bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of five Peptostreptococcus species isolated predominantly from the female genital tract by using the rapid ID32A system
Authors: Ng J , Ng L K , Chow A W , Dillon J A
Published: January 2020

The rapid ID32A kit (bioMérieux Vitek, Inc., Hazelwood, Mo.) was evaluated for its ability to identify Peptostreptococcus species compared with conventional biochemical tests and gas-liquid chromatography (Virginia Polytechnic Institute), the current "gold standard" method. A total of 5 Peptostreptococcus American Type Culture Collection strains and 95 clinical isolates comprising Peptostreptococcus anaerobius, P. asaccharolyticus, P. magnus, P. micros, and P. prevotii isolates were included for analysis. Overall, the sensitivity and specificity of the rapid ID32A kit in the identification for five Peptostreptococcus species compared with the Virginia Polytechnic Institute method were 93 and 80%, respectively. All P. anaerobius (n = 20) and P. asaccharolyticus (n = 25) isolates were identified with 100% sensitivity and 100% specificity. For the identification of P. magnus (n = 24) and P. micros (n = 19), the rapid ID32A kit was 100% sensitive for both species; the specificity for P. magnus was 95.8% and that for P. micros was 57.9%. The sensitivity and specificity of the rapid ID32A kit for identification of P. prevotii (n = 12) were poor (41.7 and 8.3%, respectively). The rapid ID32A kit is a useful method for the rapid differentiation of P. anaerobius and P. asaccharolyticus from other Peptostreptococcus spp. Conventional methods should be used to identify to the species level isolates of P. magnus, P. micros, and P. prevotii.
Effective use of frozen donor peripheral blood mononuclear cells for human immunodeficiency virus type 1 isolation from vertically infected pediatric patients
Authors: Paul M O , Tetali S , Pahwa S
Published: January 2020

In this study, we examined variables related to human immunodeficiency virus (HIV) isolation utilizing samples from 51 HIV-infected (153 plasma and 122 peripheral blood mononuclear cell [PBMC] samples) and 57 uninfected (182 plasma and 163 PBMC samples) infants. Our chief observation was that cryopreservation of donor PBMCs does not significantly alter their sensitivity or specificity for isolation of HIV from patient PBMCs or plasma.
Detection of BK virus DNA in nasopharyngeal aspirates from children with respiratory infections but not in saliva from immunodeficient and immunocompetent adult patients
Authors: Sundsfjord A , Spein A R , Lucht E , Flaegstad T , Seternes O M , Traavik T
Published: January 2020

Our understanding of important stages in the pathogenesis of the human polyomavirus BK virus (BKV) and JC virus (JCV) infections is limited. In this context, nasopharyngeal aspirates from 201 children with respiratory diseases and saliva from 60 human immunodeficiency virus type 1-infected adults and 10 healthy adult controls were collected and analyzed for the presence of BKV and JCV DNA by PCR. Neither BKV nor JCV DNA was detected in the saliva specimens. We demonstrated BKV DNA, but no infectious BKV, in 2 of 201 nasopharyngeal aspirates. Each sample contained one unique rearranged noncoding control region variant of BKV. The results indicate that (i) BKV and JCV are not regularly associated with respiratory infections in children requiring hospitalization, (ii) nasopharyngeal cells are not an important site for primary replication of human polyomavirus BKV and JCV, and (iii) the salivary glands and oropharyngeal cells seem not to be involved in BKV and JCV persistence. We propose that for the polyomaviruses BKV and JCV the alimentary tract should be considered as a portal of entrance to the human organism.
Screening of respiratory tract specimens for the presence of Mycobacterium tuberculosis by using the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test
Authors: Bodmer T , Gurtner A , Schopfer K , Matter L
Published: January 2020

A prospective 2-month trial involving 617 respiratory tract specimens was conducted to compare sensitivity, specificity, and predictive values of the newly developed Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test kit (AMTDT; Gen-Probe, Inc., San Diego, Calif.) for the rapid detection of Mycobacterium tuberculosis and of fluorescent acid-fast staining versus combined BACTEC 12B and solid-medium cultures as the "gold standard." A total of 590 specimens were culture and AMTDT negative. Twenty-one (3.4%) cultures yielded M. tuberculosis. Of these, 15 (71.4%) were detected by AMTDT, whereas 6 (28.6%) were missed. M. tuberculosis did not grow in six (28.6%) of AMTDT-positive specimens derived from three patients under treatment for tuberculosis. AMTDT exhibited a sensitivity, a specificity, a negative predictive value, and a positive predictive value of 71.4, 99, 99, and 71.4%, respectively. In comparison, the same values for fluorescent microscopy were 66.7, 98.3, 98.8, and 58.3%, respectively. AMTDT was easy to perform and highly specific. However, a screening test would require an improved sensitivity and, when feasible, the implementation of an internal amplification control.
Viability of Bordetella pertussis in four suspending solutions at three temperatures
Authors: Cassiday P K , Sanden G N , Kane C T , M'Boup S , Barbaree J M
Published: January 2020

We studied the survival of Bordetella pertussis in four suspending solutions (Casamino Acids broth, deionized water, phosphate-buffered saline, and serum inositol), subjected to three storage temperatures (4, -20, and -70 degrees C) and two freezing methods (direct freezing and fast-freezing in an ethanol-dry-ice bath). Recovery rates were higher for longer periods for suspensions stored at -70 degrees C than those stored at -20 or 4 degrees C. Serum inositol showed the highest recovery rates for all experimental conditions, followed by Casamino Acids, deionized water, and phosphate-buffered saline. Cell viability was significantly reduced in phosphate-buffered saline suspensions fast-frozen before storage. These results identify optimal conditions for storing B. pertussis cells and are applicable to the collection, transport, and storage of aspirated nasopharyngeal samples for use in the laboratory diagnosis of pertussis.
Comparison of PCR and other diagnostic techniques for detection of Helicobacter pylori infection in dyspeptic patients
Authors: Weiss J , Mecca J , da Silva E , Gassner D
Published: January 2020

A sensitive and specific PCR-based assay to detect the Helicobacter pylori 16S rRNA gene present in formalin-fixed paraffin-embedded gastric biopsy specimens has been developed. A total of 95 patients with dyspepsia were evaluated for the presence of chronic active gastritis and an infection with H. pylori through the use of diagnostic assays based on biopsy specimens and serology. The "gold standard" for the presence of the bacteria was direct detection in histological sections of biopsy specimens by Giemsa stain. The results obtained with the PCR assay performed on the biopsy specimens (94% sensitivity and 100% specificity) were equivalent to the detection of H. pylori immunoglobulin G antibodies by the commercially available second-generation Cobas Core anti-H. pylori immunoglobulin G enzyme immunoassay (94% sensitivity and 98% specificity) for the diagnosis of H. pylori infection. Urease testing and bacterial culture of the biopsy specimens were inferior (88 and 70% sensitivity and 96% and 98% specificity, respectively). A Western blot (immunoblot) analysis had slightly greater sensitivity (96%), although specificity was reduced to 93%. This research prototype PCR assay was shown to be highly reliable for the detection of infection with H. pylori and the presence of chronic active gastritis in the population studied.
Characterization of rotavirus strains from newborns in New Delhi, India
Authors: Das B K , Gentsch J R , Cicirello H G , Woods P A , Gupta A , Ramachandran M , Kumar R , Bhan M K , Glass R I
Published: January 2020

Between 1986 and 1993, 72% of rotavirus strains isolated from newborns at five hospitals in New Delhi, India, had long electropherotypes, subgroup II VP6 antigens, and G and P genotypes (G9P11) identical to those of prototype strain 116E. A novel strain with a G9P6 genotype, representing 13% of the isolates, was identified. These results demonstrate that G9P11 and G9P6 rotavirus strains are common in nurseries in New Delhi.
Evaluation of Etest for rapid susceptibility testing of Mycobacterium chelonae and M. fortuitum
Authors: Hoffner S E , Klintz L , Olsson-Liljequist B , Bolmström A
Published: January 2020

Etest is a new concept for MIC determinations for antimicrobial agents that is based on a predefined antibiotic gradient on a plastic strip calibrated with a continuous logarithmic MIC scale covering 15 twofold dilutions. Etest was compared with a reference agar dilution method for susceptibility testing of clinical isolates of Mycobacterium chelonae and M. fortuitum. Results read after 3 days showed good agreement between MICs obtained with Etest and those obtained with the reference method within +/- 2 dilutions for 90% of all test combinations. All but one of the strains were inhibited by low concentrations of ciprofloxacin or amikacin. Susceptibility to clarithromycin, erythromycin, imipenem, rifampin, doxycycline, and fusidic acid was variable, and all strains were resistant to ceftazidime and trimethoprim. The results suggest that Etest is well suited for studies of drug resistance in rapidly growing mycobacteria.
Gas chromatographic determination of D-arabinitol/L-arabinitol ratios in urine: a potential method for diagnosis of disseminated candidiasis
Authors: Larsson L , Pehrson C , Wiebe T , Christensson B
Published: January 2020

A gas chromatographic procedure was developed to determine the relative amounts of D- and L-arabinitol in urine. Samples were filtered, diluted, purified through extractions, evaporated, and treated with trifluoroacetic anhydride; the arabinitol derivatives thus obtained were separated on a chiral stationary phase and registered by using an electron-capture detector. Urine samples from a patient with disseminated candidiasis had higher D-arabinitol/L-arabinitol ratios (referred to as D/L-arabinitol ratios)--up to 19.0--than samples from 96 study individuals with no signs of deep Candida infections (range, 1.1 to 4.5). D/L-Arabinitol ratios in urine samples from hospitalized patients without Candida infections were slightly higher than those in samples from healthy individuals; ratios in urine from children were slightly higher than those in adult urine samples. The D/L-arabinitol ratios in several urine samples culture positive for Candida albicans, but from patients without symptoms of disseminated candidiasis, did not differ from those in the urine of healthy individuals. The described gas chromatographic method is straightforward and can be implemented clinically to determine urine D/L-arabinitol ratios as a means of diagnosing disseminated candidiasis.
Detection of type A, B, and E botulism neurotoxin genes in Clostridium botulinum and other Clostridium species by PCR: evidence of unexpressed type B toxin genes in type A toxigenic organisms
Authors: Franciosa G , Ferreira J L , Hatheway C L
Published: January 2020

We studied the effectiveness of the PCR in detecting the type A, B, and E botulism neurotoxin genes in 209 strains of Clostridium botulinum and 29 strains of other Clostridium spp. All 79 strains that produced type A toxin, 77 strains that produced type B toxin, and 51 organisms that produced type E toxin (46 C. botulinum and 5 C. butyricum) were PCR positive in reactions with primers targeting sequences specific for their respective toxin genes. The PCR for type A toxin was positive for one type B toxin-producing strain that produced a small amount of type A toxin in addition to a large amount of type B toxin. Surprisingly, the type B toxin gene was detected in addition to the type A toxin gene in 43 type A toxin-producing strains, only 1 of which could be shown by bioassay to produce biologically active type B toxin in culture. The type B gene was also detected in two strains of C. subterminale, which were determined to be nontoxigenic by bioassay. While the PCR was sensitive and specific in detecting the neurotoxin genes, the discovery of unexpressed toxin genes indicates that PCR results may not be adequate for establishing type B neurotoxigenicity.
Application of PCR to a clinical and environmental investigation of a case of equine botulism
Authors: Szabo E A , Pemberton J M , Gibson A M , Thomas R J , Pascoe R R , Desmarchelier P M
Published: January 2020

PCR for the detection of botulinum neurotoxin gene types A to E was used in the investigation of a case of equine botulism. Samples from a foal diagnosed with toxicoinfectious botulism in 1985 were reanalyzed by PCR and the mouse bioassay in conjunction with an environmental survey. Neurotoxin B was detected by mouse bioassay in culture enrichments of serum, spleen, feces, and intestinal contents. PCR results compared well with mouse bioassay results, detecting type B neurotoxin genes in these samples and also in a liver sample. Other neurotoxin types were not detected by either test. Clostridium botulinum type B was shown to be prevalent in soils collected from the area in which the foal was raised. Four methods were used to test for the presence of botulinum neurotoxin-producing organisms in 66 soil samples taken within a 5-km radius: PCR and agarose gel electrophoresis (types A to E), PCR and an enzyme-linked assay (type B), hybridization of crude alkaline cell lysates with a type B-specific probe, and the mouse bioassay (all types). Fewer soil samples were positive for C. botulinum type B by the mouse bioassay (15%) than by any of the DNA-based detection systems. Hybridization of a type B-specific probe to DNA dot blots (26% of the samples were positive) and PCR-enzyme-linked assay (77% of the samples were positive) were used for the rapid analysis of large numbers of samples, with sensitivity limits of 3 x 10(6) and 3,000 cells, respectively. Conventional detection of PCR products by gel electrophoresis was the most sensitive method (300-cell limit), and in the present environmental survey, neurotoxin B genes only were detected in 94% of the samples.
Genetic analysis of equine rotavirus by RNA-RNA hybridization
Authors: Imagawa H , Ishida S , Uesugi S , Masanobu K , Fukunaga Y , Nakagomi O
Published: January 2020

Serotype G3 equine rotaviruses isolated in Japan made up a common genogroup and were classified into two different genotypes. The genomes of serotype G3 equine rotaviruses with an identical electropherotype (isolated from 1982 to 1989) were very closely related to each other regardless of the year in which they were isolated. Serotype G3 equine rotavirus BI originating from England belonged to the same genogroup of serotype G3 equine rotaviruses isolated in Japan, although BI was classified as having a different genotype. The genomes of both serotype G10 equine rotavirus R-22 and serotype G10 calf rotavirus were closely related to each other.
Concordance of endotoxemia with gram-negative bacteremia in patients with gram-negative sepsis: a meta-analysis
Authors: Hurley J C
Published: January 2020

The Limulus amebocyte lysate (LAL) assay is a sensitive method for detecting endotoxin. Using gram-negative (GN) bacteremia as the basis for comparison, concordance with endotoxemia in 45 studies could be expressed as an odds ratio. Calculation of summary odds ratios by the Mantel-Haenszel-Peto method indicated that the concordance of the results was no higher by the chromogenic LAL assay than by the gelation version, and the sensitivity was improved by only 11% (62 versus 51%). Endotoxemia was detected in 77 (68%) of 114 patients with bacteremia caused by an organism that was not a member of the family Enterobacteriaceae, whereas endotoxemia was detected in only 120 (45%) of 269 patients with bacteremia caused by a member of the family Enterobacteriaceae or an anaerobe (P < 0.001). This difference was also apparent for patients with GN bacteremia for whom a fatal outcome had been recorded. The prevalence of GN bacteremia in the tested population and the type of etiological agent are critical and previously unrecognized variables which affect the interpretation of the LAL test in patients with suspected sepsis.
Rendering of mycobacteria safe for molecular diagnostic studies and development of a lysis method for strand displacement amplification and PCR
Authors: Zwadyk P , Down J A , Myers N , Dey M S
Published: January 2020

Two criteria must be met before mycobacterial specimens can be tested by DNA amplification methods: (i) the sample must be rendered noninfectious, and (ii) the organisms must be lysed to free the DNA. Previous publications reporting DNA amplification of mycobacteria have concentrated on lysis and amplification procedures and have not addressed the issue of sample safety. We have shown that heating of samples below 100 degrees C may not consistently kill mycobacteria; however, heating at 100 degrees C in a boiling-water bath or a forced-air oven for a minimum of 5 min kills mycobacteria, including Mycobacterium thermoresistibile. Furthermore, heating at 100 degrees C for 30 min consistently lyses mycobacteria to produce short fragments of DNA that are suitable for amplification by PCR and strand displacement amplification. This procedure works with clinical samples digested by the n-acetyl cysteine-NaOH method as well as with suspensions of organisms in phosphate buffer. This paper also demonstrates the feasibility of using strand displacement amplification with clinical specimens.
Evaluation of two BBL Crystal systems for identification of some clinically important gram-negative bacteria
Authors: Holmes B , Costas M , Thaker T , Stevens M
Published: January 2020

The BBL Crystal system (Becton Dickinson Microbiology Systems, Cockeysville, Md.) is a miniaturized bacterial identification method employing modified conventional and chromogenic substrates. Two products are currently available, the Rapid Stool/Enteric ID Kit and the Enteric/Nonfermenter ID Kit, each comprising thirty tests. We report an evaluation of both systems (using database version 1.1 for both) in the identification of 51 gram-negative taxa likely to be encountered commonly in the clinical laboratory. In all, 266 strains were tested in the Enteric/Nonfermenter ID Kit, and these represented 36 taxa of the family Enterobacteriaceae (188 strains), 5 oxidase-positive fermentative taxa (26 strains), and 10 nonfermentative taxa (52 strains). The majority of these same strains (203 of 266) were also tested in the Rapid Stool/Enteric ID Kit. The Enteric/Nonfermenter ID Kit performed as follows: Enterobacteriaceae, 93% correct, 6% not identified, and 1% incorrect; oxidase-positive fermenters, 88, 12, and 0%, respectively; and nonfermenters, 100% correct, although several only to the genus or group level. The Rapid Stool/Enteric ID Kit gave the following results: Enterobacteriaceae, 91% correct, 7% not identified, and 2% incorrect; oxidase-positive fermenters, 80, 13, and 7%, respectively (but results were based on only 15 strains); and nonfermenters, 100% correct (but results were based on only 11 strains). We found the systems extremely easy and rapid to use, and for the Enteric/Nonfermenter ID Kit an identification rate of 100% in 40 of 51 taxa was achieved, with corresponding figures of 29 of 39 taxa for the Rapid Stool/Enteric ID Kit.
Nosocomial infection by Staphylococcus haemolyticus and typing methods for epidemiological study
Authors: Degener J E , Heck M E , van Leeuwen W J , Heemskerk C , Crielaard A , Joosten P , Caesar P
Published: January 2020

A patient with chronic myelogenous leukemia became colonized with a Staphylococcus haemolyticus strain and experienced a septic episode caused by this strain during a cytostatic course. The strain was multiply resistant to antibiotics; the MIC and MBC of vancomycin were 2 and 4 mg/liter, and the MIC and MBC of teicoplanin were 4 and 16 mg/liter, respectively. We performed a surveillance study on the carriage of S. haemolyticus in medical and nursing staff of the hospital ward where the patient was treated. S. haemolyticus was isolated from 18 sites on 12 of the 39 people tested. A number of typing methods were performed in order to investigate the possible relationships among the isolates. Methods used were immunoblotting of staphylococcal peptides, plasmid analysis, restriction fragment length polymorphism of chromosomal DNA, and pulsed-field gel electrophoresis of total DNA. Compared with the immunoblotting technique, the molecular methods were more discriminative. The strain colonizing the patient showed a consistent pattern by all typing methods during isolation. When the immunoblot technique was used, similar patterns were found with isolates from hospital staff and isolates from unrelated sources. With the molecular techniques, no evidence of a local spread of the patient's strain was found. However, plasmid profiles and restriction fragment length polymorphism and pulsed-field gel electrophoresis patterns showed that S. haemolyticus isolates collected from hospital ward personnel were related, which was not the case with isolates collected from unrelated sources. Restriction fragment length polymorphism analysis was more discriminative when IS431 was used as a DNA probe instead of a probe based on the 16S rRNA gene. S. haemolyticus, as in this case, may develop resistance to vancomycin and teicoplanin. These antibiotics are considered the last-resort drugs for the therapy of nosocomial gram-positive infections. Thus, local spread of staphylococci resistant to these drugs is an important problem, which should be prevented by strict hygienic measures and antibiotic policy.
Analysis of hepatitis C virus isolates among healthy blood donors and drug addicts in Chiang Mai, Thailand
Authors: Apichartpiyakul C , Chittivudikarn C , Miyajima H , Homma M , Hotta H
Published: January 2020

Hepatitis C virus (HCV) isolates obtained from 25 anti-HCV antibody-positive healthy blood donors and 29 drug addicts in Chiang Mai, Thailand, were analyzed. HCV RNA was detected in 23 blood donor samples (92%) and 24 drug addict blood samples (83%) by PCR for a portion of the NS5 region. Subtype analysis revealed that HCV type 3a (HCV-3a) was the prevailing subtype (30%), which was followed in prevalence by HCV-1a (21%), -1b (13%), -3b (13%), and -6a (2%). Six (13%) of the 47 isolates showed low sequence similarities with known types and subtypes. The sequence variants could be grouped into four branches in a molecular evolutionary phylogenetic tree.
Sensitivity and specificity of rapid diagnostic tests for detection of group B streptococcal antigen in bacteremic neonates
Authors: Greenberg D N , Ascher D P , Yoder B A , Hensley D M , Heiman H S , Keith J F
Published: January 2020

Latex particle agglutination (LPA) testing for antigen to group B streptococcus (GBS) has been useful in the diagnosis of GBS sepsis in newborns. However, recent reports have demonstrated that the sensitivity of LPA assays may be as low as 27 to 54%. The purposes of the present study were to directly compare the abilities of four urine antigen assays to detect GBS antigen with clinical urine samples from neonates with GBS bacteremia and to evaluate the effect of the urine concentration on the sensitivities and specificities of these assays. Urine samples were collected serially from neonates with blood cultures positive for GBS or on admission from healthy full-term infants. One milliliter of urine was removed, and the remainder was concentrated to a volume of 1 ml. Unconcentrated samples were serially diluted with normal saline and were assayed to determine the highest dilution which would produce a positive test result. The Wellcogen, Bactigen, and Directigen LPA tests and ICON immunoassay were directly compared by using concentrated and unconcentrated urine specimens and urine specimens with known titers. A total of 94 urine specimens, including 61 concentrated and 75 unconcentrated specimens, from bacteremic infants were available for sensitivity testing, and 220 urine specimens from uninfected infants were available for specificity testing. There were significant differences in sensitivity among the four assays when they were performed on concentrated urine specimens, as follows: Directigen, 98%; Bactigen, 92%; ICON, 89%; Wellcogen, 68%. When the assays were performed on unconcentrated urine specimens, the Directigen (84%) and Bactigen (76%) assays were each significantly more sensitive than the ICON (59%) or Wellcogen (43%) assay. All four assays were significantly more sensitive in detecting GBS antigen in concentrated than in unconcentrated urine. The Directigen assay detected antigen in higher dilutions (geometric mean titer, 1:5) than the ICON (1:3), Bactigen (1.2), or Wellcogen (1:1) assay. The specificity was 99.5% for all four assays when concentrated urine was used and for the Bactigen, Directigen, and ICON assays when unconcentrated urine was used; the Wellcogen assay was 100% specific when unconcentrated urine was used. We conclude that there are significant differences in sensitivity but not specificity among the commercially available assays for the detection of GBS antigenuria when concentrated and unconcentrated urine specimens are tested. These differences in sensitivity may affect the abilities of clinicians to accurately diagnose GBS sepsis before culture results are available.
Evaluation of new medium with chromogenic substrates for members of the family Enterobacteriaceae in urine samples
Authors: Kodaka H , Ishikawa M , Iwata M , Kashitani F , Mizuochi S , Yamaguchi K
Published: January 2020

A new medium containing 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide cyclohexylammonium salt (Glu agar) for Escherichia coli and a new medium containing 5-bromo-3-indolyl-beta-D-galactoside (Gal agar) for beta-galactosidase-positive members of the family Enterobacteriaceae were compared with MacConkey agar in a diagnostic trial with 3,562 urine specimens. The isolation rates of E. coli and beta-galactosidase-positive Enterobacteriaceae were increased 8.4 and 19.5%, respectively. The sensitivities and specificities of Glu agar and Gal agar were 98.5 and 100% and 99.2 and 99.5%, respectively.
Serological reactivity and viral genotypes in hepatitis C virus infection
Authors: Maggi F , Vatteroni M L , Pistello M , Avio C M , Cecconi N , Panicucci F , Bendinelli M
Published: January 2020

Patients infected with hepatitis C virus (HCV) were examined with four commercial HCV immunoblotting assays and for anti-GOR antibody to ascertain whether serological findings varied with the genotype of the infecting virus. The results indicate that patients infected with different HCV genotypes tend to show different immunoblotting profiles, mainly due to a low prevalence of antibodies to the viral region NS4 in patients infected with genotypes III and IV. Differences were more evident with second- than with third-generation assays. Patients infected with genotype IV exhibited a lower prevalence of anti-GOR antibody than patients infected with other genotypes.
Interpretive criteria and quality control parameters for testing bacterial susceptibility to the fluoroquinolone PD131628
Authors: Barry A L , Fuchs P C , Allen S D , Tenover F C , Jorgensen J H , Reller L B
Published: January 2020

For testing bacterial susceptibility to PD131628, a 5-micrograms disk and the following tentative interpretive criteria may be used: > or = 19 mm for susceptible (MIC, < or = 1.0 micrograms/ml), 16 to 18 mm for intermediate (MIC, 2.0 micrograms/ml), and < or = 15 mm for resistant (MIC, > or = 4.0 micrograms/ml). For standard quality control strains, the following limits are proposed: for Escherichia coli ATCC 25922, zones of 31 to 41 mm or a MIC of 0.002 to 0.016 micrograms/ml; for Pseudomonas aeruginosa ATCC 27853, zones of 26 to 34 mm or a MIC of 0.12 to 0.5 micrograms/ml; for Staphylococcus aureus ATCC 25923, zones of 27 to 33 mm; for Staphylococcus aureus ATCC 29213, a MIC of 0.03 to 0.12 micrograms/ml; and for Enterococcus faecalis ATCC 29212, a MIC of 0.12 to 0.5 micrograms/ml.
Ability of commercial identification systems to identify newly recognized species of Citrobacter
Authors: O'Hara C M , Roman S B , Miller J M
Published: January 2020

The genus Citrobacter was recently determined to contain 11 genetically distinct species. In addition, the International Committee on Systematic Bacteriology no longer recognizes C. diversus and has, instead, validated the name C. koseri in its place. The 11 species are C. freundii, C. koseri, C. amalonaticus, C. farmeri, C. youngae, C. braakii, C. werkmanii, C. sedlakii, and three unnamed groups, genomospecies 9, 10, and 11. To determine the ease with which some identification systems could respond to these changes, we evaluated five systems for their potential ability to recognize current species in the genus Citrobacter. A simple dichotomous key using conventional biochemicals is presented that may be helpful to presumptively identify Citrobacter strains.
Comparison of three methods for extraction of viral nucleic acids from blood cultures
Authors: Espy M J , Patel R , Paya C V , Smith T F
Published: January 2020

Reliable nucleic acid extraction techniques for blood specimens are required for the sensitive detection of viral DNA. Standardized procedures for processing blood specimens for the molecular detection of herpesviruses (cytomegalovirus [CMV], herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus [EBV]) have not been established. Three methods were used to extract DNA from blood specimens from healthy donors and asymptomatic immunocompromised patients: (i) IsoQuick treatment of whole blood, (ii) extraction of the peripheral blood leukocytes by lysis (lysis buffer and proteinase K), and (iii) extraction of peripheral blood leukocytes with phenol-chloroform (sodium docecyl sulfate solution and proteinase K). All blood specimens from 25 healthy blood donors were negative for CMV, herpes simplex virus and varicella-zoster virus nucleic acid sequences, regardless of the extraction method, while three samples (12%) extracted by the lysis technique were positive for EBV DNA. Of 25 blood samples from asymptomatic immunocompromised patients, CMV and EBV each were detected in nine specimens by lysis extraction, four each by IsoQuick and four (CMV) and six (EBV) by the phenol-chloroform method. Our results indicate that the lysis method is optimal for the detection of CMV and EBV DNA sequences by PCR from the leukocytic fraction of blood specimens. DNA of these viruses is frequently present in blood specimens from asymptomatic immunocompromised patients and occasionally from healthy donors.
Detection of foot-and-mouth disease virus-infected cattle by assessment of antibody response in oropharyngeal fluids
Authors: Archetti I L , Amadori M , Donn A , Salt J , Lodetti E
Published: January 2020

The detection of foot-and-mouth disease virus (FMDV)-persistent carriers among convalescent ruminants is of paramount importance in the aftermath of a field outbreak. To this purpose, FMDV-specific antibody should be investigated first, since virus isolation procedures from such carriers are seriously constrained. The complexity of the overall picture may be compounded by possible emergency vaccinations in the affected areas at the beginning of the outbreak. In this case, it is suggested that mucosal rather than serum antibody be investigated. In fact, we showed that FMDV-infected cattle regularly mount an antibody response in oropharyngeal fluids, in contrast to vaccinated cattle. Antibody could be revealed by neutralization assays and/or an immunoglobulin A (IgA)-specific kinetic enzyme-linked immunosorbent assay (ELISA). Cattle vaccinated once seldom showed a mucosal antibody response, which could be only detected by a total immunoglobulin-specific kinetic ELISA. Very few, if any, cattle showed a mucosal IgA response after repeated vaccinations. Our kinetic, IgA-specific ELISA generally allowed an early detection of FMDV-infected cattle; in particular, it proved to be more sensitive than the usual indirect, antigen-trapping ELISA in experiments on saliva samples.
Controlled clinical comparison of Isolator and BACTEC 9240 Aerobic/F resin bottle for detection of bloodstream infections
Authors: Pohlman J K , Kirkley B A , Easley K A , Washington J A
Published: January 2020

A controlled clinical comparison was carried out with the BACTEC 9240 Aerobic/F resin bottle and the Isolator system with adult patients suspected of having bloodstream infections. A total of 10,500 paired specimens were collected, of which 1,122 from 520 patients were positive. There were 68 false-positive BACTEC bottles; 259 positive cultures that were excluded from analysis because the bottle, the Isolator, or both failed to meet the minimum volume criterion of 8 ml of blood; and 207 positive cultures that were excluded because the isolates were found to be clinically insignificant or of indeterminate clinical significance on the basis of patient assessment. A total of 656 positive cultures from 258 patients formed the basis of the analysis. Significantly more Staphylococcus aureus isolates (P = 0.03), Staphylococcus epidermidis isolates (P = 0.03), members of the family Enterobacteriaceae (P = 0.03), and Pseudomonas aeruginosa isolates (P = 0.04) were recovered from the resin bottle, and there was no category of organism that was recovered significantly more frequently from the Isolator system. With patients receiving antibiotics at the time of blood culture, S. aureus, S. epidermidis, and gram-negative bacilli were recovered significantly more frequently from the resin bottle. No significant differences between systems were found with cultures from patients not receiving antibiotics at the time of blood culture. Only 12 clinically significant organisms were recovered from the bottle on terminal subcultures, and only 1 of these had not been previously isolated from another blood culture set (10 of the 12) or from the companion Isolator (1 of 12). The Aerobic/F resin bottle continuously monitored in the BACTEC 9240 instrument proved to be superior to the Isolator in overall yield of organisms causing bloodstream infection in adults and required less technician time for specimen processing and examination than the Isolator system.
Recombinant mono- and polyantigens to detect cytomegalovirus-specific immunoglobulin M in human sera by enzyme immunoassay
Authors: Landini M P , Lazzarotto T , Maine G T , Ripalti A , Flanders R
Published: January 2020

Serological detection of human cytomegalovirus (HCMV)-specific antibody varies greatly because of antigen composition and the lack of antigen standardization. Antigenic materials composed of single well-characterized viral proteins or portions of them, produced via molecular biology, have proven to be promising tools in improving serodiagnosis. We constructed a recombinant protein containing two regions of ppUL32 (p150) and half of ppUL44 (p52) and compared the immunoglobulin M (IgM) reactivity of this triple-antigen fusion protein with that of a double-antigen fusion protein containing the two ppUL32 fragments and that of a monoantigen fusion protein containing half of ppUL44. We also constructed and tested two other monoantigen fusion proteins containing a large fraction of ppUL80a and a fraction of ppUL83. More than 700 serum samples from different groups of immunocompetent and immunosuppressed subjects were tested for the presence of HCMV IgM by recombinant enzyme immunoassay (rec-EIA) and by a commercially available EIA. Western blotting (immunoblotting) and (in the case of immunosuppressed individuals) antigenemia tests by immunofluorescence and PCR of polymorphonuclear leukocytes were also carried out. The results obtained demonstrate that (i) the triple-antigen fusion protein can replace the individual proteins; (ii) the triple-antigen fusion protein cannot be used alone to replace the virus or infected cells in the serological detection of anti-CMV IgM; (iii) the addition of the fusion proteins containing portions of ppUL83 and ppUL80a is essential for the formation of an antigenic mixture that can replace the virus for the search of HCMV-specific IgM; (iv) rec-EIA is very specific and is more sensitive than the commercially available EIA, and the results obtained are consistent with those obtained by Western blotting; and (v) rec-EIA can reliably be used to detect HCMV-specific IgM in different groups of patients with active HCMV infection.
Monoclonal antibodies to detect capsular diversity among Bacteroides fragilis isolates
Authors: Pantosti A , Colangeli R , Tzianabos A O , Kasper D L
Published: January 2020

The capsular polysaccharide complex (CPC) of Bacteroides fragilis is composed of two distinct polysaccharides, designated PS A and PS B, and is a major virulence factor of this microorganism. In order to investigate the antigenic diversity of the CPCs of B. fragilis strains, we generated and characterized 10 monoclonal antibodies (MAbs) directed to the CPCs of three reference strains. The specificities of the MAbs were determined by enzyme-linked immunosorbent assay and dot-immunobinding assay. At least one MAb was specific for each PS A and PS B of the three strains. The MAbs were used to detect capsular antigens on the surface of 231 B. fragilis isolates from different geographical areas by a whole-cell dot-immunobinding assay. Over half of the strains, regardless of the country of origin, reacted with at least one MAb. Clinical extraintestinal infection isolates were significantly more reactive than fecal isolates, suggesting an association between capsular composition and the propensity to cause clinical infections. The patterns of reactivity of the isolates with the 10 MAbs were very different and sometimes extremely complex and indicated a sharing of epitopes among different capsular polysaccharides. The reactive strains could be grouped according to 32 different patterns; some patterns were relatively common, while others were rarer and were shown by only one or two strains. These results show that B. fragilis capsular polysaccharides are antigenically extremely diverse. This complexity and the large number of nonreactive strains indicate that a typing system based on B. fragilis capsular antigens will be difficult to establish.
An Indian strain of hepatitis E virus (HEV): cloning, sequence, and expression of structural region and antibody responses in sera from individuals from an area of high-level HEV endemicity
Authors: Panda S K , Nanda S K , Zafrullah M , Ansari I H , Ozdener M H , Jameel S
Published: January 2020

Hepatitis E virus (HEV) is responsible for a majority of sporadic and epidemic viral hepatitides in India and other developing countries. Even though the genomes of four geographically distinct strains of HEV have been cloned and sequenced, the Indian strain of HEV remains largely uncharacterized. We have cloned and sequenced about 2.2 kb of the HEV genome constituting the structural region from an Indian strain of HEV. The nucleotide and amino acid sequences show a high degree of conservation with sequences from other HEV strains. Open reading frames (ORF) 2 and 3 were expressed in Escherichia coli as N-terminal hexahistidine epitope fusions. The purified proteins were then used in an immunoblot assay to evaluate the antibody status in sera from individuals from an area of high-level HEV endemicity. The anti-ORF2 antibodies were found to be nonspecific and could not be correlated to clinical disease. The immunoglobulin M anti-ORF3 was found to be specific for the presence of acute disease. The implications of these findings in HEV diagnosis and vaccine development are discussed.
Genomic and proteinic characterization of strain S, a rickettsia isolated from Rhipicephalus sanguineus ticks in Armenia
Authors: Eremeeva M , Balayeva N , Roux V , Ignatovich V , Kotsinjan M , Raoult D
Published: January 2020

Strain S, a spotted fever group (SFG) rickettsia isolated from Rhipicephalus sanguineus ticks collected in Armenia, was identified. Microimmunofluorescence, sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis and Western immunoblotting, PCR and then restriction fragment length polymorphism analysis, pulsed-field gel electrophoresis, and 16S rRNA gene sequencing were used to compare strain S with reference isolates. Strain S was found to possess proteinic, antigenic, and genomic patterns which were unique among SFG rickettsiae. Strain S is characterized by its high degree of pathogenicity for experimental animals, but its role as a potential human pathogen should be determined. The role of R. sanguineus ticks in the epidemiology of SFG rickettsiae is discussed.
Molecular epidemiology of toxigenic Vibrio cholerae in Bangladesh studied by numerical analysis of rRNA gene restriction patterns
Authors: Faruque S M , Roy S K , Alim A R , Siddique A K , Albert M J
Published: January 2020

Cholera is endemic in Bangladesh, and a regular seasonal pattern of cholera epidemics occurs. We examined the clonal relationships among 103 clinical and environmental Vibrio cholerae isolates belonging to O1, O139, or non-O1 non-O139 serogroups isolated during epidemic and interepidemic periods in Bangladesh and compared them with those of 51 V. cholerae isolates from four countries in Asia and Africa. These studies were done by a computer-assisted numerical analysis of the restriction endonuclease cleavage patterns of rRNA genes (ribotypes). Unweighed pair-group cluster analysis of BglI- and HindIII-generated band patterns revealed 16 clusters. Ribotypes were defined as clusters of strains possessing > 98% similarity. The results showed that 154 isolates could be differentiated into 15 different ribotypes, and strains belonging to 3 of these ribotypes (ribotypes I, V, and VIIIA and VIIIB) were isolated more frequently during the epidemic periods than during interepidemic periods in Bangladesh. Classical vibrios belonged to six different ribotypes (ribotypes I to VI), with a mean similarity coefficient of 0.84, and the El Tor vibrios belonged to five different ribotypes (ribotypes VIIIA and IX to XII), with a mean similarity coefficient of 0.82. A single clone of El Tor vibrios (ribotype XII) was resident in Tanzania, whereas Nigeria, Syria, and India shared toxigenic El Tor strains with Bangladesh. Cholera toxin (CT)-positive O139 vibrios isolated from Bangladesh and India belonged to a single ribotype (ribotype VIIIB) and were > 98% similar to one of the ribotypes of El Tor vibrios (ribotype VIIIA), but a CT-negative O139 vibrio from Argentina (ribotype XIII) was < 75% similar to the same cluster of El Tor vibrios, thus suggesting more than one possible origin for O139 vibrios. Strains belonging to the same ribotypes (ribotypes VIII to X) were isolated from both patients and surface water in Bangladesh, indicating possible transmission through surface water. A clone of a CT-positive environmental isolate of non-O1 V. cholerae (ribotype VII) was found to be closely related (76.3% similarity) to a clone of classical vibrios (ribotype I) and was only between 27.2 and 56.1% similar to clusters of El Tor, O139, and two other non-O1 nontoxigenic clones.
PCR and direct fluorescent-antibody staining confirm Chlamydia trachomatis antigens in swabs and urine below the detection threshold of Chlamydiazyme enzyme immunoassay
Authors: Krepel J , Laur I , Sproston A , Luinstra K , Jang D , Mahony J , Chernesky M
Published: January 2020

In order to test the hypothesis that specimens blocking with a neutralizing reagent below the cutoff of the Chlamydiazyme enzyme immunoassay represent infected patients, we used direct fluorescent-antibody staining for elementary bodies (EBs) and PCR to confirm results for cervical swabs collected from 55,963 women and urethral swabs or first-void urine (FVU) samples collected from 5,781 men attending physicians' offices in the Toronto, Canada, area. Within a grey zone arbitrarily selected to represent values up to 40% below the positive threshold of the test run, 134 cervical swabs, 44 urethral swabs, and 39 FVU specimens exhibited a blocking response ( > 50% reduction in signal). Three or more EBs were observed in each of 98 cervical swabs (73.1%), 38 urethral swabs (86.4%), and 21 FVU specimens (53.8%). Of the 36 cervical swabs with fewer than three EBs, 33 were PCR positive; the positive PCR results for male specimens were 6 of 6 urethral swabs and 17 of 18 FVU samples. Application of the blocking test to specimens negative in the Chlamydiazyme enzyme immunoassay but having optical densities within 40% of the cutoff added 14.2% (217 of 1,531 specimens) more positive results to the survey. A total of 213 of 217 samples (98.2%) were reconfirmed as having EBs or DNA.
Immunomagnetic PCR and DNA probe for detection and identification of Porphyromonas gingivalis
Authors: Benkirane R M , Guillot E , Mouton C
Published: January 2020

The aim of the study that we describe was to combine an immunomagnetic separation and a PCR followed by dot blot hybridization with a DNA probe for the detection and identification of Porphyromonas gingivalis. Immunomagnetic particles were coated with monoclonal antibody specific for P. gingivalis and were incubated with a suspension containing seven oral bacterial species spiked with various dilutions of P. gingivalis. Beads with their load of bound bacterial were boiled in water, and the target DNA in the supernatant was amplified with a primer pair to generate a 593-bp PCR fragment specific for P. gingivalis. Finally, the product of amplification was detected by dot blot hybridization with a digoxigenin-labeled 593-bp probe. The detection limit was determined to be 100 bacterial cells per ml. The immunomagnetic-PCR/DNA probe procedure described here should be useful for the rapid, specific, and sensitive detection and identification of P. gingivalis in clinical samples.
Physiological traits associated with success of Candida albicans strains as commensal colonizers and pathogens
Authors: Schmid J , Hunter P R , White G C , Nand A K , Cannon R D
Published: January 2020

DNA fingerprinting with the moderately repetitive sequence Ca3 has repeatedly identified groups of genetically similar strains of Candida albicans that are more frequently isolated than other groups of strains from human hosts in a geographical locale. Members of these groups are found in approximately 30% of healthy individuals and in up to 70% of patients suffering from candidiasis. The high prevalence of these strains implies that they are more successful in colonizing human hosts and in causing disease than other strains (J. Schmid, Clin. Adv. Treatment Fungal Infect. 4(6):12-16, 1993). In the present study, we have compared one such group of highly prevalent strains with other strains from the same locale to identify physiological traits a larger number of chemicals than other strains in a resistogram assay. When resistance to individual chemicals used in the resistogram assay was analyzed, strains from the group of highly prevalent strains were significantly more often resistant to boric acid, cetrimide, chlorhexidine, 5-fluorocytosine, and high sodium chloride concentrations than other strains. Strains from the group of highly prevalent strains also adhered significantly (1.5 times) better to saliva-coated surfaces than did other strains. Because members of highly prevalent groups of strains are the most common infectious agents in candidiasis, these physiological traits may be involved in determining not only the success of C. albicans in colonizing human hosts in general but also its ability to cause disease. Sodium chloride resistance and increased adherence were also associated with infectious isolates outside the group of highly prevalent strains, indicating that they may be of particular importance in pathogenesis.