bluuebunny/crossref_metadata_2025 huggingface dataset:
PCR-based study of conserved and variable DNA sequences of Tritrichomonas foetus isolates from Saskatchewan, Canada
Authors: Riley D E , Wagner B , Polley L , Krieger J N
Published: January 2020

The protozoan parasite Tritrichomonas foetus causes infertility and spontaneous abortion in cattle. In Saskatchewan, Canada, the culture prevalence of trichomonads was 65 of 1,048 (6%) among 1,048 bulls tested within a 1-year period ending in April 1994. Saskatchewan was previously thought to be free of the parasite. To confirm the culture results, possible T. foetus DNA presence was determined by the PCR. All of the 16 culture-positive isolates tested were PCR positive by a single-band test, but one PCR product was weak. DNA fingerprinting by both T17 PCR and randomly amplified polymorphic DNA PCR revealed genetic variation or polymorphism among the T. foetus isolates. T17 PCR also revealed conserved loci that distinguished these T. foetus isolates from Trichomonas vaginalis, from a variety of other protozoa, and from prokaryotes. TCO-1 PCR, a PCR test designed to sample DNA sequence homologous to the 5' flank of a highly conserved cell division control gene, detected genetic polymorphism at low stringency and a conserved, single locus at higher stringency. These findings suggested that T. foetus isolates exhibit both conserved genetic loci and polymorphic loci detectable by independent PCR methods. Both conserved and polymorphic genetic loci may prove useful for improved clinical diagnosis of T. foetus. The polymorphic loci detected by PCR suggested either a long history of infection or multiple lines of T. foetus infection in Saskatchewan. Polymorphic loci detected by PCR may provide data for epidemiologic studies of T. foetus.
Rapid diagnosis of Argentine hemorrhagic fever by reverse transcriptase PCR-based assay
Authors: Lozano M E , Enría D , Maiztegui J I , Grau O , Romanowski V
Published: January 2020

Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus. This report demonstrates that a reverse transcriptase (RT) PCR-based assay developed in our laboratory to detect Junín virus in whole blood samples is sensitive and specific. The experiments were conducted in a double-blinded manner using 94 clinical samples collected in the area in which AHF is endemic. The RT-PCR-based assay was compared with traditional methodologies, including enzyme-linked immunosorbent assay, plaque neutralization tests, and occasionally viral isolation. The calculated parameters for RT-PCR diagnosis, with seroconversion as the "gold standard," were 98% sensitivity and 76% specificity. It is noteworthy that 94% of the patients with putative false-positive results (RT-PCR positive and no seroconversion detected) exhibited febrile syndromes of undefined etiology. These results could be interpreted to mean that most of those patients with febrile syndromes were actually infected with Junín virus but did not develop a detectable immune response. Furthermore, 8 laboratory-fabricated samples and 25 blood samples of patients outside the area in which AHF is endemic tested in a similar way were disclosed correctly (100% match). The RT-PCR assay is the only laboratory test available currently for the early and rapid diagnosis of AHF. It is sensitive enough to detect the low viremia found during the period in which immune plasma therapy can be used effectively, reducing mortality rates from 30% to less than 1%.
Double-repetitive-element PCR method for subtyping Mycobacterium tuberculosis clinical isolates
Authors: Friedman C R , Stoeckle M Y , Johnson W D , Riley L W
Published: January 2020

We describe a rapid method for subtyping Mycobacterium tuberculosis based on PCR amplification of segments located between two distinct DNA repetitive elements. This method, double-repetitive-element PCR, classified 46 clinical isolates as having 25 distinct patterns; the conventional restriction fragment length polymorphism analysis classified the same isolates as having 23 distinct patterns. The double-repetitive-element PCR is a rapid subtyping method that has a discriminating power similar to that of the restriction fragment length polymorphism method.
Detection of provisional serovars of Shigella dysenteriae and designation as S. dysenteriae serotypes 14 and 15
Authors: Ansaruzzaman M , Kibriya A K , Rahman A , Neogi P K , Faruque A S , Rowe B , Albert M J
Published: January 2020

In initial studies of slide and tube agglutination tests of shigella-like isolates from diarrheal stools from Bangladesh, 10 and 8 strains of Shigella dysenteriae were identified as provisional serovars (serovars that have been defined but not given a number in the antigenic schema) E22383 and E23507, respectively. Further screening of diarrheal stool isolates in a slide agglutination test with antisera (made with the international reference strains) to the two provisional serovars of S. dysenteriae identified an additional 36 isolates as belonging to E22383 and 8 isolates as belonging to E23507. All strains had properties typical of invasive Shigella strains in that they were positive in the Sereny test and had the 120- to 140-MDa plasmid associated with invasiveness. On the basis of these markers of pathogenicity and isolation from various geographical locations, now we recommend that provisional serovars E22383 and E23507 be designated S. dysenteriae serotypes 14 and 15, respectively.
Reverse transcription multiplex PCR for differentiation between polio- and enteroviruses from clinical and environmental samples
Authors: Egger D , Pasamontes L , Ostermayer M , Bienz K
Published: January 2020

For the rapid detection of polioviruses and their differentiation from nonpoliovirus enteroviruses, we developed a protocol in which clinical or environmental specimens are first inoculated onto cell cultures in tubes. After overnight incubation, the cultures are subjected to reverse transcription multiplex PCR with a primer pair which detects all enteroviruses (T. Hyypiä, P. Auvinen, and M. Maaronen, J. Gen. Virol. 70:3261-3268 1989) and two newly designed primer pairs specific for all 36 poliovirus strains tested. The PCR products can unequivocally be identified by their lengths in agarose gels, whereas the genetic heterogeneity of the poliovirus strains precludes identification by back-hybridization with internal probes. The proposed protocol is highly insensitive to the inhibitory effects of substances in the sample (stool, sewage). It allows for the detection of polioviruses and for polioviruses to be distinguished from nonpoliovirus enteroviruses within 24 h, and it allows for the concomitant isolation of a viable strain suitable for further typing.
Comparison of four DNA-based methods for strain delineation of Candida lusitaniae
Authors: King D , Rhine-Chalberg J , Pfaller M A , Moser S A , Merz W G
Published: January 2020

Four methods for the accurate delineation of epidemiologically related and unrelated strains of Candida lusitaniae were compared. Three pulsed-field electrophoretic methods, including two contour-clamped homogeneous field gel electrophoresis methods (EKP-1 and EKP-2) yielding electrophoretic karyotype patterns of intact chromosomal DNA and a method in which the chromosomal DNA was macrodigested with the endonuclease SfiI prior to pulsed-field electrophoresis (MDP), and a random amplified polymorphic DNA (RAPD) assay were evaluated. A selected panel of 21 well-characterized isolated representing 13 strains of C. lusitaniae, including 7 epidemiologically related isolates of one strain (group I-A), 3 epidemiologically related isolates of another strain (group I-B), and 11 epidemiologically unrelated isolates (group II), were tested. All isolates were coded and tested in a blinded manner. All seven group I-A isolates were confirmed to be a single strain by the EKP-1 and MDP methods, and the three group I-B isolates were shown to be a single strain by the EKP-1, EKP-2, MDP, and RAPD methods. Subtle differences were noted with two of the group I-A isolates by the EKP-2 method, whereas three of these isolates were different by the RAPD method. Each group II isolate had distinct patterns by all four methods. These data support the fact that the three pulsed-field electrophoretic methods and the RAPD method can be used to delineate strains of C. lusitaniae. The EKP-1, EKP-2, and MDP gave results that correlated with the epidemiologic characteristics of the isolates tested in the study, whereas the RAPD method was perhaps too sensitive in detecting DNA changes for epidemiologic studies.
Serologic detection of infection with cagA+ Helicobacter pylori strains
Authors: Cover T L , Glupczynski Y , Lage A P , Burette A , Tummuru M K , Perez-Perez G I , Blaser M J
Published: January 2020

Approximately 60% of Helicobacter pylori isolates possess the cagA gene and express its 120- to 140-kDa product (CagA). In this study, the cagA gene was detected in H. pylori isolates from 26 (81.3%) of 32 patients with duodenal ulcers (DU), 17 (68.0%) of 25 patients with gastric ulcers, and 23 (59.0%) of 39 patients with nonulcer dyspepsia (NUD). By Western blotting (immunoblotting) with antiserum to CagA, in vitro CagA expression was demonstrated for 95.5% of cagA+ strains compared with 0% of strains lacking cagA. Sera from patients infected with cagA+ strains (n = 66) reacted with recombinant CagA in an enzyme-linked immunosorbent assay to a significantly greater extent than either sera from patients infected with strains lacking cagA (n = 30) or sera from uninfected persons (n = 25) (P < 0.001). A strain lacking cagA was isolated from eight patients who had serum immunoglobulin G antibodies to CagA, which suggests that these patients were infected with multiple strains. Serum immunoglobulin G antibodies to CagA were present in 87.5, 76.0, and 56.4% of patients with DU, gastric ulcers, and NUD, respectively (odds ratio, 5.41; 95% confidence interval, 1.44 to 24.72; P = 0.004 [DU versus NUD]). These data demonstrate an association between infection with cagA+ H. pylori and the presence of duodenal ulceration and indicate that serologic testing is a sensitive method for detecting infection with cagA+ strains.
Inhibitory activity in saliva of cell-to-cell transmission of human T-cell lymphotropic virus type 1 in vitro: evaluation of saliva as an alternative source of transmission
Authors: Yamamoto T , Terada K , Nishida N , Moriuchi R , Shirabe S , Nakamura T , Tsuji Y , Miyamoto T , Katamine S
Published: January 2020

Human T-cell lymphotropic virus type 1 (HTLV-1) is known to be transmitted vertically through breastfeeding and horizontally by blood transfusion and sexual contact. Our intervention study has suggested the presence of additional alternative maternal transmission pathways. To explore the possibility of transmission through saliva, we used PCR to quantify the HTLV-1 provirus in saliva samples from 18 carrier mothers and 10 patients with HTLV-1-associated myelopathy/tropical spastic paraparesis. The provirus was detected in 60 and 90%, respectively, of the samples, with estimated copy numbers in the range of 10 to 10(4)/ml. However, the saliva, regardless of the presence or absence of antibodies to the virus, showed a strong tendency to inhibit the cell-to-cell transmission of HTLV-1 in vitro, as examined by a syncytium inhibition assay. The natural inhibitory activity in saliva of seronegative volunteers was heat sensitive, and most of the activity was recovered by ultrafiltration in the fraction of macromolecules with a molecular weight of more than 100,000. In addition to this natural activity, saliva of HTLV-1-infected individuals contained immunoglobulin G molecules capable of neutralizing syncytium formation. These results strongly suggested that HTLV-1-infected cells in the carriers' saliva, which contains neutralizing antibodies in addition to the natural activity inhibiting cell-to-cell viral infection, barely transmit the virus. Transmission of HTLV-1 through the saliva would thus seem to be rare, if it occurs at all.
Correlation between phenotypic characteristics and DNA relatedness within Enterococcus faecium strains
Authors: Teixeira L M , Facklam R R , Steigerwalt A G , Pigott N E , Merquior V L , Brenner D J
Published: January 2020

We noted that a number of enterococcal strains isolated from human clinical specimens resembled Enterococcus faecium but were able to produce acid from glycerol, raffinose, and/or sorbitol, while others failed to form acid from mannitol. An additional concern was that many of these strains with atypical phenotypic characteristics also appeared to acquire vancomycin resistance. In order to determine if such atypical strains were variants of E. faecium or new Enterococcus species, 35 E. faecium or E. faecium-like strains (grouped into 10 phenotypes on the basis of the results of the following tests: capacity to form acid from glycerol, mannitol, raffinose, or sorbitol and susceptibility to vancomycin) and four strains of Enterococcus faecalis were taken from our culture collection, analyzed for their whole-cell protein profiles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and identified to the species level by DNA-DNA reassociation experiments. All E. faecium-like strains, including four mannitol-negative variants, conformed to at least two of three DNA-DNA relatedness criteria: they were 70% or more related to the type strain of E. faecium at optimal conditions, they had less than 5% divergence within the related sequences, and they had a relatedness of 60% or greater under stringent conditions. The protein profiles of atypical strains were similar to those of typical strains and were easily distinguishable from those of E. faecalis and other enterococcal species. The five E. faecalis strains were 12 to 16% related to the E. faecium type strain. These results indicate that the phenotypic description of E. faecium should include all of these variable characteristics.
Clinical and epidemiological investigations of Acinetobacter genomospecies 3 in a neonatal intensive care unit
Authors: Horrevorts A , Bergman K , Kollée L , Breuker I , Tjernberg I , Dijkshoorn L
Published: January 2020

A prospective study of Acinetobacter isolates from a neonatal intensive care unit was performed for 24 months. Fifty-six isolates were obtained from 21 patients, and another eight were obtained from environmental specimens. Infection due to Acinetobacter organisms was established for 16 patients, 6 with septicemia, 9 with pneumonia, and 1 with a wound infection. Further investigations were performed with 38 representative isolates. Twenty-nine isolates were identified as unnamed DNA-DNA hybridization group (genomospecies) 3, three were identified as genomospecies 2 (Acinetobacter baumannii), one was identified as genomospecies 5 (Acinetobacter junii), three were identified as genomospecies 14, and two were unclassified. Eight distinguishable protein profiles, coded I through VIII, were found by cell envelope protein electrophoresis. Profile V, a common profile, was observed for 17 isolates that had been recovered from 11 patients and 1 dust specimen. These isolates, all of which belonged to genomospecies 3, had similar antibiograms and biotypes. This study has revealed that genomospecies 3 can be associated with infection and be spread in hospitals.
Relevance of culturing Candida species from intravascular catheters
Authors: Khatib R , Clark J A , Briski L E , Wilson F M
Published: January 2020

The significance of isolating Candida species from intravascular catheters was examined retrospectively. Our findings demonstrate that these organisms are encountered among patients with proven invasive candidiasis or as an isolated finding. Febrile patients with yeasts in two or more additional sites may be at risk for invasive cadidiasis and may benefit from treatment.
Comparison of several fixation methods for cytomegalovirus antigenemia assay
Authors: Pérez J L , De Oña M , Niubò J , Villar H , Melón S , García A , Martín R
Published: January 2020

Two fixation methods based on formaldehyde or acetone for qualitative cytomegalovirus antigenemia assay were evaluated on 405 consecutive blood samples. Cytomegalovirus was detected in 40 samples by the antigenemia assay: 36 were detected by formaldehyde fixation; 22, by acetone; and 18, by both methods. Differences were statistically significant (P = 0.0043). In addition, four fixation methods (two based on formalin [with and without permeabilization] and two using acetone at different fixation times) for quantitative antigenemia assay in a different set of 32 samples from known viremic patients were evaluated. Formalin-based methods were superior to acetone-based methods, showing statistically significant differences in either the number of positive samples detected (P < 0.02; McNemar test) or the mean positive cell counts (P < 0.003; two-tailed Student's t test for paired samples). No differences between the two formalin-based methods were found. We recommend the formaldehyde fixation procedure without subsequent permeabilization because of its simplicity and sensitivity.
Cloning and nucleotide sequence of a specific DNA fragment from Paracoccidioides brasiliensis
Authors: Goldani L Z , Maia A L , Sugar A M
Published: January 2020

We cloned and sequenced a species-specific 110-bp DNA fragment from Paracoccidioides brasiliensis. The DNA fragment was generated by PCR with primers complementary to the rat beta-actin gene under a low annealing temperature. Comparison of the nucleotide sequence, after excluding the primers, with those in the GenBank database identified approximately 60% homology with an exon of a major surface glycoprotein gene from Pneumocystis carinii and a fragment of unknown function in Saccharomyces cerevisiae chromosome VIII. By Southern hybridization analysis, the 32P-labelled fragment detected 1.0- and 1.9-kb restriction fragments within whole-cell genomic DNA of P. brasiliensis digested with HindIII and PstI, respectively, but failed to hybridize to genomic DNAs from Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Aspergillus fumigatus, Saccharomyces cerevisiae, Pneumocystis carinii, rat tissue, or humans under low-stringency hybridization conditions. Additionally, the specific DNA fragment from three different P. brasiliensis isolates (Pb18, RP18, RP17) was amplified by PCR with primers mostly complementary to nonactin sequences of the 110-bp DNA fragment. In contrast, there were no amplified products from other fungus genomic DNAs previously tested, including Histoplasma capsulatum. To date, this is the first species-specific DNA fragment cloned from P. brasiliensis which might be useful as a diagnostic marker for the identification and classification of different P. brasiliensis isolates.
Isolation and characterization of a new strain of Ehrlichia chaffeensis from a patient with nearly fatal monocytic ehrlichiosis
Authors: Dumler J S , Chen S M , Asanovich K , Trigiani E , Popov V L , Walker D H
Published: January 2020

Ehrlichia chaffeensis is the causative agent of human monocytic ehrlichiosis, a disease that ranges in severity from asymptomatic infection to death. Only one isolate of E. chaffeensis has been made, the Arkansas strain, upon which all characterizations of the agent of human monocytic ehrlichiosis have been based. We report the isolation and characterization of a new strain of E. chaffeensis, the 91HE17 strain, which was cultivated from a patient with a nearly fatal illness. The new isolate grows best in culture with careful control of pH. The two isolates are nearly identical as determined by light and electron microscopy and have significant antigenic identity in fluorescent-antibody and immunoblot assays using polyclonal antisera and the E. chaffeensis-specific monoclonal antibody 1A9. Isolate 91HE17 had 99.9% nucleotide sequence identity with the Arkansas strain in the 16S rRNA gene. Parts of the Escherichia coli GroE operon homologs had identical restriction enzyme digestion patterns, and a 425-bp region of the GroEL gene had at least 99.8% sequence identity between the E. chaffeensis Arkansas and 91HE17 strains. Isolate 91HE17 lacked an epitope identified in E. chaffeensis Arkansas by the monoclonal antibody 6A1. This new E. chaffeensis isolate is very similar to the Arkansas strain and provides the opportunity to substantiate the existence of diversity among ehrlichiae which infect humans. Specific factors which differ among strains may then be compared to assess their potential contributions toward cellular pathogenicity and ultimately toward the development of disease in humans.
Rapid assay for mycobacterial growth and antibiotic susceptibility using gel microdrop encapsulation
Authors: Ryan C , Nguyen B T , Sullivan S J
Published: January 2020

Effective control of tuberculosis transmission in vulnerable population groups is dependent on rapid identification of the infectious agent and its drug susceptibility. However, the slow growth rate of mycobacteria has undermined the ability to quickly identify antimicrobial resistance. These studies describe a mycobacterial growth assay based on microencapsulation technology used in conjunction with flow cytometric analysis. Mycobacteria were encapsulated in agarose gel microdrops approximately 25 microns in diameter, and colony growth was monitored by using flow cytometry to evaluate the intensity of auramine staining after culture for various times at 37 degrees C. By this method, colony growth of Mycobacterium bovis and M. smegmatis could be quantified within 1 to 3 days after encapsulation. Inhibition of growth by rifampin and isoniazid was also evaluated in this time period, and the presence of an isoniazid-resistant subpopulation representing 3% of the total microorganisms could be detected. This use of encapsulation and flow cytometry has the potential to facilitate rapid and automated evaluation of inhibition of growth by antimicrobial agents and shorten the time frame for analysis of clinical specimens.
Leish-KIT, a stable direct agglutination test based on freeze-dried antigen for serodiagnosis of visceral leishmaniasis
Authors: Meredith S E , Kroon N C , Sondorp E , Seaman J , Goris M G , van Ingen C W , Oosting H , Schoone G J , Terpstra W J , Oskam L
Published: January 2020

In order to increase the application potential of the direct agglutination test (DAT) for the detection of anti-Leishmania antibodies in human serum samples, we developed an antigen based on stained and freeze-dried Leishmania donovani promastigotes. We describe here the evaluation of the performance of the DAT based on this freeze-dried antigen. It was shown that the freeze-dried antigen remains fully active, even after storage at 56 degrees C for 18 months. With a cutoff value of 1:1,600, the sensitivity of the DAT was shown to be 92% and the specificity of the test was 99.7%, which were comparable with the results found for the DAT based on liquid antigen. The major advantages of the freeze-dried antigen are that the production of a large batch of this antigen allows reproducible results in the DAT over a long period of time and that the freeze-dried antigen can be stored at ambient temperature, which, as was shown, makes the test a valuable diagnostic tool for use in the field.
Concurrence of Clostridium difficile toxin A enzyme-linked immunosorbent assay, fecal lactoferrin assay, and clinical criteria with C. difficile cytotoxin titer in two patient cohorts
Authors: Schleupner M A , Garner D C , Sosnowski K M , Schleupner C J , Barrett L J , Silva E , Hirsch D , Guerrant R L
Published: January 2020

The accurate and sensitive diagnosis of Clostridium difficile-related diarrhea, normally treated with vancomycin, has become increasingly important in light of the emergence of dangerous new strains of vancomycin-resistant enterococci. In order to improve the threshold for C. difficile diagnosis and treatment, a number of commonly used assays for the diagnosis of C. difficile diarrhea were examined. These included an enzyme-linked immunosorbent assay for C. difficile toxin A (ToxA), a CHO cell culture assay for fecal C. difficile (cyto)toxin B, and a lactoferrin latex agglutination assay for fecal lactoferrin (LFLA). We studied 722 fecal specimens submitted by physicians for C. difficile toxin testing at the Salem, Va., Veterans' Affairs Hospital and at the University of Virginia Medical Center in Charlottesville. Charts were reviewed from 123 Veterans' Hospital patients and 114 University of Virginia patients for clinical criteria indicative of C. difficile diarrhea. An increasing titer of CHO cell cytotoxicity was correlated with an increasing likelihood of ToxA positivity (5 to 90%), LFLA positivity (39 to 77%), and clinical agreement (28 to 85%). However, some data indicate that the CHO cell cytotoxicity assay may be nonspecific when positive only at low titers. When the CHO assay result is positive at high titers, it remains the best diagnostic tool. Yet, when it is positive at a low titer, careful interpretation of the results in conjunction with other assays and the clinical setting is warranted, especially in light of new drug-resistant strains of microorganisms.
Evaluation of Etest for susceptibility testing of rapidly growing mycobacteria
Authors: Biehle J R , Cavalieri S J , Saubolle M A , Getsinger L J
Published: January 2020

MICs of amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, and imipenem were determined by Etest for 100 clinical strains of rapidly growing mycobacteria and compared with MICs determined by a reference agar dilution method. Etest MICs were also determined by an alternative inoculum application (agar overlay) method and compared with MICs determined by the inoculum application method recommended by the manufacturer (swabbing). Agreement between Etest and agar dilution MICs within +/- 1 log2 dilution was 85% (511 of 600), and agreement within +/- 2 log2 dilutions was 97% (580 of 600). The rate of complete category agreement was 88%, and rates of major and minor errors were 2.2 and 11.7%, respectively. No very major errors were detected for Etest MICs. Interlaboratory agreement between MICs determined at two separate laboratories was 81% (121 of 149) within +/- 1 log2 dilution and 92% (137 of 149) within +/- 2 log2 dilutions. Agreement between laboratories by interpretive category was 92%. Exact agreement between agar overlay and swab application MICs was 52.3%, and agreement within +/- 1 log2 dilution was 82.3%. Diffuse ellipse edges and trailing growth were still a problem with the overlay method, and in some cases results were more difficult to interpret than they were with the corresponding swab-prepared plate. In summary, our data suggest that Etest may be an accurate and reproducible method for determining susceptibility of rapidly growing mycobacteria.
Diversity of DNA fingerprints in Cryptococcus neoformans
Authors: Varma A , Swinne D , Staib F , Bennett J E , Kwon-Chung K J
Published: January 2020

DNA fingerprint patterns of 156 Cryptococcus neoformans isolates (26 AIDS patients, 46 non-AIDS patients, and 40 environmental sources) from both varieties (126 C. neoformans var. neoformans and 30 C. neoformans var. gattii isolates) and from seven countries were analyzed by using the DNA probe UT-4p. Nine and twelve distinct DNA fingerprint patterns were observed for isolates of the C. neoformans var. neoformans and var. gattii, respectively. No pattern was unique to AIDS patients, non-AIDS patients, or the environment. Pattern II was observed more often in non-AIDS patients (8 of 23) than in AIDS patients (0 of 25). Pattern V was the most prevalent pattern (42 of 82) in clinical and environmental isolates. Isolates from three AIDS patients in Burundi and Zaire exhibited patterns identical to each other but different from those of isolates collected from their houses (i.e., dust of floors, walls, etc.) or a nearby pigeon coop. DNA fingerprint stability was determined for 53 isolates from nine non-AIDS patients at different time intervals during 5 to 128 weeks of antifungal therapy. For eight patients, the fingerprint pattern was stable while the ninth may have had a mixed infection. Pattern II was observed in 4 of 9 patients, which is similar to 4 of 14 in other non-AIDS patients as reported here. In spite of the extensive pattern heterogeneity among 15 C. neoformans var. gattii isolates in Australia, the patterns observed in seven California isolates were quite different from those in Australia. Among isolates of C. neoformans var. gattii, one fingerprint pattern (designated b) was observed in several countries of the Far East. The fingerprint patterns of two of three environmental isolates from Eucalyptus camaldulensis trees in Australia were identical to those of 2 of the 12 clinical isolates from the country.
High incidence of Haemophilus influenzae in nasopharyngeal secretions and middle ear effusions as detected by PCR
Authors: Ueyama T , Kurono Y , Shirabe K , Takeshita M , Mogi G
Published: January 2020

PCR was used to detect Haemophilus influenzae in samples of nasopharyngeal secretion and middle ear effusion (MEE). Nasopharyngeal secretions were collected from 102 patients with otitis media with effusion and from 111 healthy subjects. Eighty samples of MEE were collected from patients with otitis media with effusion. A pair of primers was designed to amplify a DNA segment of the gene encoding P6 outer membrane protein of H. influenzae. The amplified PCR product was detected with an internal probe that hybridized specifically to the P6 DNA of H. influenzae. Samples of MEE and nasopharyngeal secretion were also examined by a conventional culture method. The incidence of P6 gene DNA in nasopharyngeal secretions detected by PCR was about two times higher than that of H. influenzae detected by the conventional culture. Culture-positive samples were all positive in the PCR test. In MEEs, the rate of detection of the P6 gene DNA target was about five times higher than that of H. influenzae detected by the culture method. All patients who had P6 gene DNA in MEEs were found to have the DNA in nasopharyngeal secretions. These findings suggest that the presence of H. influenzae in MEEs and in nasopharyngeal secretions is more common than previously reported.
In vivo stability and discriminatory power of methicillin-resistant Staphylococcus aureus typing by restriction endonuclease analysis of plasmid DNA compared with those of other molecular methods
Authors: Hartstein A I , Phelps C L , Kwok R Y , Mulligan M E
Published: January 2020

We evaluated test discriminatory power and DNA type alterations among methicillin-resistant Staphylococcus aureus strains by testing 199 sequential isolates from 39 patients collected over 30 to 228 days. Isolates were typed by one or three different methods (restriction endonuclease analysis of plasmid DNA [REAP] with or without pulsed-field gel electrophoresis of genomic DNA [PFGE] and immunoblotting [IB]). REAP was highly discriminatory compared with PFGE and IB. However, the initial isolates from 4 of the 39 patients lacked detectable plasmid DNA and could not be typed by REAP. Typing of individual patient isolates showed that a different REAP type was identified only once every 138 days. Among 25 comparisons, seven sequential isolate pairs demonstrating REAP differences were also different by PFGE and IB. This likely represented the presence of more than one strain. Eighteen other pairs with REAP differences were identical or related to one another by PFGE and IB typing, and 17 of these differences were likely caused by a single genetic alteration within the same strain or clone. The rate of PFGE differences explicable by single genetic alterations among sequential isolates identical by REAP was similar to the overall rate for REAP differences in the whole collection. We conclude that REAP and PFGE typing differences explicable by single genetic alterations are relatively infrequent but not rare. These isolates should be examined by alternative typing systems to further support or refute clonality.
Unusually large number of methicillin-resistant Staphylococcus aureus clones in a Portuguese hospital
Authors: Couto I , Melo-Cristino J , Fernandes M L , Garcia T , Serrano N , Salgado M J , Torres-Pereira A , Sanches I S , de Lencastre H
Published: January 2020

Methicillin-resistant Staphylococcus aureus (MRSA) has been endemic in Hospital de Santa Maria, a 1,300-bed teaching hospital in Lisbon, Portugal, since the mid-1980s with a prevalence of 30% in 1993. A total of 54 MRSA and 93 methicillin-susceptible S. aureus (MSSA) isolates recovered during the first 3 months of 1993 were analyzed for the particular mecA polymorphs and Tn554 attachment sites (in the case of MRSA) and for pulsed-field gel electrophoretic patterns. While all MRSA isolates shared a very similar multidrug resistance antibiogram, molecular methods allowed the identification of an unusually large number of genetic backgrounds (24 different pulsed-field gel electrophoresis patterns in 54 isolates) and three different mecA polymorphs among the MRSA strains. Similar large variation in the genetic backgrounds of MSSA was observed. The most frequent mecA polymorph (mecA type I) was found in association with three different Tn554 patterns. Among the MRSA strains of Hospital Santa Maria, we found two clonal types previously described in Portugal: one corresponding to the dominant clone in an MRSA outbreak at the pediatric ward of the Lisbon Hospital Dona Estefânia and another one identical to the Iberian epidemic clone identified in several Portuguese hospitals and in MRSA outbreaks in Barcelona and Madrid. This suggests that MRSA clones of Hospital de Santa Maria may have been a reservoir for staphylococcal strains over the past decade.
Detection of human immunodeficiency virus type 1 proviral DNA by PCR using an electrochemiluminescence-tagged probe
Authors: Schutzbank T E , Smith J
Published: January 2020

We have developed a rapid, pseudohomogeneous assay for the detection of PCR amplicons, based on the use of electrochemiluminescence generated from a Tris-bipyridine ruthenium(II) label. PCR amplification of highly conserved human immunodeficiency virus type 1 (HIV-1) gag gene sequences was performed with SK38 and SK39 primers, the latter of which was 5' biotinylated. Post-PCR reaction mixtures were combined with 10(12) copies of the SK19 probe-Tris-bipyridine ruthenium(II) conjugate, denatured by heating at 100 degrees C for 5 min, and hybridized at 55 degrees C for an additional 15 min. Hybridization to the biotinylated strand of the amplified DNA was determined by the addition of streptavidin-conjugated magnetic particles and analyzed by using an Origen-1 electrochemiluminescence analyzer. Our results demonstrated a sensitivity of fewer than five copies of HIV-1 (pre-PCR), by using either purified plasmid DNA containing one complete copy of the HIV-1 cDNA genome or lysed, proteinase K-treated 8E5 cells as the starting material. In an evaluation of actual clinical specimens (peripheral blood monocytes from both healthy and HIV-1-infected children), the electrochemiluminescent detection assay correlated 100% with both our standard method (solution hybridization with a radiolabeled probe followed by polyacrylamide gel electrophoresis [PAGE] and autoradiography) and a commercial method (Roche Amplicor). The electrochemiluminescent method was substantially easier to perform than either the PAGE or microtiter plate assays and was considerable faster to perform than either of these alternative formats.
Inactivation of human immunodeficiency virus type 1 by the amine oxidase-peroxidase system
Authors: Klebanoff S J , Kazazi F
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) is rapidly inactivated by exposure to a naturally occurring antimicrobial system consisting of peroxidase, H2O2, and a halide. Among the potential sources of H2O2 is the amine oxidase system in which mono-, di-, and polyamines are oxidatively deaminated with the formation of H2O2. The polyamine spermine is present at exceptionally high concentrations in semen. We report here that spermine, spermidine, and, to a lesser degree, the synthetic polyamine 15-deoxyspergualin are viricidal to HIV-1 when combined with amine oxidase and myeloperoxidase. Antiviral activity required each component of the spermine-amine oxidase-peroxidase system and was inhibited by azide (a peroxidase inhibitor) and by catalase but not by superoxide dismutase. Heat treatment of catalase largely abolished its inhibitory effect. These findings implicate H2O2 formed by the amine oxidase system in the antiviral effect and raise the possibility that the polyamine-amine oxidase-peroxidase system influences the survival of HIV-1 in semen and in the vaginal canal.
Auxotrophy of Burkholderia (Pseudomonas) cepacia from cystic fibrosis patients
Authors: Barth A L , Pitt T L
Published: January 2020

The nutritional status of 89 isolates of Burkholderia cepacia from 81 cystic fibrosis (CF) patients was evaluated. Forty of the isolates, from 38 patients, were not able to grow in a minimal medium containing glucose and mineral salts only and were thus auxotrophs. In contrast, all of 29 isolates from non-CF (clinical and environmental) sources were prototrophic. Addition of a pool of amino acids to the minimal medium was sufficient to promote growth of all tested CF auxotrophic isolates. Indeed, phenylalanine, tyrosine, cysteine, methionine, and histidine alone or in combination were required for growth by the majority of the nutritionally deficient B. cepacia isolates. Furthermore, extracts of sputum from CF patients, when added to minimal medium, promoted growth of 29 auxotrophic B. cepacia isolates regardless of their amino acid requirements. Finally, auxotrophic and prototrophic isolates from the same patient exhibited a conserved genotype, as determined by macrorestriction analysis of chromosomal DNA. These results suggest that the auxotrophic mutants are selected from the prototrophic population and maintained by the nutritionally rich environment of the CF airways.
Evaluation of mupirocin E-test for determination of isolate susceptibility: comparison with standard agar dilution techniques
Authors: Simpson I N , Gisby J , Hemingway C P , Durodie J , Macpherson I
Published: January 2020

Mupirocin E-test strips have been evaluated for their ease of use and accuracy in determining the susceptibilities of 171 strains of Staphylococcus spp., Streptococcus spp., Haemophilus influenzae, and Moraxella catarrhalis. The susceptibility of each strain was determined on two occasions, using parallel E-test and agar dilution methodologies each time. To ensure similar precisions for statistical analyses, E-test MICs were rounded up to a standard twofold agar dilution scale. Clear, elliptical zones were obtained against Staphylococcus spp. M. catarrhalis also gave clear zones, but the scale intercept was often difficult to interpret because of the irregular shape of the inhibition zone. Poor growth sometimes resulted in less-distinct zones of inhibition against Streptococcus spp. and H. influenzae. Excellent correlation was observed between the the E-test and agar dilution against Staphylococcus spp. and H. influenzae, with > 95% of the E-test values falling within one log2 dilution of the corresponding agar MIC. The correlation was lower for Streptococcus spp. and M. catarrhalis, with 86 and 83%, respectively, of E-test results falling within one log2 dilution of the agar MIC. When E-test MICs did not agree exactly with the corresponding agar MIC against Staphylococcus spp. or Streptococcus spp., there was a tendency for the E-test to give a lower MIC. This bias has little effect upon individual MICs in staphylococci or in the generation of susceptibility interpretation errors ( < 1.5% overall), but it could reduce population geometric mean MICs by factors of 0.78 to 0.83. This effect was more marked for Streptococcus spp., reducing the population mean by a factor of 0.73 and resulting in 0.7% major and 8% very major errors. In contrast, the E-test tended to give higher MICs against M. catarrhalis, resulting in 7.3% major errors and increasing the population geometric mean MIC by a factor of 1.60.
Simplified procedure for preparation of sensitized latex particles to detect capsular polysaccharides: application to typing and diagnosis of Actinobacillus pleuropneumoniae
Authors: Inzana T J
Published: January 2020

A novel, inexpensive method for obtaining immunoglobulin G (IgG) specific for capsular antigen is described for use in latex agglutination tests. Hyperimmune rabbit serum against encapsulated Actinobacillus pleuropneumoniae was thoroughly adsorbed with a nonencapsulated mutant. The capsule titer of the absorbed serum was unaffected, whereas reactivity to nonencapsulated cells was reduced to background levels, as determined by enzyme immunoassay. The IgG component of the adsorbed serum was recovered by protein A chromatography and was covalently coupled through a water-soluble carbodiimide to carboxylate latex beads. The sensitized latex particles (SLP) were agglutinated by 10 ng of homologous capsule or more per ml, were not agglutinated by heterologous capsules at concentrations of < 10 micrograms/ml, and were stable for over 1 year at 4 degrees C without loss of sensitivity. There was no difference in the sensitivity or specificity of latex particles coupled with IgG purified by capsule affinity chromatography. The SLP were agglutinated by all strains of bacteria of the homologous serotype but not by heterologous serotypes or strains of Pasteurella multocida, Actinobacillus suis, or Haemophilus parasuis tested at a density equivalent to a 0.5 McFarland standard. The SLP detected homologous capsule in lung tissue, nasal swabs, and concentrated urine samples from all pigs culture positive for A. pleuropneumoniae but one. Precoating of carboxylate latex particles with avidin followed by conjugation of biotin-hydrazide-labelled IgG to capsule increased the sensitivity of the assay approximately 10-fold. Adsorption of serum with nonencapsulated mutants may be used to prepare SLP with optimum sensitivity and specificity without the need to purify capsule or couple capsule to affinity columns.
Clinical and microbiological features of necrotizing fasciitis
Authors: Brook I , Frazier E H
Published: January 2020

The microbiological and clinical characteristics of 83 patients with necrotizing fasciitis (NF) treated over a period of 17 years are presented. Bacterial growth was noted in 81 of 83 (98%) of specimens from patients with NF. Aerobic or facultative bacteria only were recovered in 8 (10%) specimens, anaerobic bacteria only were recovered in 18 (22%) specimens, and mixed-aerobic-anaerobic floras were recovered in 55 (68%) specimens. In total, there were 375 isolates, 105 aerobic or facultative bacteria and 270 anaerobic bacteria, for an average of 4.6 isolates per specimen. The recovery of certain bacteria from different anatomical locations correlated with their distribution in the normal flora adjacent to the infected site. Anaerobic bacteria outnumbered aerobic bacteria at all body sites, but the highest recovery rate of anaerobes was in the buttocks, trunk, neck, external genitalia, and inguinal areas. The predominant aerobes were Staphylococcus aureus (n = 14 isolates), Escherichia coli (n = 12), and group A streptococci (n = 8). The predominant anaerobes were Peptostreptococcus spp. (n = 101), Prevotella and Porphyromonas spp. (n = 40), Bacteroides fragilis group (n = 36), and Clostridium spp. (n = 23). Certain clinical findings correlated with some bacteria: edema with B. fragilis group, Clostridium spp., S. aureus, Prevotella spp. and group A streptococci; gas and crepitation in tissues with members of the family Enterobacteriaceae and Clostridium spp.; and foul odor with Bacteroides spp. Certain predisposing conditions correlated with some organisms: trauma with Clostridium spp.; diabetes with Bacteroides spp., members of the family Enterobacteriaceae, and S. aureus; and immunosuppression and malignancy with Pseudomonas spp. and members of the family Enterobacteriaceae. These data highlight the polymicrobial nature of NF.
Outer surface protein C gene sequence analysis of Borrelia burgdorferi sensu lato isolates from Japan
Authors: Fukunaga M , Hamase A
Published: January 2020

The nucleotide sequences of the outer surface protein C gene (ospC) from Borrelia burgdorferi sensu lato isolates representing six different restriction fragment length polymorphism (RFLP) ribotype groups were determined, and the deduced amino acid sequences were aligned in comparison with the previously published OspC protein sequences. The sequence similarity analysis revealed the high sequence variability of OspC protein, and the degree of amino acid similarity ranged from 53.8 to 100% among 25 isolates. It has been reported that the representatives belonging to the three species of B. burgdorferi sensu lato showed a species-specific amino acid sequence motif at positions 23 to 35 (B. Wilske, S. Jauris-Heipke, R. Lobentanzer, I. Pradel, V. Preac-Mursic, D. Rössler, E. Soutschek, and R.C. Johnson, J. Clin. Microbiol. 33:103-109, 1995). Alignment with the OspC sequences of RFLP ribotype group IV, V, and VI isolates revealed that a sequence motif of all the isolates was quite similar to that of Borrelia garinii. A phylogenetic analysis based on OspC protein sequences also showed that most of the Japanese isolates were closely related to the species B. garinii. THe RFLP ribotype group IV species is predominant among clinical isolates of Lyme disease patients, reservoir rodents, and adult ticks in Japan. Although the isolates differed from type strains of the three delineated genospecies in genetic and immunological characteristics, it is likely that the spirochetes diverged within the species level. Therefore, the representatives of ribotype groups IV, V, and VI appear to have evolved within B. garinii and to have adapted to an Asiatic habitat, and there appeared to be a sufficient ecological pressure to allow bacterial species level development.
Detection of Toxoplasma gondii by PCR and tissue culture in cerebrospinal fluid and blood of human immunodeficiency virus-seropositive patients
Authors: Dupon M , Cazenave J , Pellegrin J L , Ragnaud J M , Cheyrou A , Fischer I , Leng B , Lacut J Y
Published: January 2020

To investigate whether both tissue culture and PCR on a sequence from the repetitive rDNA could contribute to the diagnosis of toxoplasmosis, blood samples and, if they were available, cerebrospinal fluid (CSF) and aqueous humor samples from 72 human immunodeficiency virus-seropositive patients with suspected toxoplasmosis were prospectively tested. For 10 patients with fever of unknown origin but without confirmed toxoplasmosis, no Toxoplasma gondii was detected. For two patients with confirmed toxoplasmic uveitis, only PCR of aqueous humor samples was positive. Of 60 patients (48 with CSF samples) with neurological signs, 25 (from 13 of whom CSF samples were available) had confirmed cerebral toxoplasmosis and 10 had a positive PCR of CSF and/or blood samples, while for 1 patient culture of the CSF sample was also positive. Unlike tissue culture, PCR of rDNA is of value for the detection of cerebral toxoplasmosis in human immunodeficiency virus-seropositive patients, provided that both CSF and blood samples are available (sensitivity, 76.9%; specificity, 100%).