Rapid Methods for the Diagnosis of Fungal Infections

Transcription

1 CE Update Microbiology I B. Laurel Elder, PhD, and Glenn D. Roberts, PhD Rapid, accurate identification of fungal agents has become essential for the laboratory, particularly in those serving the increasingly frequent compromised patient. Rapid diagnosis can currently be achieved by using direct fungal smears whenever appropriate, by taking advantage of new fungal isolation systems, and by judicious selection of rapid identification tests for organism identification. In addition, knowledge of the fungi seen most frequently in each particular laboratory setting will allow the microbiologist to intensify efforts at identification for those organisms that are deemed most important or that occur most commonly. D uring the past 25 years, changes in medicine and society have forced the microbiologist to become adept in the isolation and identification of organisms previously considered rare, unusual, or nonpathogenic. In no area is this more true than in clinical mycology. Increased travel and mobility require every laboratory to consider organisms such as Coccidioides immitis or Histoplasma capsulatum, among others, as possible etiologic agents. Our laboratory, although located distant to the desert southwest, regularly makes the diagnosis of coccidioidomycosis in 15 to 20 patients each year. Many of these patients have spent the winter in an endemic area, avoiding snow and ice, and have encountered the arthroconidia of C immitis. The development of therapeutic modalities for previously fatal neofrom the Section of Clinical Microbiology, Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN plastic diseases, the widespread use of corticosteroids and antibiotics, and the necessity for invasive techniques in diagnosis and therapy have resulted in a growing population of patients susceptible to life-threatening diseases produced by organisms such as Aspergillus or Candida. The emergence of immunocompromising diseases, such as the acquired immune deficiency syndrome (AIDS), has given the laboratory specimens from patients susceptible to multiple fungal agents. Therefore, it becomes important for every clinical laboratory to be able to recognize the presence of fungi in a clinical specimen and report it as soon as possible. In these severely ill patients, rapid diagnosis becomes essential. Unless a fungal etiology has been specifically considered, the patient generally will not be treated with the required antifungal agents. In some cases, the differentiation between organisms is important for selecting the correct antifungal agent. In addition, clini- cians are often forced to use a larger number of antimicrobial agents when a patient first presents with an unexplained, possibly infectious illness, and identification of the etiologic agents can allow these drugs to be changed to best suit the needs of a particular patient. For these reasons, clinical mycology laboratories have been faced with the task of providing accurate diagnostic service rapidly in a clinically relevant period. Steps involved in the laboratory diagnosis of fungal infections may be divided into three stages: (1) the direct demonstration of mycelia, yeast, or fungal antigens in the clinical specimen, (2) the inoculation and growth of the organisms on culture media, and (3) the identification of the fungal etiologic agent. The focus of this article will be to present techniques that may be used at each of these stages to provide a rapid diagnosis for the clinician. Direct Demonstration of Fungi in Clinical Specimens Rapid Methods for the Diagnosis of Fungal Infections A number of methods are available to the microbiologist or technologist for demonstrating fungi in clinical specimens (Table I). With the exception of the latex test for cryptococcal antigen, most methods detect either intact yeast, spherules, or mycelial fragments in the specimen or histologic section. It is important to recognize that the detection of fungi is LABORATORY MEDICINE VOL. 17, NO. 10, OCTOBER

2 Table I: Characteristics of Methods Available for the Direct Detection of Fungi in Clinical Specimens* Method Time Required Use Advantages Disadvantages Detection of fungi 1 min Can be mixed with potassium hydroxide; detects fungi rapidly due to bright fluorescence Requires use of a fluorescence microscope; background fluorescence prominent, but fungi exhibit more intense fluorescence; vaginal secretions are difficult to interpret Gram's stain Detection of bacteria 3min Is commonly performed on most clinical specimens submitted for bacteriology and will detect most fungi, If present Some fungi stain well; however, others, eg, Cryptococcus sp, stain weakly in some instances India Ink Detection of Cryptococcus neoformans in CSF 1 min When positive in CSF, it is diagnostic for meningitis Positive in less than 50% of cases of meningitis; not reliable Cryptococcal antigen Detection of antigens of C neoformans in CSF or serum 40 min Extremely sensitive method for detection of meningeal cryptococcus; titers may have prognostic values False-positive results can occur in patients with rheumatoid factor; interference can be removed with pronase treatment Potassium hydroxide Clearing of specimen to make fungi more readily visible 5 min; if clearing is not Rapid detection of fungal elecomplete, an additional 5 ments to 10 min Is necessary Experience required since background artifacts are often confusing; clearing of some specimens may require an extended time Methenamine silver stain Detection of fungi In histologic section 1h Best stain to detect fungal elements Requires a specialized staining method that is not usually readily available to microbiology laboratories Papanicolaou stain Examination of secretions for presence of malignant cells 30 min Cytotechnologist can detect fungal elements Periodic acid-schiff (PAS) stain Detection of fungi 20 min; 5 min additional if Stains fungal elements well; hycounterstaln is employed phae of molds and yeasts can be readily distinguished Wright's stain Examination of bone marrow or peripheral blood smears Detects Hlstoplasma capsulatum Detection is limited to H capsulatum "Modified from Lennette E, Balows A, Hausler W, et al (eds): Manual of Clinical Microbiology. Washington, DC, American Society for Microbiology, 1985, p 501. not limited to traditionally recognized mycologic methods, such as the potassium hydroxide (KOH) preparation, but that fungi may be detected by the cytotechnologist, the hematologist, or the bacteriologist in the course of his or her work. Thus, it is important for all laboratorians to be familiar with the appearance of fungi found in the clinical specimens with which th"y work and to recognize the imr,rtance of rapid diagnosis to the clinician and patient. The KOH preparation is the most widely used direct preparation for identification of fungal elements in clinical specimens. A small amount of specimen is placed on a slide, a drop of 10% KOH is added, and the coverslipped slide is gently heated. The KOH acts to disrupt cellular sheets or clumps of proteinaceous debris that may be present and clears the specimen for easier fungal detection. Although this preparation is technically simple and inexpensive to perform, it requires training in distinguishing background materials and artifacts from fungal elements. Bergman and colleagues1 reported a sensitivity of only 19% for KOH detection of Candida albicans in vaginal specimens when compared with culture on Sabouraud's medium; all KOH preparations were read by a nurse practitioner at a family medicine clinic. In contrast, when experienced technologists have examined vaginal secretions for fungal elements, the sensitivity of the KOH preparation was 70.6% compared with culture.2 In our laboratory, the overall detection rate of the KOH preparation was found to be 42.4% when 29,764 specimens LABORATORY MEDICINE VOL. 17, NO. 10, OCTOBER 1986 Nocardla spp do not stain well; B dermatitidis appears pleomorphic Calcofluor white from various sources were examined. Since many of these cultures later yielded only small numbers of saprophytic fungi, the actual detection rate of significant pathogens was higher. The KOH preparation, when positive, may also provide valuable clues as to the etiologic agent of infection. For example, visualization of broad-based budding yeast, endospore-filled spherules, or budding yeast with pseudohyphae or hyphae should suggest a diagnosis, respectively, of blastomycosis, coccidioidomycosis, or candidiasis to the technologist and clinician. A modification of the KOH preparation has recently been used to improve detection of fungal elements in clinical specimens. Calcofluor white (a cotton whitener that fluoresces on excitation with ultraviolet light3) can

3 pretation of these results is confusing to clinicians, reducing the effectiveness of the test. Several methods have been proposed to detect and eliminate interference, including parallel testing of the sample with normal globulin-coated latex particles or removal of the interfering proteins by boiling, chemical, or enzymatic methods. Stockman and Roberts8 have developed a simple method for the destruction of rheumatoid and other interference factors that involves adding as little as 150 xl of sample (CSF or serum) to a lyophilized pronase solution, followed by heating at 56 C for 15 minutes and immersion in a boiling water bath for an additional five minutes. The specimen is then retested for cryptococcal antigens. Routine pronase treatment of all specimens, which initially yields a positive cryptococcal antigen test result, obviates the necessity for testing or titering specimens with the control latex particles, and allows determination of a final antigen titer that has not been diluted by reagent addition. Overall, because of the sensitivity, speed, and specificity of the cryptococcal antigen assay, it should be a replacement for the India ink preparation in the clinical laboratory. Histologic stains have traditionally been used for the detection of fungi in tissue sections but may also be used by the laboratory for examination of body fluids and exudates. Haley and Callaway 9 recommended routine staining of KOH-negative specimens with a periodic acid-schiff stain to increase the yield of positive direct smears by up to 15%. The procedure takes approximately 25 minutes to stain polysaccharides in the fungal wall and allows one to observe organisms in magenta. Occasionally, bacteria (generally cocci) or polymorphonuclear neutrophilic granules will also retain the basic fuchsin of the stain; however, these can be distinguished from fungi on the basis of size and shape. Another commonly used histopathologic stain for fungal detection is methenamine silver. With this excellent fungal stain, organisms appear dark brown to black against a pink or green cellular background, depending on the counterstain used. Since performance of the stain is fairly time-consuming (approximately one hour) and involves a number of spe- cialized solutions, it is generally not offered by the microbiology laboratory. However, the methenamine silver stain is a superior stain for detecting fungi in tissue. If the microbiologist or technologist is asked to examine stained histologic sections for the presence of fungi, it is important that the fungal elements in tissue do not appear different than those seen in preparations such as those with KOH, and they may be easier to distinguish because of their differential staining reaction. Thus, the laboratorian should be able to aid significantly in the interpretation of these smears once familiarity has been gained with the staining reactions of the method used. Examination of patient specimens for fungi need not involve only those stains traditionally recognized as "fungal." The Wright or Giemsa stains performed by the hematology laboratory will stain Histoplasma capsulatum in bone marrow or peripheral blood smears and may provide the first clue of disseminated histoplasmosis. In our institution, several cases of blastomycosis have first been detected by the cytotechnologist reading Papanicolaou stained respiratory tract specimens for the presence of tumor cells. Since Blastomyces dermatitidis may present with a clinical picture consistent with malignancy, these specimens may be among the first examined. Observation of the typical yeast form of B dermatitidis can provide a rapid, possibly unsuspected diagnosis and prevent further invasive diagnostic procedures. Papanicolaou stained sputum smears can also detect the yeast cells of H capsulatum and C neoformans, as well as the spherules of C immitis and hyphae of Aspergillus and Zygomycetes. Another stain that is frequently overlooked as an aid to rapid diagnosis of fungal infections is the Gram's stain. Nearly every patient will have specimens collected for bacterial cultures and Gram's stain; unfortunately, fungal cultures and stains are often overlooked. Observation of yeast or hyphal fragments on the Gram's stain can provide the first clue to the physician that a fungal etiologic agent may be present. For this reason, the bacteriologist should be attuned to the appearance and possible significance of fungi as seen with the Gram's stain. be added to supplement the KOH solution, and the specimen can be prepared as described for the KOH. Calcofluor white will stain fungi, resulting in bright, sharply delineated organisms when the preparation is viewed with a fluorescent microscope. Although background tissue elements also stain with Calcofluor white, in general the fungi are much brighter and simple to discern. Fungal septae are particularly bright, facilitating preliminary fungal identification. Although the method requires a fluorescent microscope, it has the advantage of allowing easier fungal detection with less search time or technologist experience. It has also been adapted for the detection of fungi in paraffin-embedded tissues,4 as well as frozen sections of lung and after soft tissues.5 In our laboratory, the Calcofluor white has been as sensitive as the standard KOH method6 but is preferred over the KOH preparation because it often allows detection of fungal elements in a shorter period of time and with greater resolution of fungal structure. Another method for direct examination of clinical material that has traditionally belonged to the mycology laboratory is the India ink preparation. Used to detect encapsulated yeast (primarily Cryptococcus neoformans), this technique is rapid, inexpensive, and simple to perform. Unfortunately, it suffers from a lack of sensitivity and is positive in only 40% to 50% of culturally proven cases of C neoformans meningitis. For this reason, many laboratories have replaced the India ink preparation with a latex test for detection of cryptococcal antigen. Cryptococcal antigen testing detects the presence of the capsular polysaccharide by the agglutination of anticryptococcal antibody-coated latex particles. The test is extremely sensitive, and detection rates as high as 99% have been reported from patients with proven meningeal cryptococcosis.7 False-positive reactions can occur, however, due to agglutination of the latex particles by rheumatoid factor or other interfering factors. Although the rate of false-positivity may be relatively low (1.96% from 27,000 cerebrospinal fluid [CSF] and serum samples tested in the Mayo Clinic Fungal Serology Laboratory8), inter- LABORATORY MEDICINE VOL. 17, NO. 10, OCTOBER

4 Fungal Growth on Laboratory Media Rapid Identification of Fungi Once a fungal isolate is growing on culture media, the identification of the Organism Aspergillus species Aspergillus fumigatus Alternaria Trichophyton rubrum Cladosporium Aspergillus niger Aspergillus versicolor Fusarium Geotrichum Scopulariopsis Acremonlum Aspergillus flavus group Trichophyton mentagrophytes Histoplasma capsulatum Epidermophyton floccosum Coccidioides intimitis Blastomyces dermatittdis Sporothrix schenckii Total No. of Isolates Total No. of Patients 4,473 3,484 3,299 3,164 2,956 1,697 1,522 1,303 1,143 1, ,850 2,466 2,888 2,612 2,508 1,454 1,417 1,140 1, Table III: Recovery of Yeasts From Clinical Specimens, Mayo Clinic Organism Total No. of Isolates Total No. of Patients 21,641 18,538 3,867 1,894 1,671 1, ,553 11,644 2, , Yeast, not Cryptococcus Candida albicans Candida glabrata Candida tropicalis Candida parapsilosis Other Candida species Cryptococcus neoformans Saccharomyces species Other Cryptococcus species Rhodotorula Trichosporon organism can often be achieved rapidly. For many of the filamentous organisms, identification will primarily require careful observation of the microscopic morphology of the organism and possible subculture to media that stimulate conidiogenesis. Definitive diagnosis of dimorphic pathogens requires either conversion from the mold to the yeast phase or demonstration of organism-specific exoantigens; the latter is recommended. For the filamentous fungi, microscopic identification can be greatly facilitated by knowledge of the organisms most commonly seen in a particular laboratory setting. With this knowledge, technologists can first concentrate efforts learning the colonial and microscopic features of the most common isolates to provide a rapid identification of these organisms. Table II lists the organisms recovered most frequently at the Mayo Clinic. Yeasts are as commonly recovered as the filamentous fungi, however, and identification methods may be as simple or elaborate as the laboratory desires. To facilitate their rapid identification of yeast isolates, the LABORATORY MEDICINE VOL. 17, NO. 10, OCTOBER 1986 Definitive identification of fungi from clinical specimens nearly always requires cultivation of the organism on artificial media. Once colonies are obtained, identification may involve simple microscopic examination of the colony or a few rapid tests, such as the urea or germ tube tests. Therefore, the greatest impediment to a rapid, definitive diagnosis is the time required to yield the organism in the laboratory. In most cases, the time necessary for colony growth is fixed and nonvariable, depending on the growth rate of the organism and the inoculum density. However, recent changes in blood culture methodology have resulted in decreased detection time for many fungi from this important source. Traditionally, fungal blood cultures have required a separate culture from those obtained for bacterial culture and have used a vented, biphasic brain-heart infusion (BHI) medium for optimal recovery. Often, detection of fungemia did not occur in a clinically relevant period, so that laboratory data generated was too late to affect patient care beneficially. The use of the lysis-centrifugation blood culture system (Isolator 8) has helped partially alleviate these problems. In a study at Mayo Clinic, Bille and colleagues10 found that the Isolator recovered 90.3% of positive fungal cultures compared with 63.4% recovered by a biphasic BHI medium. Recovery times for yeasts were shortened from an average of 4.90 days to 2.12 days. The greatest difference in recovery time was seen with H capsulatum, in which the mean time changed from days (range, 15 to 40 days) with the BHI medium to 8.0 days (range seven to 14 days) with the Isolator. Clinical evaluation of this system indicated that in 53% of the clinically significant episodes of fungemia, the earlier detection was directly helpful in patient management.11 Because of these findings, the Isolator is recommended as the optimal fungal blood culture system. Table II: Recovery of Filamentous or Dimorphic Fung From Clinical Specimens, Mayo Clinic laboratory should also be familiar with the yeasts most frequently encountered. In part, this is influenced by the patient population the institution serves. In our tertiary care center, for example, the most frequently encountered yeasts are Candida albicans, followed by C glabrata, C tropicalis, and C parapsilosis. The next most common isolate is Cryptococcus neoformans, which reflects, in part, the immunocompromised population of our patients (Table III). These figures indicate that for our laboratory, on the basis of numbers alone, tests that rapidly identify C albicans and C glabrata would be useful. When tests for identification are selected, a second point each laboratory should consider is the possible significance of an isolate from a particular location. For example, with the exception of C neoformans, we have not found it necessary to identify routinely yeasts from respiratory secretions. Thus, a screening method for Cryptococcus allows rapid identification of nearly all respiratory yeast isolates as "yeast, not Cryptococcus" (Table III). Cryptococcus neoformans,

5 despite its relatively infrequent occurrence, is often an extremely significant isolate; therefore, a battery of tests that allows at least a rapid preliminary identification of this organism should be available. After considering the numbers of isolates and their clinical significance, a laboratory should be able to design or select a battery of tests that will prove most rapid and cost-effective for its particular setting. A number ofsimple, rapid tests have been developed that allow identification of many of the more frequently encountered yeast isolates. Foremost among these is the germ tube test.12 This test involves a light suspension of a yeast colony made in approximately 0.5 ml of serum and incubated for three hours at 37 C. Then the suspension is examined microscopically for the presence of short hyphal filaments protruding from the blastoconidia. If these are present, the yeast can be identified as Candida albicans. False-positive germ tube test results are most often due to misinterpretation of the pseudogerm tubes produced by Candida tropicalis. Careful examination of these structures, however, reveals a constriction at the junction between the yeast cell and the putative germ tube, indicating that the structure is not a true hyphal tube. Inclusion of C tropicalis as the negative control for the test can help obviate this problem. Yeasts not producing germ tubes have traditionally required more extensive and time-consuming testing for their identification. Stockman and Roberts13 have recently described a rapid trehalose screening test to distinguish Candida glabrata, the second most frequently recovered yeast in our laboratory. In this test, a yeast colony is emulsified in a broth containing trehalose, yeast nitrogen base, cycloheximide, and bromocresol green and incubated at 35 C for one hour. Trehalose-positive organisms change the indicator from green to bright yellow. Evaluation of 362 yeast isolates indicated that the test was 89.3% sensitive and 96.8% specific for C glabrata. Thus, use of the germ tube and rapid trehalose tests allows our laboratory to rapidly, simply, and inex- dimethyl-1-naphthylamine and observed for the development of red, which indicates the presence of nitrate reductase. Cryptococcus neoformans does not use an inorganic nitrate substrate, while some other species in the genus may reduce nitrate. A final rapid test for the presumptive identification of C neoformans is designed to detect phenoloxidase,17 an enzyme produced only by C neoformans. In this test, filter paper discs that have been impregnated with a solution of Levodopa-ferric citrate are moistened and inoculated with several colonies of the yeast to be tested. The discs are incubated for three to six hours and examined at 30-minute intervals for the development of a dark pigment, which indicates phenoloxidase production. Kaufman and Merz,17 in their initial evaluation of this assay, found no false-negative or falsepositive results with any of 37 clinical isolates of Cryptococcus, or 1,800 other clinical yeast isolates using this method. In many instances, however, a plate of niger seed agar can be heavily inoculated and incubated at 37 C and a brown pigment will be produced by C neoformans within four hours. Thus, use of a phenoloxidase test, along with the rapid nitrate and urease tests on yeasts suspected to be Cryptococcus, can provide a rapid preliminary identification in three to four hours. Commonly Available Identification Systems Many commercially available yeast identification systems have been available to the microbiologist for a number of years. Most of these systems offer convenience to the laboratory by providing a panel of tests (primarily carbohydrate assimilations) that can be rapidly inoculated at one time. However, the majority of these systems continue to require 48 to 72 hours of incubation before a final identification is available, removing them from the category of "rapid tests." Several companies, however, have recently introduced identification systems that can be read in four to 24 hours, and may be useful as rapid identification systems. These systems have the advantage of identifying a wider spectrum of organisms than the individual tests previously discussed; Individual Tests pensively identify the vast majority of the yeast isolates. Several rapid tests can also be used to provide a preliminary identification of C neoformans. These include the rapid urease, nitrate reduction and phenoloxidase tests. Urease detection can also provide a simple screen for the differentiation of Cryptococcus from other yeasts present in respiratory secretions. One simple method for rapid detection of urease has been described by Roberts and colleagues.14 A heavy inoculum of yeast (less than seven days old) is suspended in 3 to 4 drops of Bacto Urea R Brothb and incubated four hours at 37 C. Urease-positive organisms change the broth color from red to purple. It is important to ensure that the suspension is not contaminated with bacteria as this may yield a false-positive result; a loopful of each yeast suspension can be examined prior to incubation so that urease-positive organisms known to be contaminated with bacteria can be isolated and retested. Another rapid urease method has been described by Zimmer and Roberts16 and detects urease from colonies grown on Sabouraud's dextrose agar within 15 minutes. While not as suitable for high-volume screening as the broth urease previously described, it can serve as a useful test in the identification of C neoformans. Of the 286 strains of C neoformans tested, 99.6% produced detectable amounts of urease within the 15-minute incubation. None of the other urease-positive (Rhodotorula, Trichosporon, other Cryptococcus species) or urease-negative yeasts yielded a positive urease reaction with this test. A second useful test for rapid identification of Cryptococcus is the nit r a t e reduction test, originally described by Hopkins and Land.16 Nitrate reductase is detected by sweeping a substrate-impregnated swab across several of the yeast colonies to be tested and incubating the swab for ten minutes at 45 C. The swab is impregnated with 1% benzalkonium chloride (which serves to disrupt the cell walls releasing the enzyme) as well as an excess of inorganic nitrate so that complete reduction of the nitrate to ammonia does not occur. After incubation, the swab is placed in a solution of sulfanilic acid and N,N- LABORATORY MEDICINE VOL. 17, NO. 10, OCTOBER

6 free Sabouraud's dextrose agar; in many cases, this will delay the laboratory diagnosis by two to three days over the four-hour incubation period. Clinical evaluations of the Yeast-Ident system are not available at this time, but tentative data indicate that additional tests are required for many isolates, lengthening the time to identification. With both the aforementioned systems, as with most commercially available products, costs are high when compared with conventional identification methods. If additional conventional tests are required for identification, the convenience of these methods is lost, and the laboratory incurs additional expense for little return benefit. Most laboratories should consider their own patient populations, assess the frequency of various isolates, and develop a battery of individual rapid tests to use. These tests can then be supplemented by commercial systems or conventional biochemical tests when necessary for a more complete identification. Conclusion The mycology laboratory has a responsibility to generate accurate reports within a clinically relevant period. This can be accomplished by making use of the direct microscopic examination of clinical specimens, if feasible, both in the specialized laboratory or clinical microbiology laboratories. It can be further accomplished by using techniques such as the lysiscentrifugation blood culture method to decrease the detection time of clinically relevant fungi. And, finally, it can be improved by being familiar with the patient population and common fungal isolates seen by each laboratory and developing a battery of tests designed to rapidly identify these common or significant isolates. References 2. Quinn PR, Roberts GD: Usefulness of the potassium hydroxide preparation for the detection of fungi in clinical specimens. Abstract F60, Abstracts of the Annual Meeting of the American Society for Microbiology, St Louis, 1984, p Darken M: Applications of fluorescent brighteners in biological techniques. Science 1961;133: Hollander H, Keilig W, Bauer J, et al: A reliable fluorescent stain for fungi in tissue sections and clinical specimens. Mycopathologia 1984;88: Monheit JE, Cowan DF, Moore DG: Rapid detection of fungi in tissues using Calcofluor white and fluorescence microscopy. Arch Pathol Lab Med 1984;108: Horstmeier CD, Roberts GD: Clinical laboratory evaluation of the Calcofluor white stain for the detection of fungi in clinical specimens. Abstract F82, Abstracts of the Annual Meeting of the American Society for Microbiology, Las Vegas, 1985, p Kaufman L, Reiss E: Serodiagnosis of fungal diseases, in Lennette E, Balows A, Hausler W, et al (eds): Manual of Clinical Microbiolo Washington, DC, American Society for Microbiology, 1985, pp Stockman L, Roberts G: Specificity of the latex test for cryptococcal antigen: A rapid, simple method for eliminating interference factors. J Clin Microbiol 1982;16: Haley LD, Callaway C: Laboratory methods in medical mycology. Atlanta, US Department of Health, Education and Welfare, Centers for Disease Control, Bille J, Stockman L, Roberts G, et al: Evaluation of a lysis-centrifugation system for recovery of yeasts and filamentous fungi from blood. J Clin Microbiol 1983;18: Bille J, Edson R, Roberts G: Clinical evaluation of the lysis-centrifugation blood culture system for the detection of fungemia and comparison with a conventional biphasic broth blood culture system. J Clin Microbiol 1984;19: MacKenzie D: Serum tube identification of Candida albicans. J Clin Pathol 1962;15: Stockman L, Roberts G: Rapid screening method for the identification of Candida glabrata. Abstract F80, Abstracts of the Annual Meeting of the American Society for Microbiology, 1985, p Roberts GD, Horstmeier CD, Land GA, et al: Rapid urea broth test for yeasts. J Clin Microbiol 1978;7: Zimmer BL, Roberts GD: Rapid selective urease test for presumptive identification ofcryptococcus neoformans. J Clin Microbiol 1979;10: Hopkins JM, Land GA: Rapid method for determining nitrate utilization by yeasts. J Clin Microbiol 1977;5: Kaufmann CS, Merz WG: Two rapid pigmentation tests for identification of Cryptococcus neoformans. J Clin Microbiol 1982;15: Lundy J, Roberts G: Clinical laboratory evaluation of the Abbott Quantum II yeast identification system. Abstract P3-17, Abstracts of the International Society for Human and Animal Mycology. 19. Kiehn T, Edwards F, Tom D, et al: Evaluation of the Quantum II yeast identification system. J Clin Microbiol 1985;22: the major disadvantage is their increased cost when compared with the simple rapid tests. A recently developed system is the Quantum II,C which consists of a cartridge with 20 chambers containing lyophilized biochemical substrates. After inoculation and a subsequent 24hour incubation period, the cartridge is read by an automated reader. A germ tube test is also done independent of the system, and the results are entered into the reader. A printout of the most likely identification, any additional tests required for identification, and other possible identifications (with probabilities) is generated by the reader. An evaluation of the Quantum II system in our laboratory indicated that the system correctly identified only 80% of all yeasts tested and frequently required the use of additional biochemical tests.18 These additions extend both the time to identification, as well as the cost of the system. Klein and co-workers19 have recently evaluated the Quantum II system and also found it correctly identified 80.4% of 245 yeasts tested, incorrectly identified 7.8%, and could not identify 11.8% of the yeasts. Most of the misidentifications were due to individual reactions that varied from standard results; these reactions occurred even with yeasts that were correctly identified and were considered primarily false-positive reactions. The authors concluded that the Quantum II system currently requires modifications in hardware, substrate, or data base to improve its accuracy. Analytab Products (API) has recently introduced its Yeast-Ident system,'1 a rapid method that requires only a four-hour incubation period. The system consists of 20 cupules containing dehydrated substrates and/or nutrient media; the reactions are dependent on the presence of preformed or constitutive enzymes. The rapid nature of the system, as with most "rapid" commercial systems, is compromised by the requirement of 48to 72-hour-old cultures on antibiotic- Suppliers 1. Bergman J, Berg A, Schneeweiss R, et al: Clin- a. E.I. DuPont, Wilmington, DE ical comparison of microscopic and culture tech- b. Difco Laboratories, Detroit, MI niques in the diagnosis of Candida vaginitis. J c. Abbott Laboratories, Irving, TX Fam Pract 1984;18: d. Analytab Products, Plainview, NY LABORATORY MEDICINE VOL. 17, NO. 10, OCTOBER 1986