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PubMed1987

beta-Glucosidase in Candida albicans and its application in yeast identification.

In this report we attempt to explain the discrepancy between beta-glucosidase (EC 3.2.1.21) activity in Candida albicans as measured by commercial kits and that found in an experimental assay. beta-Glucosidase activity in American and Israeli isolates of C. albicans was evaluated with the API ZYM and YeastIdent systems (Analytab Products) and with experimental biochemical assays. Activity was found with whole cells and cell extracts of isolates from both sources. The greatest beta-glucosidase activity was found at pH 5.0 and with p-nitrophenyl-beta-glucopyranoside (PNP-BDG) as the substrate. In assays with beta-naphthyl-beta-D-glucopyranoside and 6-bromo-2-naphthyl-beta-D-glucopyranoside (6-Br-2-naphthyl-BDG), no enzyme activity was detected in whole cells and only limited activity was found in cell extracts of isolates from both sources. In studies with PNP-BDG at pH 5.0 and 7.5, 29 to 38% less activity was found at both pHs with American whole cells, and minor activity (20%) was found at pH 7.5 with isolates from both sources. Because assays with PNP-BDG in cell extracts of isolates from both sources showed no significant differences in activity, the more limited beta-glucosidase activity in American whole cells was most likely due to less efficient transport. Because the API ZYM system uses 6-Br-2-naphthyl-BDG as the substrate and because the substrate is buffered at pH 7.5 in the API YeastIdent kit, both systems appear to be of limited value for the detection of beta-glucosidase activity in C. albicans.

Candida albicans↗
PubMed1991

Evaluation of the MicroScan Rapid Yeast Identification panel.

The MicroScan Rapid Yeast Identification (RYI) panel is a 4-h microdilution system for identification of clinical yeastlike isolates. Its accuracy was evaluated by using 357 isolates encompassing 11 genera and 30 species. The RYI panel identifications were compared with those obtained by the API 20C system assisted with morphological characterization on cornmeal-Tween 80 agar. The panels were read both visually and with the AutoScan-4, a computer-controlled microplate reader. Both the RYI panel and the API 20C system correctly identified 78% of the strains within 4 and 72 h, respectively, with no additional tests. Supplementary tests recommended by the manufacturers made it possible to identify up to 96.6% (AutoScan-4) and 98.9% (API 20C) of the strains. The accuracy of the RYI panel was 99.5% with common strains and 92.1% with less common strains. The RYI panel misidentified 10 or 12 strains and failed to identify 2 or 3 strains, depending on whether it was read with the AutoScan-4 or visually. Errors occurred with one strain of Torulopsis glabrata and the less common yeasts T. candida, Candida lusitaniae, C. lambica, C. rugosa, C. stellatoidea, Cryptococcus albidus, C. laurentii, and C. uniguttulatus. Overall, the RYI panel appears to be a reliable system for identification of the more common clinical yeast isolates.

Candida↗
PubMed1992

Modified MacConkey medium which allows simple and reliable identification of Providencia stuartii.

This work describes a modified MacConkey medium (MCP medium) enabling the simple identification of Providencia stuartii, an emerging nosocomial pathogen. A total of 813 strains, belonging to the families Enterobacteriaceae and Pseudomonadaceae, were tested on MCP medium; all P. stuartii strains were phosphatase positive, as were 97.5% of Morganella morganii strains, in contrast with all other tested organisms. A simple discriminating test, such as the ornithine or citrate test, allowed identification of strains of these species. We have also compared the reliabilities of P. stuartii identification by commercial kits (API 20E system) by using a standard MacConkey or MCP medium. Sixteen and three-tenths percent of P. stuartii strains were misidentified by using the former procedure, while with the latter all strains were correctly identified. Finally, the MCP medium was used over a 6-month period in our routine clinical laboratory. Of a total of 1,278 seeded urine samples from elderly patients, we isolated 103 P. stuartii strains which were all correctly identified by coupling MCP medium and the API 20E system. Seventeen and one-half percent of these strains were misidentified when the API 20E system was used in combination with standard MacConkey medium.

Aged↗
PubMed1995

Evaluation of Staph ID 32 system and Staph-Zym system for identification of coagulase-negative staphylococci.

The purpose of the investigation was to evaluate two commercially available identification systems: a new modification of the Staph-Zym system (Rosco, Tåstrup, Denmark) and the Staph ID 32 API system (API System, BioMérieux, Paris, France). A local standard method to be used in routine laboratories was also evaluated. A total of 200 staphylococcal isolates, including strains from both the American Type Culture Collection and the Czechoslovak Collection of Microorganisms as well as 89 clinical isolates, were used in tests of all three identification systems. The Staph ID 32 API system identified from 50 to 100% of the reference strains and 82.1% of the clinical isolates correctly. The Staph-Zym system identified from 90 to 100% of the reference strains and 82.1% of the clinical isolates correctly. Most misidentifications were of minor importance, but in both systems major failures appeared (Staphylococcus aureus was identified as a coagulase-negative staphylococcus). Both systems needed backup from a reference laboratory to determine if two isolates were of the same strain.

Bacteriological Techniques↗
PubMed1998

Rapid identification of Candida species with species-specific DNA probes.

Rapid identification of Candida species has become more important because of an increase in infections caused by species other than Candida albicans, including species innately resistant to azole antifungal drugs. We previously developed a PCR assay with an enzyme immunoassay (EIA) format to detect amplicons from the five most common Candida species by using universal fungal primers and species-specific probes directed to the ITS2 region of the gene for rRNA. We designed probes to detect seven additional Candida species (C. guilliermondii, C. kefyr, C. lambica, C. lusitaniae, C. pelliculosa, C. rugosa, and C. zeylanoides) included in the API 20C sugar assimilation panel, five probes for species not identified by API 20C (C. haemulonii, C. norvegica, C. norvegensis, C. utilis, and C. viswanathii), and a probe for the newly described species C. dubliniensis, creating a panel of 18 Candida species probes. The PCR-EIA correctly identified multiple strains of each species tested, including five identified as C. albicans by the currently available API 20C database but determined to be C. dubliniensis by genotypic and nonroutine phenotypic characteristics. Species identification time was reduced from a mean of 3.5 days by conventional identification methods to 7 h by the PCR-EIA. This method is simple, rapid, and feasible for identifying Candida species in clinical laboratories that utilize molecular identification techniques and provides a novel method to differentiate the new species, C. dubliniensis, from C. albicans.

Base Sequence↗
PubMed1999

Simple strategy for direct identification of medically important yeast species from positive blood culture vials.

We compared direct inoculation of the Auxacolor yeast identification system from positive blood culture vials to standard identification with the API 20C AUX (API 20C), using 44 prospectively collected clinical specimens and 25 seeded blood culture vials. Direct inoculation of the Auxacolor system was accurate and more rapid than standard identification with the API 20C.

Blood↗
PubMed1999

Identification of Burkholderia spp. in the clinical microbiology laboratory: comparison of conventional and molecular methods.

Cystic fibrosis (CF) predisposes patients to bacterial colonization and infection of the lower airways. Several species belonging to the genus Burkholderia are potential CF-related pathogens, but microbiological identification may be complicated. This situation is not in the least due to the poorly defined taxonomic status of these bacteria, and further validation of the available diagnostic assays is required. A total of 114 geographically diverse bacterial isolates, previously identified in reference laboratories as Burkholderia cepacia (n = 51), B. gladioli (n = 14), Ralstonia pickettii (n = 6), B. multivorans (n = 2), Stenotrophomonas maltophilia (n = 3), and Pseudomonas aeruginosa (n = 11), were collected from environmental, clinical, and reference sources. In addition, 27 clinical isolates putatively identified as Burkholderia spp. were recovered from the sputum of Dutch CF patients. All isolates were used to evaluate the accuracy of two selective growth media, four systems for biochemical identification (API 20NE, Vitek GNI, Vitek NFC, and MicroScan), and three different PCR-based assays. The PCR assays amplify different parts of the ribosomal DNA operon, either alone or in combination with cleavage by various restriction enzymes (PCR-restriction fragment length polymorphism [RFLP] analysis). The best system for the biochemical identification of B. cepacia appeared to be the API 20NE test. None of the biochemical assays successfully grouped the B. gladioli strains. The PCR-RFLP method appeared to be the optimal method for accurate nucleic acid-mediated identification of the different Burkholderia spp. With this method, B. gladioli was also reliably classified in a separate group. For the laboratory diagnosis of B. cepacia, we recommend parallel cultures on blood agar medium and selective agar plates. Further identification of colonies with a Burkholderia phenotype should be performed with the API 20NE test. For final confirmation of species identities, PCR amplification of the small-subunit rRNA gene followed by RFLP analysis with various enzymes is recommended.

Bacterial Typing Techniques↗
PubMed1976

Evaluation of commercial systems for the identification of clinical yeast isolates.

The Analytab Products Inc. (API), Micro-Drop (MD), and Uni-Yeast-Tek (UYT) systems for the presumptive identification of common clinical yeast isolates were compared with the oxidation-fermentation (OF) and a conventional procedure. With 229 coded isolates, the identification accuracies were API 94, MD 83, OF 82, and UYT 99%. The API system required the greatest technical ability. The MD materials were prone to malfunction. OF media, if incubated beyond 14 days, gave an accuracy of 87%, but this offered no advantage over the conventional procedure. The UYT system was the easiest to use.

Carbohydrate Metabolism↗
PubMed2003

Experience with the MicroSeq D2 large-subunit ribosomal DNA sequencing kit for identification of commonly encountered, clinically important yeast species.

Experience with a MicroSeq D2 large-subunit (LSU) ribosomal DNA (rDNA) sequencing kit for identification of yeast species commonly encountered in the mycology laboratory at Mayo Clinic is described here. A total of 131 isolates of yeasts recovered from clinical specimens were included in the study. Phenotypic methods used for initial identification included germ tube formation, urease production, microscopic morphological features on cornmeal agar, and an API 20C AUX system; all isolates were sequenced using a MicroSeq D2 LSU rDNA sequencing kit. Nucleic acid sequencing identified 93.9% of the isolates to the correct genus and species. A total of 100 of the isolates (representing 19 species of Candida) were sequenced, and 98% gave results concordant with identifications made by the API 20C AUX system; distance scores ranged from 0 to 1.88%, with an average value of 0.23%. Candida dubliniensis was not included in the MicroSeq database and was identified as Candida albicans. A total of 32 isolates representing 9 other genera (including Cryptococcus, Filobasidium, Kloeckera, Malassezia, Pichia, Sporidiobolus, Rhodotorula, Zygosaccharomyces, and Trichosporon) were included, and 81.3% showed concordant results when phenotypic and sequencing results were compared. Most discrepancies were attributed to the lack of inclusion of the species in the MicroSeq or API 20C AUX database. The MicroSeq D2 LSU rDNA sequencing kit appears to be accurate and useful for the identification of yeasts that might be seen in a clinical laboratory.

Base Sequence↗
PubMed2003

Evaluation of three rapid methods for the direct identification of Staphylococcus aureus from positive blood cultures.

Staphylococci represent the most commonly encountered blood culture isolates. Differentiating Staphylococcus aureus from coagulase-negative staphylococci (CoNS) is important in guiding empirical therapy, especially since the majority of CoNS are contaminants. This study evaluated three rapid methods for the direct identification of S. aureus from blood cultures. A total of 157 patient blood cultures with gram stains showing gram-positive cocci in clusters were included. The following assays were evaluated: API RAPIDEC staph (API) (bioMerieux, Durham, N.C.), the tube coagulase test (TCT) read at 4 h, and peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) (AdvanDx, Woburn, Mass.). All assays yielded results of S. aureus or non-S. aureus. The direct rapid results were compared to results obtained with isolated colonies using the AccuProbe Staphylococcus aureus Culture Identification Test (Gen-Probe, San Diego, Calif.). API, TCT, and PNA FISH exhibited sensitivities of 96, 84, and 99% and specificities of 99, 100, and 100%, respectively. Direct identification testing by any of these three assays yielded acceptable performance and timely results. This ability to accurately detect S. aureus in blood cultures gives the physician information with which to initiate or tailor antimicrobial therapy. Coupled with direct susceptibility testing of positive blood culture broths, the patient and institution may experience improved outcomes.

Bacteremia↗
PubMed1979

Biochemical, enzymatic, and serological differentiation of Peptococcus indolicus (Christiansen) Sørensen from Peptococcus asaccharolyticus (Distaso) Douglas.

Peptococcus indolicus (formerly Micrococcus indolicus) is an asaccharolytic anaerobic coccus that is frequently isolated from udder secretions from cases of heifer and dry-cow mastitis (summer mastitis). To facilitate better identification and its differentiation from Peptococcus asaccharolyticus, a variety of biochemical, enzymatic, and serological properties were studied. Seventy-nine strains of P. indolicus of bovine origin and 10 strains of P. asaccharolyticus of human origin were examined using the API 20A and API-ZYM test kit systems. In addition, production of extracellular enzymes by using sensitive substrate-containing agar plate tests, production of peptocoagulase (a plasma-clotting factor), hemolytic properties, metabolic end products by gas chromatography, and serological characteristics with a set of P. indolicus typing antisera were investigated. P. indolicus and P. asaccharolyticus were not satisfactorily differentiated solely by the API 20A system. P. indolicus differed from P. asaccharolyticus in producing H(2)S, reducing nitrate to nitrite, producing peptocoagulase, possessing alkaline phosphatase, and producing large amounts of propionate from lactate. Moreover, none of the strains of P. asaccharolyticus was typable with the P. indolicus typing antisera. The majority (88%) of P. indolicus strains also gave weak hydrolysis of ribonucleic acid, and 6 out of 79 produced deoxyribonuclease. All strains in this study were sensitive to metronidazole (5 mug) by disk diffusion tests.

Anaerobiosis↗
PubMed1979

Identification of Pseudomonas pseudomallei in the clinical laboratory.

Ninety-one strains of Pseudomonas pseudomallei were tested in the API 20E system and in equivalent conventional tests. Except for utilisation of citrate there was good correlation between API and conventional tests. Seven-digit profiles were constructed from each strain after 48 hours' incubation, and numerical codes 2 006 727, 2 206 706, 2 206 707, and 2 206 727 accounted for 77% of strains tested. API 20 E, in combination with tests for heat stability of alkaline phosphatase, resistance to colistin and gentamicin, oxidative attack only of glucose, and acid from maltose, was found to provide a simple method for positive identification of all strains of this organism in two days.

Bacteriological Techniques↗
PubMed1991

Incidence of penicillin tolerance among blood culture isolates of Streptococcus sanguis, 1987-88.

Laboratories that reported isolations of Streptococcus sanguis from blood cultures to the Communicable Disease Surveillance Centre (CDSC) Colindale were requested to submit strains to Bath Public Health Laboratory to allow the prevalence of penicillin tolerance within different biotypes of this species to be studied. One hundred and fifty one Streptococcus spp were received from 78 United Kingdom laboratories in one year. Strains were identified using the API 20 Strep, and minimum inhibitory concentrations (MICs) of penicillin were determined using the spiral gradient plate method. Penicillin tolerance was detected by spraying beta-lactamase over inoculated gradient plates, reincubating for 48 hours and counting the number of surviving organisms represented by colonies. There were 57 different API identification profiles encountered in the survey. Most S sanguis I/1 strains were penicillin tolerant, most S sanguis II strains were non-tolerant. The overall geometric mean MIC of penicillin was considerably lower for S sanguis I/1 than for all other biotypes. The distribution of biotypes and the geometric mean MIC of penicillin for each biotype were not significantly different for infective endocarditis strains than for all strains tested, suggesting little or no association between penicillin tolerance and the seeding of endocardium. When the reactions obtained using API 20 Strep were compared with a recent taxonomic study of viridans streptococci, 22 of 38 S sanguis I/1 strains could be reclassified as S gordonii; all these strains were penicillin tolerant. Such reclassification would allow likely penicillin tolerant strains to be predicted.

Blood↗
PubMed1991

Evaluation of the Microring YT system for identifying clinical yeast isolates.

The Microring YT is a commercial system for identifying clinical yeast isolates. The system uses a series of discs impregnated with inhibitory agents mounted on a ring. The pattern of growth and inhibition produced provides a six digit code which can be compared with a table provided by the manufacturer. The performance of this system was compared with the API 32C in the identification of 606 yeast isolates (355 clinical and 251 environmental strains). The Microring YT system was in 72.6% agreement with the API 32C system. The sensitivity of identification of different species varied from 38% to 100%. The API 32C system has a more extensive database than the Microring YT and is thus more reliable for use, but it is considerably more expensive. It is concluded that although the Microring YT is cheap, easy, and convenient to use, it is inadequate for many common Candida species.

Candida↗
PubMed2003

Gemella bacteraemia characterised by 16S ribosomal RNA gene sequencing.

AIMS: To define epidemiology, clinical disease, and outcome of gemella bacteraemia by 16S rRNA gene sequencing. To examine the usefulness of the Vitek, API, and ATB systems in identifying two gemella species. METHODS: All alpha haemolytic streptococci other than Streptococcus pneumoniae isolated from blood cultures during a six year period were identified by conventional biochemical methods, the Vitek system, and the API system. 16S rRNA gene sequencing was performed on all isolates identified by both kits as gemella with >or= 95% confidence or by either kit as any bacterial species with < 95% confidence. The ATB expression system was used to identify the two isolates that were defined as gemella species by 16S rRNA gene sequencing. RESULTS: Of the 302 alpha haemolytic streptococci other than S pneumoniae isolated, one was identified as Gemella morbillorum, and another as Gemella haemolysans by 16S rRNA gene sequencing. The patient with monomicrobial G morbillorum bacteraemia was a 66 year old man with community acquired infective endocarditis with septic thromboemboli. The patient with G haemolysans bacteraemia was a 41 year old woman with hospital acquired polymicrobial bacteraemia during the neutropenic period of an autologous bone marrow transplant for non-Hodgkin's lymphoma, the first case of its kind in the English literature. The API and ATB expression systems only identified the second strain as G haemolysans at 94% and 99% confidence, respectively, whereas the Vitek system identified none of the two strains correctly at > 70% confidence. CONCLUSIONS: Gemella bacteraemia is uncommon. 16S rRNA gene sequencing is the method of choice for identification of gemella and gemella-like isolates.

Adult↗
PubMed1982

Lack of time-dependent augmentation of carotid sinus baroreflex system after vagotomy.

The purpose of this experiment was to study whether the carotid sinus baroreflex system (CS system) increases its gain with time after vagotomy in compensation for the loss of the vagally mediated arterial pressure control system (V system). In 7 dogs anesthetized with pentobarbital sodium we determined the responsiveness of the V system by repeatedly measuring the overall open-loop gain (G) of the negative feedback control system. G was assessed as (delta API/delta APS) -- 1, where delta API and delta APS are, respectively, the immediate and steady-state falls in arterial pressure at the aortic arch following a stepwise reduction in blood volume. delta API, delta APS, and G in intact condition were -12.0 +/- 1.8 mmHg, -1.1 +/- 0.2 mmHg, and 10.1 +/- 0.7 (SD), respectively. delta API, delta APS, and G after vagotomy, i.e., G of the CS system (GCS), were -15.6 +/- 3.6 mmHg, -6.4 +/- 1.9 mmHg, and 1.6 +/- 0.4 GCS did not change with time over 4 h after vagotomy. We conclude that the CS system cannot augment its ability to restore arterial pressure in compensation for the lost function of the V system within 4 h after vagotomy in the anesthetized dog.

Animals↗
PubMed1999

Reactivity of 77 antibodies to prostate-specific antigen with isoenzymes and complexes of prostate-specific antigen.

Seventy-seven antibodies submitted to the ISOBM TD-3 PSA Workshop (TD-3.1 and TD-3.2) were characterized by measuring their reactivity with isoenzymes of free prostate-specific antigen (PSA), PSA complexed to alpha1-antichymotrypsin (PSA-ACT) and alpha1-proteinase inhibitor (PSA-API). Antibodies were classified into 15 distinct groups according to their reaction profiles with the various isoenzymes. Some antibodies recognizing both free and complexed PSA were inaccurate in measuring total PSA. Eight of the 9 free PSA-specific antibodies cross-reacted more with PSA-API than with PSA-ACT, while 1 antibody reacted less with PSA-API than PSA-ACT. From the panel of antibodies 39 reacted with both free and complexed PSA and were classified as total PSA antibodies.

Antibodies, Monoclonal↗