
Authors: Kordick D L , Wilson K H , Sexton D J , Hadfield T L , Berkhoff H A , Breitschwerdt E B
Published: January 2020

Recent evidence supports a causal relationship between Bartonella (Rochalimaea) henselae, cat-scratch disease (CSD), and bacillary angiomatosis. Cats appear to be the primary reservoir. Blood from 19 cats owned by 14 patients diagnosed with CSD was cultured. Blood samples from cats owned by veterinary students (n = 25) having no association with CSD or bacillary angiomatosis were cultured as controls. Eighty-nine percent (17 of 19) of cats associated with CSD patients and 28% (7 of 25) of controls were bacteremic with Bartonella species (chi-square = 16.47; P < 0.001). Twenty-three isolates were characterized as B. henselae, while one isolate from the cat of a CSD patient appeared to be a new Bartonella species. Thirteen cats remained culture positive during the ensuing 12-month period. Our results support the conclusion that B. henselae is the predominant species involved in CSD and is transmitted by cats. The incidence of Bartonella bacteremia in control cats suggests that B. henselae bacteremia is prevalent among the domestic cat population in the United States.
Recovery of mycobacteria from blood in mycobacteria growth indicator tube and Lowenstein-Jensen slant after lysis-centrifugation
Authors: Hanna B A , Walters S B , Bonk S J , Tick L J
Published: January 2020

Five hundred five blood samples for culture were processed in the Isolator lysis-centrifugation system and were then inoculated into a Mycobacteria Growth Indicator Tube (MGIT) and onto a Lowenstein-Jensen (L-J) slant. Forty-nine isolates of Mycobacterium avium complex and three isolates of Mycobacterium tuberculosis were recovered from 50 of the blood culture specimens. Forty-five isolates from 43 specimens were recovered in the MGIT, with a mean time to detection of 21 days. Forty-one isolates from 40 specimens were recovered in the L-J slants, and the mean time to detection was 36 days. Nine specimens were positive in the MGIT alone, while seven specimens were positive only in L-J medium.
Inhibitory effect of sodium dodecyl sulfate in detection of Mycobacterium tuberculosis by amplification of rRNA
Authors: Manterola J M , Gamboa F , Lonca J , Matas L , Ruiz Manzano J , Rodrigo C , Ausina V
Published: January 2020

The concentrations of sodium dodecyl (lauryl) sulfate (SDS) found in the sediments of the respiratory samples pretreated with SDS-NaOH varied between 3.36 and 12.42 mg/ml. These concentrations of SDS were higher than the level considered critical (> or = 0.16 mg/ml) to obtain negative results with the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test. Extensive washing, after neutralization of sediment, is sufficient to remove all traces of detergent which interfere with the enzymes used by the Mycobacterium Tuberculosis Direct Test assay.
Detection and subsequent sequencing of Puumala virus from human specimens by PCR
Authors: Hörling J , Lundkvist A , Persson K , Mullaart M , Dzagurova T , Dekonenko A , Tkachenko E , Niklasson B
Published: January 2020

A sensitive method based on PCR was developed for the detection of Puumala virus (PUU) in human samples. The assay was found to be specific for PUU-like strains and distinguished between these and hantaviruses of other serotypes. The detection limit was found to be 10(-5) focus-forming units. Clinical samples were collected from patients with nephropathia epidemica in Sweden and western Russia. Five whole blood samples collected from patients in Russia with the acute phase of disease were found to be positive by the PCR. All samples were negative for PUU antigen when examined by enzyme-linked immunosorbent assay. Virus isolation on Vero E6 cells from several of the acute-phase samples, including the 5 PCR-positive samples, was not successful. The amplified samples were subjected to direct nucleic acid sequencing for confirmation of identity. The sequences differed from each other and were closely related to the Russian bank vole isolate CG-1820, thereby indicating the origin of nephropathia epidemica. The PCR was used for amplification and subsequent nucleotide sequencing of eight PUU-like isolates with different geographic origins. The Swedish strains were more closely related to the Finnish PUU prototype strain, Sotkamo, than to the Russian isolates. Interestingly, a Belgian isolate, CG-13891, differed markedly from all other PUU strains.
Rapid detection of bovine viral diarrhea virus by using RNA extracted directly from assorted specimens and a one-tube reverse transcription PCR assay
Authors: Hamel A L , Wasylyshen M D , Nayar G P
Published: January 2020

We describe a simple method for the rapid detection of bovine viral diarrhea virus (BVDV) that uses a one-tube reverse transcription PCR (RT-PCR) and total RNA extracted directly from a variety of bovine specimens, including whole blood and tissues. Reagents for both RT and PCR were combined in a one-tube, single-buffer system, and amplification was performed with a single uninterrupted thermal cycling program. Using the novel cationic surfactant tetradecyltrimethylammonium oxalate (Catrimox-14), we consistently extracted RT-PCR-quality RNA from specimens containing blood. Amplification with primers derived from conserved sequences within the BVDV 5'-untranslated region yielded a 244-bp product. Assay specificity was confirmed by ethidium bromide-stained gel electrophoresis and by chemiluminescence-assayed Southern blot hybridizations involving BVDV 5'-untranslated region-specific digoxigenin-labelled cDNA probes. The assay detection level was 0.1 50% tissue culture infectious dose of BVDV when ethidium bromide-stained gel electrophoresis was used and 0.01 50% tissue culture infectious dose of BVDV when Southern blot hybridization was used. Our method is an alternative to the conventional cell culture assays used in a diagnostic laboratory and is an improvement over existing RT-PCR assays for BVDV.
Molecular population genetic analysis of Staphylococcus aureus recovered from cows
Authors: Kapur V , Sischo W M , Greer R S , Whittam T S , Musser J M
Published: January 2020

Staphylococcus aureus is one of the most common causes of bovine mastitis. To estimate genetic relationships among S. aureus strains recovered from cows, 357 isolates from milk samples from worldwide localities were examined for electrophoretic variation at 13 metabolic-enzyme loci. Thirty-nine electrophoretic types which represented distinctive multilocus enzyme genotypes were identified, and nearly 90% of all isolates were assigned to one of eight clones. Genetic heterogeneity was found among organisms recovered from dairy herds from which multiple isolates were obtained, indicating that the S. aureus population in a single herd can be multiclonal. Although humans and cows shared 7 of the 39 S. aureus clones, each clone was predominantly associated with one of these host species. These results are consistent with the concept of host specialization among S. aureus clones and imply that successful transfer of bacteria between humans and cows is limited.
Evaluation of SHARP signal system for enzymatic detection of amplified hepatitis B virus DNA
Authors: Valentine-Thon E
Published: January 2020

The sensitivity, specificity, reproducibility, detection level, and quantification potential of the SHARP Signal System for enzymatic detection of amplified hepatitis B virus (HBV) DNA in clinical samples were evaluated by testing 104 samples in parallel in a SHARP PCR, an in-house HBV PCR, and a dot blot hybridization assay for semiquantification. SHARP PCR showed a sensitivity of 100%, a specificity of 92.3% (resolved, 100%), a reproducibility of 92.3% (all discrepant serum samples involved very low levels of HBV DNA), and a detection level of at least 3.5 pg/ml. Clinically relevant quantification of the amplified products was not feasible.
Characterization of Paracoccidioides brasiliensis isolates by random amplified polymorphic DNA analysis
Authors: Soares C M , Madlun E E , da Silva S P , Pereira M , Felipe M S
Published: January 2020

We initially used 25 different random primers in order to test their ability to generate random amplified polymorphic DNA fragments from the dimorphic human pathogenic fungus Paracoccidioides brasiliensis. From the tested primers we chose five to distinguish between seven isolates of this microorganism. The DNA amplification patterns allowed clear differentiation of the seven isolates into two distinct groups with only 35% genomic identity. One of these groups contained two subgroups with 81% genetic similarity. The random amplified polymorphic DNA analysis method proved to be a good tool for analyzing and comparing different genomes of P. brasiliensis isolates.
Direct detection of verotoxin-producing Escherichia coli in stool samples by PCR
Authors: Ramotar K , Waldhart B , Church D , Szumski R , Louie T J
Published: January 2020

A method for the rapid detection of verotoxin-producing Escherichia coli in stool samples by PCR was evaluated. Verotoxin-1 and verotoxin-2 genes in DNA extracted directly from stool samples were amplified with oligonucleotide primers. Stools spiked with control organisms, E. coli C600 (H19B) (verotoxin-1) or E. coli C600 (933W) (verotoxin-2), demonstrated that verotoxin-1-containing organisms could be detected at 10(2) CFU per 0.1 g of stool and verotoxin-2-containing organisms could be detected at 10(7) CFU per 0.1 g of stool. Testing of stool samples from patients with diarrhea showed a high concordance between PCR positivity and the presence of verotoxin-producing E. coli, determined by isolation of serotype O157:H7 on sorbitol-MacConkey medium (34 of 35 stool samples) or by colony blots with gene probes (19 of 21 stool samples). Conversely, only 1 of 20 (5.0%) stool samples that were O157:H7 culture negative and colony blot negative and that contained free verotoxin only was positive by PCR. As well, only 4 of 145 (2.8%) stool samples that were negative for serotype O157:H7 or free verotoxin were PCR positive. PCR of DNA extracted directly from stool samples provides a rapid method for the detection of stool samples containing verotoxin-producing E. coli compared with colony blot testing.
Investigation of Candida albicans transmission in a surgical intensive care unit cluster by using genomic DNA typing methods
Authors: Voss A , Pfaller M A , Hollis R J , Rhine-Chalberg J , Doebbeling B N
Published: January 2020

An apparent outbreak of serious Candida albicans infections (n = 6) occurred in a surgical intensive care unit over a 4-week period. Four patients developed C. albicans bloodstream infections. An additional patient developed catheter-related C. albicans infection; the sixth patient developed an infection of cerebrospinal fluid. C. albicans was isolated from the hands of five health care workers (17%) and the throat of one health care worker (3%) during the outbreak investigation. Karyotyping and restriction endonuclease analysis of genomic DNA with BssHII of 23 C. albicans isolates from patients and the 6 health care worker isolates revealed 9 and 12 different patterns, respectively. Three of six patients appeared to be infected with the same C. albicans strain (two bloodstream infections and one cerebrospinal fluid infection). The hands of a health care worker were colonized with strain that appeared identical to an isolate from a patient prior to infection of the patient. However, restriction endonuclease analysis with SfiI found differences among the isolates determined to be identical by the other two methods. Karyotyping alone does not appear to be sufficient to differentiate between outbreak and control isolates. Restriction endonuclease analysis typing may be a more sensitive method than karyotyping alone in the investigation of a cluster of C. albicans infections. Furthermore, the use of more than one restriction enzyme may be necessary for optimal strain discrimination in restriction endonuclease analysis of genomic DNA.
Comparative evaluation of commercial Premier EIA and microimmunodiffusion and complement fixation tests for Coccidioides immitis antibodies
Authors: Kaufman L , Sekhon A S , Moledina N , Jalbert M , Pappagianis D
Published: January 2020

A total of 409 serum and cerebrospinal fluid specimens from human subjects with proven coccidioidomycosis, with other infections, or with no apparent illness were tested for antibodies to Coccidioides immitis by the Premier EIA (Meridian Diagnostics, Inc., Cincinnati, Ohio), which tests for immunoglobulin G (IgG) and IgM responses to coccidioidal antigens, and by the conventional complement fixation (CF) or immunodiffusion (ID) assays for antibodies corresponding to those detected by the tube precipitin (TP) or CF tests. Of the 409 specimens, 47 were from persons with confirmed coccidioidomycosis and all were positive for C. immitis antibodies in IDCF tests and enzyme immunoassays (EIAs) for both IgG and IgM. The EIA for detecting both IgG and IgM antibodies proved to be sensitive for detecting coccidioidomycosis case sera positive by the IDCF, IDTP, and CF tests. Maximal sensitivity for diagnosing coccidioidomycosis is dependent upon detection of both IgG and IgM antibodies in the EIA. The EIA, however, was not absolutely specific, since some sera from patients with confirmed blastomycosis and some from patients with noncoccidioidal disease produced false-positive reactions.
Nonradioactive PCR-enzyme-linked immunosorbent assay method for detection of human cytomegalovirus DNA
Authors: Allen R D , Pellett P E , Stewart J A , Koopmans M
Published: January 2020

We developed a rapid, sensitive, and specific PCR-based assay for human cytomegalovirus (HCMV). The assay includes primer and probe sequences derived from conserved HCMV nucleotide sequences and nonradioactive hybridization-confirmation. The assay detected between 10 and 100 viral genomes. All HCMV clinical isolates tested (39 of 39) gave positive reactions.
Direct reverse transcriptase PCR to determine virulence potential of influenza A viruses in birds
Authors: Horimoto T , Kawaoka Y
Published: January 2020

A reverse transcriptase PCR (RT-PCR) was used for rapid determination of the hemagglutinin (HA) cleavage site sequence, a marker for the virulence potential of avian influenza viruses. When applied to specimens from chickens experimentally infected with either a virulent or an avirulent virus, RT-PCR uniformly detected the HA gene, even in specimens that were negative for virus by standard testing in eggs. This technique, combined with sequencing of the HA cleavage site, offers a rapid and sensitive way to assess the virulence potential of avian influenza viruses. Early detection of field isolates with virulence-associated structural motifs at the HA cleavage site would allow better control of influenza among large poultry populations.
Colorimetric microwell plate hybridization assay for detection of amplified Mycobacterium tuberculosis DNA from sputum samples
Authors: Cho S N , van der Vliet G M , Park S , Baik S H , Kim S K , Chong Y , Kolk A H , Klatser P R , Kim J D
Published: January 2020

We developed a colorimetric microwell plate hybridization assay (CoMPHA) for the specific detection of 5'-biotinylated amplified Mycobacterium tuberculosis DNA. The optical densities of the CoMPHA corresponded to the initial amounts of purified template DNA. Here, we show that the CoMPHA is useful in distinguishing the PCR-positive and PCR-negative samples.
Evaluation of CMV-vue antigenemia assay for rapid detection of cytomegalovirus in mixed-leukocyte blood fractions
Authors: Erice A , Holm M A , Sanjuan M V , Dunn D L , Gill P C , Balfour H H
Published: January 2020

The CMV-vue antigenemia assay was evaluated by using mixed-leukocyte (ML) blood fractions. Of 234 ML fractions studied, 32 (14%), 23 (10%), and 20 (8.5%) were positive in the CMV-vue assay and conventional and shell vial cultures, respectively. The CMV-vue assay was more sensitive than shell vial cultures for rapid detection of cytomegalovirus. ML fractions are appropriate specimens for the cytomegalovirus antigenemia assay.
Detection of vancomycin resistance in enterococci by the Alamar MIC system
Authors: Zabransky R J , Dinuzzo A R , Woods G L
Published: January 2020

The ability of the Alamar microdilution MIC system to detect vancomycin resistance in enterococci was evaluated by comparing the results with an agar dilution screen method. Of 100 strains tested, 41 were resistant and 47 were susceptible by both tests. Five strains were intermediate and one was resistant by the Alamar MIC system but susceptible by the agar screen. Three strains each were susceptible or intermediate by the Alamar MIC system but resistant by the agar screen. The predictive values for the Alamar MIC system were 94% (susceptible) and 88% (combined intermediate and resistant). The Alamar MIC system does not appear to have sufficient accuracy for the detection or confirmation of vancomycin resistance in enterococci.