Rapid identification ofStreptococcus pyogenes by flow cytometry

Flow cytometry combined with immunofluorescence ofStreptococcus pyogenes was used to assay bacteria suspended in buffer solution and in saliva derived from throat swabs of healthy volunteers. The method allowed the enumeration of as few as 5 × 10³ and 5 × 10⁴ CFU per milliliter of buffer and saliva respectively. Controls includingStreptococcus salivarius instead ofStreptococcus pyogenes or buffer instead of specific antibodies confirmed the specificity of the detection ofStreptococcus pyogenes in the samples. The results suggest that flow cytometry may serve as a basis for an automated reliable method for the diagnosis of streptococcal infections.     

Antifungal susceptibility testing of Candida species by flow cytometry

The feasibility of flow cytometric antifungal susceptibility testing has been studied using the fluorescent anionic membrane potential probe, bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. The in vitro antifungal susceptibility testing of amphotericin B was performed on 8 Candida isolates from clinical specimens and 2 ATCC strains by flow cytometry with the results compared to those of the National Committee of Clinical Laboratory Standards (NCCLS) M27-T, broth macrodilution method. The flow cytometric method is based on an increase of fluorescence given out by DiBAC4(3) in fungi when they are killed by antifungal agents. Minimum inhibitory concentration (MIC) of amphotericin B ranged from 0.25 to 1 microg/mL. All results agreed within +/-2 dilution between the flow cytometric method and the M27-T method. MIC with ATCC strains were within recommended ranges of M27-T. The new flow cytometric method revealed a clear and distinct reproducible test end point. A four hr of incubation was sufficient for the test. In conclusion, flow cytometry using DiBAC4(3) is a rapid and accurate in vitro antifungal susceptibility testing method.     

Rapid identification of Candida species in blood cultures by a clinically useful PCR method.

Widespread use of fluconazole for the prophylaxis and treatment of candidiasis has led to a reduction in the number of cases of candidemia caused by Candida albicans but has also resulted in the emergence of candidemias caused by innately fluconazole-resistant, non-C. albicans Candida species. Given the fulminant and rapidly fatal outcome of acute disseminated candidiasis, rapid identification of newly emerging Candida species in blood culture is critical for the implementation of appropriately targeted antifungal drug therapy. Therefore, we used a PCR-based assay to rapidly identify Candida species from positive blood culture bottles. This assay used fungus-specific, universal primers for DNA amplification and species-specific probes to identify C. albicans, C. krusei, C. parapsilosis, C. tropicalis, or C. glabrata amplicons. It also used a simpler and more rapid (1.5-h) sample preparation technique than those described previously and used detergent, heat, and mechanical breakage to recover Candida species DNA from blood cultures. A simple and rapid (3.5-h) enzyme immunosorbent assay (EIA)-based format was then used for amplicon detection. One hundred fifty blood culture bottles, including 73 positive blood culture bottle sets (aerobic and anaerobic) from 31 patients with candidemia, were tested. The combined PCR and EIA methods (PCR-EIA) correctly identified all Candida species in 73 blood culture bottle sets, including bottles containing bacteria coisolated with yeasts and 3 cultures of samples from patients with mixed candidemias originally identified as single-species infections by routine phenotypic identification methods. Species identification time was reduced from a mean of 3.5 days by routine phenotypic methods to 7 h by the PCR-EIA method. No false-positive results were obtained for patients with bacteremias (n = 18), artificially produced non-Candida fungemias (n = 3), or bottles with no growth (n = 20). Analytical sensitivity was 1 cell per 2-microl sample. This method is simpler and more rapid than previously described molecular identification methods, can identify all five of the most medically important Candida species, and has the potential to be automated for use in the clinical microbiology laboratory.     

Rapid susceptibility testing of Candida albicans by flow cytometry.

The emerging magnitude of human fungal infections has renewed interest in developing rapid and standardized methods for susceptibility testing. We demonstrated that susceptibility testing of Candida albicans can be accomplished rapidly by using flow cytometry. Test results were available within 8 to 24 h after C. albicans isolates were incubated with amphotericin B, itraconazole, and flucytosine. This is an improvement of 24 to 60 h in the time to availability of susceptibility test results compared to the time to availability of National Committee for Clinical Laboratory Standards-recommended broth macrodilution test results. In addition, the flow cytometric endpoints, mean channel fluorescence, and number of fluorescence-labeled C. albicans cells were easy to interpret for greater sensitivity and reliability. Flow cytometry provides a more accurate means of obtaining antifungal susceptibility test results.     

CHROMagar Candida, a new differential isolation medium for presumptive identification of clinically important Candida species.

CHROMagar Candida is a novel, differential culture medium that is claimed to facilitate the isolation and presumptive identification of some clinically important yeast species. We evaluated the use of this medium with 726 yeast isolates, including 82 isolated directly on the medium from clinical material. After 2 days of incubation at 37 degrees C, 285 C. albicans isolates gave distinctive green colonies that were not seen with any of 441 other yeast isolates representing 21 different species. A total of 54 C. tropicalis isolates also developed distinctive dark blue-gray colonies with a halo of dark brownish purple in the surrounding agar. C. krusei isolates (n = 43) also formed highly characteristic rough, spreading colonies with pale pink centers and a white edge that was otherwise encountered only rarely with isolates of C. norvegensis. Trichosporon spp. (n = 34) formed small, pale colonies that became larger and characteristically rough with prolonged incubation. Most of the other 310 yeasts studied formed colonies with a color that ranged from white to pink to purple with a brownish tint. The only exceptions were found among isolates identified as Geotrichum sp. or Pichia sp., some of which formed colonies with a gray to blue color and which in two instances formed a green pigment or a dark halo in the agar. The specificity and sensitivity of the new medium for the presumptive identification of C. albicans, C. krusei, and C. tropicalis exceeded 99% for all three species. A blinded reading test involving four personnel and 57 yeast isolates representing nine clinically important species confirmed that colonial appearance after 48 h of incubation on CHROMagar Candida afforded the correct presumptive recognition of C. albicans, C. tropicalis, C, krusei, and Trichosporon spp. None of nine bacterial isolates grew on CHROMagar Candida within 72 h, and bacteria (Escherichia coli) grew from only 4 of 104 vaginal, 100 oral, and 99 anorectal swabs. The new medium supported the growth of 19 of 23 dermatophyte fungi tested and 41 of 43 other molds representing a broad range of fungal pathogens and contaminants. In parallel cultures of 348 clinical specimens set up on Sabourand agar and CHROMagar Candida, both media grew yeasts in the same 78 instances. CHROMagar Candida is recommended as a useful isolation medium capable of the presumptive identification of the yeast species most commonly isolated from clinical material and facilitating recognition of mixed yeast cultures.     

Comparison of the new API Candida system to the ID 32C system for identification of clinically important yeast species.

API Candida was evaluated in comparison with the ID 32C system for the identification of 619 yeast isolates. The sensitivity of API Candida for the identification of the 15 species it claims to identify with and without additional tests was 97.4% (593 of 609) and 75.2% (458 of 609), respectively. The API Candida system is easy to use and rapid (result in 18 to 24 h).     

Rapid identification of Candida species by confocal Raman microspectroscopy

Candida species are important nosocomial pathogens associated with high mortality rates. Rapid detection and identification of Candida species can guide a clinician at an early stage to prescribe antifungal drugs or to adjust empirical therapy when resistant species are isolated. Confocal Raman microspectroscopy is highly suitable for the rapid identification of Candida species, since Raman spectra can be directly obtained from microcolonies on a solid culture medium after only 6 h of culturing. In this study, we have used a set of 42 Candida strains comprising five species that are frequently encountered in clinical microbiology to test the feasibility of the technique for the rapid identification of Candida species. The procedure was started either from a culture on Sabouraud medium or from a positive vial of an automated blood culture system. Prior to Raman measurements, strains were subcultured on Sabouraud medium for 6 h to form microcolonies. Using multivariate statistical analyses, a high prediction accuracy (97 to 100%) was obtained with the Raman method. Identification with Raman microspectroscopy may therefore be significantly faster than identification with commercial identification systems that allow various species to be identified and that often require 24 to 48 h before a reliable identification is obtained. We conclude that confocal Raman microspectroscopy offers a rapid, accurate, and easy-to-use alternative for the identification of clinically relevant Candida species.     

Rapid yeast DNA staining method for flow cytometry

The variation of the fluorescence intensity of olivomycin-stained yeast cells as a function of the concentration of olivomycin, NaCl, and MgCl2 in the staining solution was studied. The best results were obtained when the staining solution contained 100 micrograms, olivomycin/ml, 40 mM MgCl2, and 1 M NaCl. A staining time of 12 min was sufficient for proper staining.     

Identification of medically important Candida and non-Candida yeast species by an oligonucleotide array

The incidence of yeast infections has increased in the recent decades, with Candida albicans still being the most common cause of infections. However, infections caused by less common yeasts have been widely reported in recent years. Based on the internal transcribed spacer 1 (ITS 1) and ITS 2 sequences of the rRNA genes, an oligonucleotide array was developed to identify 77 species of clinically relevant yeasts belonging to 16 genera. The ITS regions were amplified by PCR with a pair of fungus-specific primers, followed by hybridization of the digoxigenin-labeled PCR product to a panel of oligonucleotide probes immobilized on a nylon membrane for species identification. A collection of 452 yeast strains (419 target and 33 nontarget strains) was tested, and a sensitivity of 100% and a specificity of 97% were obtained by the array. The detection limit of the array was 10 pg of yeast genomic DNA per assay. In conclusion, yeast identification by the present method is highly reliable and can be used as an alternative to the conventional identification methods. The whole procedure can be finished within 24 h, starting from isolated colonies.     

Evaluation of the new chromogenic medium Candida ID 2 for isolation and identification of Candida albicans and other medically important Candida species

The usefulness of Candida ID 2 (CAID2) reformulated medium (bioMérieux, France) has been compared with that of the former Candida ID (CAID; bioMérieux), Albicans ID 2 (ALB2; bioMérieux), and CHROMagar Candida (CAC; Chromagar, France) chromogenic media for the isolation and presumptive identification of clinically relevant yeasts. Three hundred forty-five stock strains from culture collections, and 103 fresh isolates from different clinical specimens were evaluated. CAID2 permitted differentiation based on colony color between Candida albicans (cobalt blue; sensitivity, 91.7%; specificity, 97.2%) and Candida dubliniensis (turquoise blue; sensitivity, 97.9%; specificity, 96.6%). Candida tropicalis gave distinguishable pink-bluish colonies in 97.4% of the strains in CAID2 (sensitivity, 97.4%; specificity, 100%); the same proportion was reached in CAC, where colonies were blue-gray (sensitivity, 97.4%; specificity, 98.7%). CAC and CAID2 showed 100% sensitivity values for the identification of Candida krusei. However, with CAID2, experience is required to differentiate the downy aspect of the white colonies of C. krusei from other white-colony-forming species. The new CAID2 medium is a good candidate to replace CAID and ALB2, and it compares well to CAC for culture and presumptive identification of clinically relevant Candida species. CAID2 showed better results than CAC in some aspects, such as quicker growth and color development of colonies from clinical specimens, detection of mixed cultures, and presumptive differentiation between C. albicans and C. dubliniensis.     

Rapid identification of Candida dubliniensis with commercial yeast identification systems.

Candida dubliniensis is a newly described species that is closely related phylogenetically to Candida albicans and that is commonly associated with oral candidiasis in human immunodeficiency virus-positive patients. Several recent studies have attempted to elucidate phenotypic and genotypic characteristics of use in separating the two species. However, results obtained with simple phenotypic tests were too variable and tests that provided more definitive data were too complex for routine use in the clinical laboratory setting. The objective of this study was to determine if reproducible identification of C. dubliniensis could be obtained with commercial identification kits. The substrate reactivity profiles of 80 C. dubliniensis isolates were obtained by using the API 20C AUX, ID 32 C, RapID Yeast Plus, VITEK YBC, and VITEK 2 ID-YST systems. The percentages of C. dubliniensis isolates capable of assimilating or hydrolyzing each substrate were compared with the percentages from the C. albicans profiles in each kit's database, and the results were expressed as percent C. dubliniensis and percent C. albicans. Any substrate that showed >50% difference in reactivity was considered useful in differentiating the species. In addition, assimilation of methyl-alpha-D-glucoside (MDG), D-trehalose (TRE), and D-xylose (XYL) by the same isolates was investigated by the traditional procedure of Wickerham and Burton (L. J. Wickerham and K. A. Burton, J. Bacteriol. 56:363-371, 1948). At 48 h (the time recommended by the manufacturer for its new database), we found that the assimilation of four carbohydrates in the API 20C AUX system could be used to distinguish the species, i.e., glycerol (GLY; 88 and 14%), XYL (0 and 88%), MDG (0 and 85%), and TRE (15 and 97%). Similarly, results with the ID 32 C system at 48 h showed that XYL (0 and 98%), MDG (0 and 98%), lactate (LAT; 0 and 96%), and TRE (30 and 96%) could be used to separate the two species. Phosphatase (PHS; 9 and 76%) and alpha-D-glucosidase (23 and 94%) proved to be the most useful for separation of the species in the RapID Yeast Plus system. While at 24 h the profiles obtained with the VITEK YBC system showed that MDG (10 and 95%), XYL (0 and 95%), and GLY (26 and 80%) could be used to separate the two species, at 48 h only XYL (6 and 95%) could be used to separate the two species. The most useful substrates in the VITEK 2 ID-YST system were TRE (1 and 89%), MDG (1 and 99%), LAT (4 and 98%), and PHS (83 and 1%). While the latter kit was not yet commercially available at the time of the study, it would appear to be the most valuable for the identification of C. dubliniensis. Although assimilation of MDG, TRE, and XYL proved to be the most useful for species differentiation by the majority of commercial systems, the results with these carbohydrates by the Wickerham and Burton procedure were essentially the same for both species, albeit following protracted incubation. Thus, it is the rapidity of the assimilation achieved with the commercial systems that allows the differentiation of C. dubliniensis from C. albicans.     

Multiplex PCR identification of eight clinically relevant Candida species.

Invasive fungal infections, specifically candidemia, constitute major public health problems with high mortality rates. Therefore, in the last few years, the development of novel diagnostic methods has been considered a critical issue. Herein we describe a multiplex PCR strategy allowing the identification of 8 clinically relevant yeasts of the Candida genus, namely C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, C. guilliermondii, C. lusitaniae and C. dubliniensis. This method is based on the amplification of two fragments from the ITS1 and ITS2 regions by the combination of 2 yeast-specific and 8 species-specific primers in a single PCR reaction. Results from the identification of 231 clinical isolates are presented pointing to the high specificity of this procedure. Furthermore, several Candida isolates were identified directly from clinical specimens which also attests to the method's direct laboratory application. The results from the multiplex reactions with other microorganisms that usually co-infect patients also confirmed its high specificity in the identification of Candida species. Moreover, this method is simple and presents a sensitivity of approximately 2 cells per ml within 5 hours. Furthermore, it allows discrimination of individual Candida species within polyfungal samples. This novel method may therefore provide a clinical diagnostic procedure with direct applicability.     

DNA probes for identification of clinically important Bacteroides species.

Conventional procedures for identifying gram-negative anaerobes such as Bacteroides spp. are cumbersome and time-consuming. A simpler approach would be to use DNA probes to identify these organisms. Since many different species of gram-negative anaerobes are isolated from clinical specimens, the most useful DNA probes would be probes that identify a few clinically significant groups of anaerobes. To obtain such probes, we cloned HindIII-digested chromosomal DNA from various Bacteroides species and then screened these probes, labeled with 32P by nick translation, for hybridization to DNA from various species of Bacteroides, Fusobacterium, and other gram-negative bacteria. We identified three DNA probes (pBFII-4, pBFII-5, and pBFII-6) that hybridized specifically to DNA from Bacteroides fragilis, the species that accounts for about half of all isolates from clinical specimens containing anaerobes. We identified one DNA probe (pBE-3) that hybridized to all members of the B. fragilis group, a group of species that resemble B. fragilis with respect to fermentation pattern and antibiotic resistances. We also identified one probe (pBO-21) that hybridized to DNA from all Bacteroides sp. strains as well as to DNA from strains of Fusobacterium necrophorum and Fusobacterium nucleatum. pBO-21, but not pBE-3, cross-hybridized with a cloned Bacteroides sp. 16S rRNA gene. The limit of detection for these probes was 10(6) bacteria. The probes could detect B. fragilis in blood culture medium and in mixed cultures with other gram-negative bacteria. Attempts to use biotin-labeled DNA probes instead of 32P-labeled probes were not successful because the Bacteroides sp. extracts contained material that bound the streptoavidin-peroxidase detection reagent.     

Rapid identification of Candida species in oral rinse solutions by PCR

AIMS: To determine the sensitivity and specificity of a multiplex PCR assay for the contemporary identification of major species involved in oral candidiasis, without extraction and purification of DNA from the samples under investigation; and evaluation of this method in comparison with routine phenotypic culture identification. METHODS: 78 oral rinse solutions were collected. The concentrated oral rinse technique was used for a quantitative and qualitative study. Research and identification of Candida spp, with routine phenotypic culture identification (germ-tube test in serum at 37 degrees C for 3 hours and sugar assimilation strip analysis), were performed. Each sample was analysed with multiplex PCR directly on oral rinse solution. Samples giving discrepant results between routine phenotypic and PCR identification methods were resubcultured on CHROMagar Candida plates. The fungus-specific primers ITS1, ITS2, CA3, and CA4 were used. For the identification of other species (C kefyr, C famata and C dubliniensis), ITS1F, ITS1K, and ITS2D primers were designed. RESULTS: Multiplex PCR correctly identified all samples, including those with single species, or with mixed species, negative samples and positive samples which appeared to be negative from routine phenotypic methods. CONCLUSION: This multiplex PCR assay provides a rapid alternative to the conventional culture based technique for the identification and speciation of the most frequently isolated Candida species. The absence of an extraction method made identification of 10 species possible in a few hours.     

Rapid identification of Candida species by DNA fingerprinting with PCR.

DNA polymorphisms in different species and strains of the genus Candida were assessed by amplifying genomic DNA with single nonspecific primers. This PCR method employed an arbitrary primer (the 10-mer AP3), a primer derived from the intergenic spacer regions (T3B), and the microsatellite primers (GTG)5 and (AC)10. Distinctive and reproducible sets of amplification products were observed for 26 different Candida and 8 other fungal species. The numbers and sizes of the amplification products were characteristic for each species. All yeast species tested could be clearly distinguished by their amplification patterns. With all primers, PCR fingerprints also displayed intraspecies variability. However, PCR profiles obtained from different strains of the same species were far more similar than those derived from different Candida species. By comparing species-specific PCR fingerprints of clinical isolates with those of reference strains, clinical isolates could be identified to the species level even if they could not be identified by routine biochemical methods.     

Rapid identification of clinically relevant Enterococcus species by fluorescence in situ hybridization

A fluorescence in situ hybridization assay for the rapid identification of clinically relevant enterococci (Enterococcus faecalis, E. faecium, E. gallinarum, the VanC-type resistance group) was developed and evaluated with 33 reference strains, 68 clinical isolates, and 58 positive blood cultures. All probes showed excellent sensitivities and specificities.     

Laccase and melanization in clinically important Cryptococcus species other than Cryptococcus neoformans

The laccase enzyme and melanin synthesis have been implicated as contributors to virulence in Cryptococcus neoformans. Since isolations of Cryptococcus species other than C. neoformans from clinical specimens have been increasing, we examined the laccase activities of C. albidus, C. laurentii, C. curvatus, and C. humicola. Incubation of cells with epinephrine produced adrenochrome color in C. albidus, C. laurentii, and C. curvatus but not in C. humicola. Activity was always less than in C. neoformans. Laccase was detected in the soluble fractions of disrupted C. albidus, C. laurentii, and C. curvatus cells. Activity staining of partially purified enzyme after nondenaturing polyacrylamide gel electrophoresis revealed that laccases from C. albidus, C. laurentii, and C. curvatus migrated more slowly than that from C. neoformans. One strain of C. curvatus exhibited two melanin bands. Thus, several clinically emerging Cryptococcus species express laccase and can synthesize melanin.     

Candida albicans serotype analysis by flow cytometry.

Candida albicans strains can be assigned to either of two major serogroups, A or B. Antigenic surface determinants present only in serotype A strains allow such a distinction, which has epidemiologic relevance. Reports have established that the relative distributions of the two serotypes can vary depending on the geographic origin of the isolates. A prevalence of susceptibility to an antifungal agent, flucytosine, was also observed with isolates of serotype A. More recently, it was suggested that the occurrence of serotype B isolates in various clinical forms of candidiasis is increasing. However, this latest finding remains controversial since serotyping results vary widely from one laboratory to another because of the lack of standardized methodologies. Difficulty in interpretation of results, which may lead to erroneous serotype identification, is the major setback associated with current methods. For this study, we thus devised a procedure that relies on flow cytometry and that may eliminate ambiguities in serotype determination. The validation of results was achieved with two types of serotype A-specific antisera, Iatron Factor 6 antiserum and an anti-C. albicans antiserum adsorbed on serotype B yeast cells. Agreement between results obtained with these two reagents was 100% with a wide array of Candida strains. These results confirmed the potential of the flow cytometric procedure as a reliable and reproducible method to establish the serotypes of C. albicans strains. Furthermore, some applications of this procedure to the epidemiological study of this human pathogen are presented.     

Rapid microbiochemical identification of Corynebacterium diphtheriae and other medically important corynebacteria.

A rapid biochemical method based on the fermentation of carbohydrates, the hydrolysis of urea, and the reduction of nitrate was used to identify Corynebacterium diphtheriae, C. ulcerans, C. pseudodiphtheriticum, C. haemolyticum, C. pseudotuberculosis, C. pyogenes, C. ovis, the Centers for Disease Control JK group, and Rhodococcus (Corynebacterium) equi. With this procedure identification was confirmed for 133 stock cultures and clinical isolates of corynebacteria. Most were identified within 1 h and all were identified within 4 h after inoculation into the test substrates.     

Molecular probe for identification of medically important Candida species and Torulopsis glabrata.

A cloned DNA fragment from Candida albicans containing the gene for the protein actin was used to probe the molecular structure of the actin gene of several medically important yeasts (C. albicans, Candida stellatoidea, Candida tropicalis, Candida pseudotropicalis, Candida krusei, Candida parapsilosis, Candida guilliermondii, and Torulopsis glabrata). Whole-cell DNA from each species was digested with restriction endonucleases, electrophoresed on agarose gels, and transferred to nitrocellulose. Radioactively labeled C. albicans actin gene was hybridized to the DNA fragments on the nitrocellulose. The C. albicans probe produced a strong signal with all of the Candida DNAs tested, indicating considerable conservation of this gene. In addition, the actin genes of all of the species tested were found to have no internal EcoRI or SalI restriction sites. With the exception of C. guilliermondii, all of the species tested had a single internal HindIII recognition site. However, the location of flanking restriction sites was found to be species specific. For all of the enzymes tested, the locations of the flanking restriction sites in C. albicans and C. stellatoidea were identical; all of the other strains yielded fragments clearly distinct from one another. These differences provide a molecular tool for the differentiation of medically important Candida species.