Journal of Medical and Veterinary Mycology (1992), 30, Supplement 1,241-248
Developments in the diagnostic mycology laboratory
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G. D. ROBERTS 1, M. A. PFALLER 2, E. GUEHO 3, T. R. ROGERS 4, C. DE VROEY s AND W. G. MERZ 6
1Mayo Clinic and Foundation, Rochester, Minnesota, and ZOregon Health Sciences University, Portland, Oregon, USA; 3Institut Pasteur, Paris, France; 4Charing Cross and Westminster Medical School, London, England; 5Instutuit Voor Tropishe Geneeskunde, Antwerpen, Belgium; and 6The Johns Hopkins Medical Institutions, Baltimore, MD, USA The laboratory diagnosis of specific human mycoses still relies on classic mycological procedures including direct examination and recovery of fungi from clinical specimens, and accurate identification of the fungal organisms. To date, the non-culture methods developed, with the exception of tests for the detection of cryptococcal polysaccharide antigen, have lacked sensitivity and/or specificity, or have not been available for widespread use. This is unfortunate since techniques for rapid detection of specific fungal antigens, chemical constituents, metabolic products, and fungal species-specific RNA and /or DNA sequences, have the potential to yield rapid diagnostic information that can guide early and appropriate use of specific antifungal chemotherapy. These methods, however, will perhaps never replace classic methods due to lack of sensitivity or specificity and the need for a wide range of reagents to be available to keep up with the increasing number of fungal species emerging as new pathogens in different clinical settings. Therefore, improvement in all aspects of laboratory testing is appropriate. The purpose of this manuscript is to present an update on selected methods that are currently being evaluated for diagnosis of the mycoses, some of which are being made available for widespread use. The methods to be discussed include improved techniques for culturing medically important fungi, improvements in methods for accurately identifying fungi, as well as improvements in several non-culture approaches to enhance our taxonomic delineation of species and to detect fungi or fungal products directly in clinical specimens.
Non-culture techniques for improved laboratory diagnosis Invasive candidiasis. Invasive candidiasis is the most frequently encountered lifethreatening human mycosis. Unfortunately, the clinical signs and symptoms of this infection are generally non-specific and specific diagnosis depends on culture and/or histological evidence from appropriately collected blood or deep tissue specimens. Problems in sensitivity and specificity of these methods and the need for invasive procedures seriously compromise diagnosis of this important infection. Therefore, rapid, non-culture methods may have a very profound impact on diagnosis. Three assays for detection of Candida antigens in serum either currently commerCorrespondence address: Dr W. G. Merz, Department of Laboratory Medicine, Meyer B1-193, The Johns Hopkins Hospital, Baltimore, MD 21205, USA. 241
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cially available or under development include a latex agglutination (LA) assay for detection of an unknown, heat-labile antigen (Cand-Tec, Ramco, Houston, TX), an enzyme immunoassay (EIA) for detection of Candida enolase (Becton Dickenson Microbiology Systems, Baltimore, MD) and an EIA being developed to detect a cell wall mannan antigen (Hybritech, San Diego, CA). With all three tests, results can be available in 15-30 min. The sensitivities and specificities of these assays are presented in Table 1. The wide range of values for the LA test is not acceptable for a test in a clinical laboratory. Although the EIA tests have a 15% false negative rate and would never replace blood cultures, they nevertheless appear promising for inclusion in a series of laboratory tests to optimize the diagnosis of invasive candidiasis. Prospective studies evaluating these tests in comparison to culture results for the rapid diagnosis and monitoring of invasive candidiasis are necessary.
T A B L E 1. Use of antigen detection tests for the diagnosis of invasive candidiasis Assay L A test E I A - enolase E I A - CWM a
Reference number
Sensitivity (%)
Specificity (%)
[2, 8] [11] [12]
19-100 85 86
29-99 96 91
aCWM = cell wall mannan.
Another exciting approach as an aid to the diagnosis of invasive candidiasis is the detection of the Candida-specific metabolite, D-arabinitol. Ongoing studies with a gas-liquid chromatography procedure to detect the enantiospecific D-arabinitol in sera from experimental animals infected with Candida albicans and from human cases of invasive candidiasis are encouraging, with a sensitivity of 80% when combined with blood cultures (44-65% detected by blood culture or o-arabinitol alone, respectively) (B. Wong, personal communication). Prospective clinical studies are necessary to evaluate the usefulness of this method. Similar to the results of antigen tests, screening for o-arabinitol will probably augment classic methods, not replace them. The use of DNA/DNA or DNA/RNA hybridization assays to detect Candidaspecific DNA sequences has not yet been formulated due to the decreased sensitivity of these assays for DNA detection in clinical specimens. A DNA-based procedure which employs amplification of a DNA sequence within the lanosterol 14 a-demethylase gene by the polymerase chain reaction and the use of specific DNA probes has been developed [1]. Prospective clinical studies are necessary to determine whether this assay or similar assays will have the sensitivity to detect fungal nucleic acids in a full range of clinical specimens.
Invasive aspergillosis. Invasive aspergillosis (IA) has become a major infection in the setting of the neutropenic patient, second only to invasive candidiasis as the most frequent opportunistic fungal pathogen. Clinical signs and symptoms of this infection are generally non-specific, making early diagnosis of IA difficult and necessitating empirical therapy with amphotericin B. Although important findings suggestive of IA can be made by various imaging procedures (CT scan, MRI), specific diagnosis
243
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presently depends on the histological examination of biopsy specimens and culture of the fungal pathogen. The detection of an Aspergillus-specific antigen(s) produced early in the infectious process, in advance of other signs of IA, would make a significant contribution to successful treatment and appropriate use of antifungal drugs. An EIA-inhibition assay using either a polyclonal serum or monoclonal antibody to galactomannan antigen has been developed and tested in 121 high risk bone marrow transplant patients [9]. The overall sensitivity was 95% and the specificity was 99% (Table 2). Important findings included: (i) positive results in this assay correlated with the development of IA; (ii) antigenaemia occurred at a sufficiently early stage in the infection to be a predictor of IA; and (iii) serial sampling was necessary since circulating levels fluctuated significantly. A commercially available LA test (Pastorex Aspergillus, Diagnostics Pasteur, Paris, France) has also been developed, and was tested with sera from the same 121 bone marrow transplant recipients. Results were comparable, although, there were more false positive tests with the latex assay than with the EIA-inhibition assay (T. Rogers, personal communication). These results are very encouraging and warrant further investigation of these assays to determine their role in the laboratory diagnosis of this extremely serious, life-threatening infection. TABLE 2. EIA-Inhibition assays for the diagnosis of invasive aspergillosis~ Patient group
Proven invasive aspergillosis Clinically suspected aspergillosis Other fungal infection No fungal infection Total
Number of patients
Number of patients with positive test
Positive antigen test preceded other diagnostic finding
19 12 8 82
18 12 0 1
16 11 NA NA
121
31
aData from [9].
Improvement in classic mycologic procedures for diagnosis Invasive candidiasis. As mentioned previously, the specific-diagnosis of invasive candidiasis depends on the recovery of the organism from deep tissue with positive histologic findings or repeated recovery from blood specimens. Unfortunately, it is widely held that Candida species are recovered from only 15-50% of patients with invasive candidiasis and cultures may become positive only immediately before death. Recent studies, however, document the improved sensitivities of newer blood culture methods for the recovery of yeasts from blood specimens. These improvements in blood culture methods include venting of broth blood cultures, commercial development and the use of agitation of biphasic blood culture bottles, improvements in non-radiometric CO2 detecting blood culture systems, and the development of the lysis-centrifugation system. The vast majority of published data indicate that the lysiscentrifugation system (Isolator, Wampole, USA) provides the most sensitive means of detecting fungaemia; however, it is clearly not 100% sensitive and a combined
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approach using the Isolator plus a broth or biphasic system always results in the detection of a greater number of fungemic episodes than either system alone (Table 3). Recent data document improved sensitivity of 50-82% for blood cultures in neutropenic patients with tissue-proven invasive candidiasis [6, 7, 11] (Pfaller et al., Abstract C25, 91st meeting of the American Society for Microbiology, Dallas, Texas, May 5-9, 1991; Telenti et al., Abstract 129, 26th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, Louisianna, Sept. 28-Oct 1, 1986). These observations suggest that improved culturing strategy (more frequent blood cultures of adequate volume) and/or improved blood culture methods may provide a sensitive means of diagnosing invasive candidiasis. TABLE 3. 1 Studies comparing lysis-centrifugation (LC) versus broth blood cultures for ability to detect fungemiaa Blood culture Total Total technique specimens positive LC vs VB LC vs AgB LC vs VBP LC vs AgBP LC vs L-BP
10,335
117
16,000
117
28,711
258
14,223
182
1,778
52
Percentage Time(days) to positiveby: detection 91 65 86 71 88 64 80 80 67 83
1.8-1.9 2.9-3.7 1.9 2.7 1.9-2.3 4.4-5.0 2.6--4.1 2.6--3.9 3.1 3.5
aData adapted from [5, 6 & 7]. bLC = lysis - centrifugation; VB = vented broth; A g B = agitated broth; VBP = vented biphasic; A g B P = agitated biphasic; L-BP = lysis biphasic (contains saponin).
Comparisons of genotypic vs. phenotypic characteristics for accurate taxonomy and species identification
Accurate identification of fungi to species level has become more important than ever, both to help clinicians determine the clinical relevance of specific species recovered from clinical specimens and also to decide the appropriate antifungal chemotherapy. The latter is emerging as an important issue as newer antifungal agents with different spectra of activity are being developed. An excellent example of the importance of fungal species identification can be made with medically important yeasts. Although many laboratories do not routinely identify Candida isolates to species level, recent reports suggest that shifts have occurred in the distribution of infections caused by various Candida species and evidence is accumulating that species identification can provide a diagnostic clue to the source of infection. Clearly C. albicans remains the most common cause of invasive candidiasis; however, there has been an increase in infections caused by Candida tropicalis, Candida parapsilosis, Candida krusei and Candida lusitaniae. Both C. tropicalis and C. krusei are important causes of fungemia in neutropenic cancer patients. Gastrointestinal colonization appears to be an important predisposing event in patients subsequently infected with either C. tropicalis or C. krusei. C. parapsilosis is well-known as a cause of fungaemia and invasive candidiasis associated
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with parenteral hyperalimentation, intravascular devices and contaminated ophthalmic solutions. More recently, C. parapsilosis has displaced C. tropicalis as the second most common cause of fungaemia in several US hospitals. Finally, C. lusitaniae is recognized as an emerging opportunistic fungal pathogen. The vast majority of patients infected with C. lusitaniae acquire the infection as a nosocomial event. The hallmark of C. lusitaniae, and the major cause for concern clinically, is its propensity to develop resistance to amphotericin B. Accurate delineation of taxa is of prime importance for laboratories and clinicians. Taxa defined solely on phenotypic characteristics may not accurately reflect phylogenic relatedness or unrelatedness. Therefore, genotypic characterizations are often necessary to determine properly delineation of species of fungi of medical importance. This can be well illustrated with the Pseudallescheria boydii/Scedosporium group of filamentous fungi that have emerged as important human pathogens. A taxonomic study of the Pseudallescheria-Scedosporium complex by means of genomic comparison and scanning electron microscopy indicated a correlation between species and epidemiology [4]. Isolates corresponding to P. boydii (Shear) McGinnis, Padhye et Ajello were separated into three groups on the basis of their genomic relationships (Fig. 1); the strains included in each group, with D N A reassociation values systematically above 80% proved to be conspecific. In contrast, between groups the homologies were in the range of 58-72% proving a relatedness at the varietal level. It is interesting to note that six out of nine strains in Group 1 were of pulmonary origin whereas none of the Group 2 strains had such an origin; they were mainly isolated from cases of sinusitis and otitis. Similarly, sexual reproduction (Pseudallescheria) was only observed in Group 1, while coremia (Graphium) were only in Group 2. FIG. 1. Genomic relationships among isolates of P. boydii and other related fungia Group 1
CDC A495 CBS 101.22 IP 1251.80 IP 1448.83 IP I698.87 IP 1945.90 IP 1947.90 IP 1988.91 IP 1986.91 IP 2002.91
Group 2
94-99% knee arthritis, P. shearii mycetoma man? abscess --58-72% -lung, leukaemia sputum, cystic fibrosis sputum, cystic fibrosis sputum, cystic fibrosis sputum, cystic fibrosis sputum, lymphoma
I I
CBS CBS CBS IP IP IP IP IP IP
297.38 895.70 987.73 1411.82 1907.90 1946.90 2003.91 2004.91 2008.91
68--70%
85-96% wood,pulp, S. borzinii sinus,swine, Acremonium sula humanotils, G. enumorphum mycetoma sinus sinus foot blood, kidney transplant bone
I I
65-66%
I
Group 3
83% CBS 108.64 soil IP 1742.88 soil aData adapted from [4]. bCDC = Center of Disease Control, Atlanta, USA; CBS = Centraalbureau voor Schimmelcultures Baarn, Netherlands; IP = Pasteur Institute, France.
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Scedosporium inflatum Malloch & Salkin is the other species of the genus Scedosporium now recognized as a human pathogen [10]. DNA homologies among clinical isolates of this species (bone or blood isolates) are 96-100% and only 0-17% with varieties of P. boydii (Fig. 2). Gueho & de Hoog [4] have also included in this study the type strain of Lomentospora prolificans Hennebert et Desai originated from soil in Belgium which gave 100% relatedness with clinical isolates. This finding, in addition to mycological features, proves that the two species are identical. Since prolificans is the oldest available epithet, the new combination Scedosporium prolificans (Hennebert et Desai) Gueho et de Hoog was proposed. This species is known only by its imperfect state. However, it is well-separated from the anamorph, Scedosporium apiospermum, by cultural (much darker) and microscopic characteristics (typically swollen conidogenous cells). S. prolificans is less frequently encountered than S. apiospermum, but it is responsible for deep mycosis as well and has a preference for bones. The distinction between these two Scedosporium species would also be supported by differences in sensitivity to antifungal drugs [3]. Scedosporium inflatum and Lomentospora prolificans
FIG. 2. Genomic relationships within
Group 1
Group 2
Pseudallescheria boydiP
0-17%
0-6%
Scedosporium inflatum"~ CBS CBS IP IP IP IP
114.90 b 467.74 c 1236.80 1883.89 1912.90 1913.90
foot osteomyelitis green house soil foot osteoarthritis blood, medullar aplasia blood, acute leukaemia blood, myeloblastic leukaemia (96-100%)
aData adapted from [4]. ~Fype culture of Scedosporium inflatum. ~Type culture of Lomentospora prolificans.
Use of DNA probes for rapid identification of medically important fungi Genotypic methods can also be used for rapid, accurate species identification in the clinical laboratory. DNA probes specific for variable regions of rRNA have been developed and are now available commercially for the identification of Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis and Cryptococcus neoforroans (Murphy Clark et al., Abstract FT; Rubin et al., Abstract F6, 90th meeting of the American Society for Microbiology, Anaheim, California, May 13-17, 1990). Specific diagnosis of histoplasmosis, blastomycosis and coccidioidomycosis frequently requires obtaining tissue through invasive procedures with culture and/or histological confirmation. Recovery of these fungi may take 3 days to 6 weeks with an additional 3 days to 2 weeks for species identification. Therefore, the dimorphic fungi are the perfect organisms for the application of rapid identification systems. The DNA hybridization assay requires less than 1 h for identification from a single small colony from any medium, even if it is not a pure culture. In addition, either the yeast or filamentous forms of fungi are appropriate for testing with the probe assay system. The results of an evaluation of four probes for the identification of these important
DIAGNOSTIC MYCOLOGY
247
human pathogenic fungi are presented in Table 4 (Stockman et al., Abstract F5, 90th meeting of the American Society for Microbiology, Anaheim, California, May 13-17, 1990.) These assays provide an accuracy of identification comparable to that obtained with exoantigen testing; however, the probe assay results are available with 1 h compared with 72 h with the exoantigen tests. The application of the DNA probe assays to direct detection of fungal pathogens in tissue has not been accomplished but studies are currently underway. TABLE 4. Use of DNA probes for rapid identification of medically important fungia
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Fungal species tested
H. B. C. C.
capsulatum dermatitidis immitis neoformans
Sensitivity (isolates positive/isolates tested)
Specificity (non-target isolates/non-target isolates tested)
111/112 (99.1%) 88/89 (98.9%) 119/119 (100%) 103/103 (100%)
164/165 (99.4%) 228/228 (100%) 164/164 (100%) 237/237 (100%)
aData adapted from Stockman et al. Abstract F5, 90th Meeting of the American Society for Microbiology, Anaheim, California, May 13-17, 1990.
In conclusion, the methods described in this manuscript have the potential to dramatically improve the laboratory diagnosis of human mycoses. The rapid tests, for the detection of Aspergillus and Candida antigens, the detection of o-arabinitol produced by Candida species, and detection of specific DNA in clinical specimens, all provide results within hours. This dramatic reduction in time for positive results compared to culture techniques should permit earlier institution of specific antifungal chemotherapy and reduce the need for performing invasive procedures. Negative results, however, may not reduce the use of empirical antifungal therapy due to the possibility of false negatives with all of these tests. Importantly, these tests need to be performed on multiple specimens from each high risk patient and it must be emphasized that these tests augment but do not replace culture methods for the important opportunistic pathogens. Widespread use of all of these tests is not possible at the present time since many reagents are not universally available. Furthermore use of these tests is limited due to the requirement for specialized equipment not currently available in clinical laboratories. The methods for accurate species delineation and for rapid identification of medically important fungi will also aid in the diagnosis of human infections. The use of DNA/DNA or DNA/RNA hybridization assays can save days to weeks for accurate identification. The non-radiolabelled probes, with a reasonable shelf-life, will permit many laboratories to expand their ability to identify fungal pathogens. ACKNOWLED GEMENTS We wish to thank Mark Kielkucki for preparation of the manuscript and all the individuals who work in the laboratories of the authors who deserve special thanks.
CONTRIBUTORS The contributors to this symposium were: M. A. Pfaller, Laboratory aids in the diagnosis of invasive canclidiasis; T. R. Rogers, Diagnosis of pubnonary and dissemi-
248
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AL.
nated aspergillosis; G. D. Roberts, The application of nucleic acid probe testing in the clinical mycology laboratory; E. Gueho, Phylogenetic relationships among species of Cryptococcus and Trichosporon as determined by partial rRNA sequences. The coconvenors were W. Merz and C. De Vroey.
REFERENCES
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1. BUCHMAN,T. G., ROSSIER,M., MERZ, W. G. & CHARACHE,P. 1990. Detection of surgical pathogens by in vitro DNA amplification. Part 1. Rapid identification of Candida albicans by in vitro amplification of a fungus-specific gene. Surgery, 108, 338-347. 2. CABEZUDO,I., PFALLER,M., GERARDEN,T., KOONTZ,F., WENZEL, R., GINGRICH,R., HECKMAN,K.
3. 4. 5. 6. 7.
8. 9. 10.
& BURNS, C. P. 1989. Value of the Cand-Tec candida antigen assay in the diagnosis and therapy of systemic candidiasis in high-risk patients. European Journal of Clinical Microbiology and Infectious Diseases, 8, 770-777. DvpoNx, B., IMVROVISl,L. & RONIN, O. 1991. Aspects epidemiologiques et cliniques des infections ~. Scedosporium et Pseudallescheria. Journal de Mycologie M(dicale, 1, 33-42. GuEuo, E. & DE HOOO, G. S. 1991. Taxonomy of the medical species of PseudaUescheria and Scedosporium. Journal de Mycologie M3dicale, 1, 3-9. JONES, J. M. 1990. Laboratory diagnosis of invasive candidiasis. Clinical Microbiology Reviews, 3, 32-45. MURRAY, P. R. 1991. Comparison of the lysis-centrifugation and agitated biphasic blood culture systems for detection of fungemia. Journal of Clinical Microbiology, 29, 96-98. MURV.AY,P. R., SPmzo, A. W. & NILES, A. C. 1991. Clinical comparison of the recoveries of bloodstream pathogens in Septi-Chek brain heart infusion broth with saponin, Septi-Chek tryptic soy broth, and the isolator lysis-centrifugation system. Journal of Clinical Microbiology, 29, 901-905. NESS, M. J., VAUGHAN,W. P. & WOODS, G. L. 1989. Candida antigen latex test for detection of invasive candidiasis in immunocompromised patients. Journal oflnfectious Diseases, 159(3), 495-502. ROGERS, T. R., HAYNES, K. A. & BAaNES, R. A. 1990. Value of antigen detection in predicting invasive pulmonary aspergillosis. Lancet, 336, 1210-1213. SALmN, I. F., MCGINNIS,M. R., DYKSTV,A, M. J. & RINALDI, M. G. 1988. Scedosporium inflatum an emerging pathogen. Journal of Clinical Microbiology, 26, 498-503.
11. WALSH,T. J., HATHORN,J. W., SOBEL,J. D., MERZ, W. G., SANCHEZ,V., MARET, S. M., BUCKLEY, S. R., PFALLER,M. A., SCHAUFELE,R., SLIVA,C., NAVARRO,E., LECCIONES,J., CHANDRASEKAR,P., LEE, J. & Ptzzo, P. A. 1991. Detection of circulating caudida enolase by immunoassay in patients with cancer and invasive candidiasis. New England Journal of Medicine, 324, 1026-1031. 12. Pfaller et al. Abstract C25, 91st meeting of the American Society for Microbiology, Dallas TX, May 5-9, 1991.
Developments in the diagnostic mycology laboratory
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G. D. ROBERTS 1, M. A. PFALLER 2, E. GUEHO 3, T. R. ROGERS 4, C. DE VROEY s AND W. G. MERZ 6
1Mayo Clinic and Foundation, Rochester, Minnesota, and ZOregon Health Sciences University, Portland, Oregon, USA; 3Institut Pasteur, Paris, France; 4Charing Cross and Westminster Medical School, London, England; 5Instutuit Voor Tropishe Geneeskunde, Antwerpen, Belgium; and 6The Johns Hopkins Medical Institutions, Baltimore, MD, USA The laboratory diagnosis of specific human mycoses still relies on classic mycological procedures including direct examination and recovery of fungi from clinical specimens, and accurate identification of the fungal organisms. To date, the non-culture methods developed, with the exception of tests for the detection of cryptococcal polysaccharide antigen, have lacked sensitivity and/or specificity, or have not been available for widespread use. This is unfortunate since techniques for rapid detection of specific fungal antigens, chemical constituents, metabolic products, and fungal species-specific RNA and /or DNA sequences, have the potential to yield rapid diagnostic information that can guide early and appropriate use of specific antifungal chemotherapy. These methods, however, will perhaps never replace classic methods due to lack of sensitivity or specificity and the need for a wide range of reagents to be available to keep up with the increasing number of fungal species emerging as new pathogens in different clinical settings. Therefore, improvement in all aspects of laboratory testing is appropriate. The purpose of this manuscript is to present an update on selected methods that are currently being evaluated for diagnosis of the mycoses, some of which are being made available for widespread use. The methods to be discussed include improved techniques for culturing medically important fungi, improvements in methods for accurately identifying fungi, as well as improvements in several non-culture approaches to enhance our taxonomic delineation of species and to detect fungi or fungal products directly in clinical specimens.
Non-culture techniques for improved laboratory diagnosis Invasive candidiasis. Invasive candidiasis is the most frequently encountered lifethreatening human mycosis. Unfortunately, the clinical signs and symptoms of this infection are generally non-specific and specific diagnosis depends on culture and/or histological evidence from appropriately collected blood or deep tissue specimens. Problems in sensitivity and specificity of these methods and the need for invasive procedures seriously compromise diagnosis of this important infection. Therefore, rapid, non-culture methods may have a very profound impact on diagnosis. Three assays for detection of Candida antigens in serum either currently commerCorrespondence address: Dr W. G. Merz, Department of Laboratory Medicine, Meyer B1-193, The Johns Hopkins Hospital, Baltimore, MD 21205, USA. 241
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ROBERTS E T A L .
cially available or under development include a latex agglutination (LA) assay for detection of an unknown, heat-labile antigen (Cand-Tec, Ramco, Houston, TX), an enzyme immunoassay (EIA) for detection of Candida enolase (Becton Dickenson Microbiology Systems, Baltimore, MD) and an EIA being developed to detect a cell wall mannan antigen (Hybritech, San Diego, CA). With all three tests, results can be available in 15-30 min. The sensitivities and specificities of these assays are presented in Table 1. The wide range of values for the LA test is not acceptable for a test in a clinical laboratory. Although the EIA tests have a 15% false negative rate and would never replace blood cultures, they nevertheless appear promising for inclusion in a series of laboratory tests to optimize the diagnosis of invasive candidiasis. Prospective studies evaluating these tests in comparison to culture results for the rapid diagnosis and monitoring of invasive candidiasis are necessary.
T A B L E 1. Use of antigen detection tests for the diagnosis of invasive candidiasis Assay L A test E I A - enolase E I A - CWM a
Reference number
Sensitivity (%)
Specificity (%)
[2, 8] [11] [12]
19-100 85 86
29-99 96 91
aCWM = cell wall mannan.
Another exciting approach as an aid to the diagnosis of invasive candidiasis is the detection of the Candida-specific metabolite, D-arabinitol. Ongoing studies with a gas-liquid chromatography procedure to detect the enantiospecific D-arabinitol in sera from experimental animals infected with Candida albicans and from human cases of invasive candidiasis are encouraging, with a sensitivity of 80% when combined with blood cultures (44-65% detected by blood culture or o-arabinitol alone, respectively) (B. Wong, personal communication). Prospective clinical studies are necessary to evaluate the usefulness of this method. Similar to the results of antigen tests, screening for o-arabinitol will probably augment classic methods, not replace them. The use of DNA/DNA or DNA/RNA hybridization assays to detect Candidaspecific DNA sequences has not yet been formulated due to the decreased sensitivity of these assays for DNA detection in clinical specimens. A DNA-based procedure which employs amplification of a DNA sequence within the lanosterol 14 a-demethylase gene by the polymerase chain reaction and the use of specific DNA probes has been developed [1]. Prospective clinical studies are necessary to determine whether this assay or similar assays will have the sensitivity to detect fungal nucleic acids in a full range of clinical specimens.
Invasive aspergillosis. Invasive aspergillosis (IA) has become a major infection in the setting of the neutropenic patient, second only to invasive candidiasis as the most frequent opportunistic fungal pathogen. Clinical signs and symptoms of this infection are generally non-specific, making early diagnosis of IA difficult and necessitating empirical therapy with amphotericin B. Although important findings suggestive of IA can be made by various imaging procedures (CT scan, MRI), specific diagnosis
243
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DIAGNOSTIC MYCOLOGY
presently depends on the histological examination of biopsy specimens and culture of the fungal pathogen. The detection of an Aspergillus-specific antigen(s) produced early in the infectious process, in advance of other signs of IA, would make a significant contribution to successful treatment and appropriate use of antifungal drugs. An EIA-inhibition assay using either a polyclonal serum or monoclonal antibody to galactomannan antigen has been developed and tested in 121 high risk bone marrow transplant patients [9]. The overall sensitivity was 95% and the specificity was 99% (Table 2). Important findings included: (i) positive results in this assay correlated with the development of IA; (ii) antigenaemia occurred at a sufficiently early stage in the infection to be a predictor of IA; and (iii) serial sampling was necessary since circulating levels fluctuated significantly. A commercially available LA test (Pastorex Aspergillus, Diagnostics Pasteur, Paris, France) has also been developed, and was tested with sera from the same 121 bone marrow transplant recipients. Results were comparable, although, there were more false positive tests with the latex assay than with the EIA-inhibition assay (T. Rogers, personal communication). These results are very encouraging and warrant further investigation of these assays to determine their role in the laboratory diagnosis of this extremely serious, life-threatening infection. TABLE 2. EIA-Inhibition assays for the diagnosis of invasive aspergillosis~ Patient group
Proven invasive aspergillosis Clinically suspected aspergillosis Other fungal infection No fungal infection Total
Number of patients
Number of patients with positive test
Positive antigen test preceded other diagnostic finding
19 12 8 82
18 12 0 1
16 11 NA NA
121
31
aData from [9].
Improvement in classic mycologic procedures for diagnosis Invasive candidiasis. As mentioned previously, the specific-diagnosis of invasive candidiasis depends on the recovery of the organism from deep tissue with positive histologic findings or repeated recovery from blood specimens. Unfortunately, it is widely held that Candida species are recovered from only 15-50% of patients with invasive candidiasis and cultures may become positive only immediately before death. Recent studies, however, document the improved sensitivities of newer blood culture methods for the recovery of yeasts from blood specimens. These improvements in blood culture methods include venting of broth blood cultures, commercial development and the use of agitation of biphasic blood culture bottles, improvements in non-radiometric CO2 detecting blood culture systems, and the development of the lysis-centrifugation system. The vast majority of published data indicate that the lysiscentrifugation system (Isolator, Wampole, USA) provides the most sensitive means of detecting fungaemia; however, it is clearly not 100% sensitive and a combined
244
ROBERTS E T A L .
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approach using the Isolator plus a broth or biphasic system always results in the detection of a greater number of fungemic episodes than either system alone (Table 3). Recent data document improved sensitivity of 50-82% for blood cultures in neutropenic patients with tissue-proven invasive candidiasis [6, 7, 11] (Pfaller et al., Abstract C25, 91st meeting of the American Society for Microbiology, Dallas, Texas, May 5-9, 1991; Telenti et al., Abstract 129, 26th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, Louisianna, Sept. 28-Oct 1, 1986). These observations suggest that improved culturing strategy (more frequent blood cultures of adequate volume) and/or improved blood culture methods may provide a sensitive means of diagnosing invasive candidiasis. TABLE 3. 1 Studies comparing lysis-centrifugation (LC) versus broth blood cultures for ability to detect fungemiaa Blood culture Total Total technique specimens positive LC vs VB LC vs AgB LC vs VBP LC vs AgBP LC vs L-BP
10,335
117
16,000
117
28,711
258
14,223
182
1,778
52
Percentage Time(days) to positiveby: detection 91 65 86 71 88 64 80 80 67 83
1.8-1.9 2.9-3.7 1.9 2.7 1.9-2.3 4.4-5.0 2.6--4.1 2.6--3.9 3.1 3.5
aData adapted from [5, 6 & 7]. bLC = lysis - centrifugation; VB = vented broth; A g B = agitated broth; VBP = vented biphasic; A g B P = agitated biphasic; L-BP = lysis biphasic (contains saponin).
Comparisons of genotypic vs. phenotypic characteristics for accurate taxonomy and species identification
Accurate identification of fungi to species level has become more important than ever, both to help clinicians determine the clinical relevance of specific species recovered from clinical specimens and also to decide the appropriate antifungal chemotherapy. The latter is emerging as an important issue as newer antifungal agents with different spectra of activity are being developed. An excellent example of the importance of fungal species identification can be made with medically important yeasts. Although many laboratories do not routinely identify Candida isolates to species level, recent reports suggest that shifts have occurred in the distribution of infections caused by various Candida species and evidence is accumulating that species identification can provide a diagnostic clue to the source of infection. Clearly C. albicans remains the most common cause of invasive candidiasis; however, there has been an increase in infections caused by Candida tropicalis, Candida parapsilosis, Candida krusei and Candida lusitaniae. Both C. tropicalis and C. krusei are important causes of fungemia in neutropenic cancer patients. Gastrointestinal colonization appears to be an important predisposing event in patients subsequently infected with either C. tropicalis or C. krusei. C. parapsilosis is well-known as a cause of fungaemia and invasive candidiasis associated
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with parenteral hyperalimentation, intravascular devices and contaminated ophthalmic solutions. More recently, C. parapsilosis has displaced C. tropicalis as the second most common cause of fungaemia in several US hospitals. Finally, C. lusitaniae is recognized as an emerging opportunistic fungal pathogen. The vast majority of patients infected with C. lusitaniae acquire the infection as a nosocomial event. The hallmark of C. lusitaniae, and the major cause for concern clinically, is its propensity to develop resistance to amphotericin B. Accurate delineation of taxa is of prime importance for laboratories and clinicians. Taxa defined solely on phenotypic characteristics may not accurately reflect phylogenic relatedness or unrelatedness. Therefore, genotypic characterizations are often necessary to determine properly delineation of species of fungi of medical importance. This can be well illustrated with the Pseudallescheria boydii/Scedosporium group of filamentous fungi that have emerged as important human pathogens. A taxonomic study of the Pseudallescheria-Scedosporium complex by means of genomic comparison and scanning electron microscopy indicated a correlation between species and epidemiology [4]. Isolates corresponding to P. boydii (Shear) McGinnis, Padhye et Ajello were separated into three groups on the basis of their genomic relationships (Fig. 1); the strains included in each group, with D N A reassociation values systematically above 80% proved to be conspecific. In contrast, between groups the homologies were in the range of 58-72% proving a relatedness at the varietal level. It is interesting to note that six out of nine strains in Group 1 were of pulmonary origin whereas none of the Group 2 strains had such an origin; they were mainly isolated from cases of sinusitis and otitis. Similarly, sexual reproduction (Pseudallescheria) was only observed in Group 1, while coremia (Graphium) were only in Group 2. FIG. 1. Genomic relationships among isolates of P. boydii and other related fungia Group 1
CDC A495 CBS 101.22 IP 1251.80 IP 1448.83 IP I698.87 IP 1945.90 IP 1947.90 IP 1988.91 IP 1986.91 IP 2002.91
Group 2
94-99% knee arthritis, P. shearii mycetoma man? abscess --58-72% -lung, leukaemia sputum, cystic fibrosis sputum, cystic fibrosis sputum, cystic fibrosis sputum, cystic fibrosis sputum, lymphoma
I I
CBS CBS CBS IP IP IP IP IP IP
297.38 895.70 987.73 1411.82 1907.90 1946.90 2003.91 2004.91 2008.91
68--70%
85-96% wood,pulp, S. borzinii sinus,swine, Acremonium sula humanotils, G. enumorphum mycetoma sinus sinus foot blood, kidney transplant bone
I I
65-66%
I
Group 3
83% CBS 108.64 soil IP 1742.88 soil aData adapted from [4]. bCDC = Center of Disease Control, Atlanta, USA; CBS = Centraalbureau voor Schimmelcultures Baarn, Netherlands; IP = Pasteur Institute, France.
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Scedosporium inflatum Malloch & Salkin is the other species of the genus Scedosporium now recognized as a human pathogen [10]. DNA homologies among clinical isolates of this species (bone or blood isolates) are 96-100% and only 0-17% with varieties of P. boydii (Fig. 2). Gueho & de Hoog [4] have also included in this study the type strain of Lomentospora prolificans Hennebert et Desai originated from soil in Belgium which gave 100% relatedness with clinical isolates. This finding, in addition to mycological features, proves that the two species are identical. Since prolificans is the oldest available epithet, the new combination Scedosporium prolificans (Hennebert et Desai) Gueho et de Hoog was proposed. This species is known only by its imperfect state. However, it is well-separated from the anamorph, Scedosporium apiospermum, by cultural (much darker) and microscopic characteristics (typically swollen conidogenous cells). S. prolificans is less frequently encountered than S. apiospermum, but it is responsible for deep mycosis as well and has a preference for bones. The distinction between these two Scedosporium species would also be supported by differences in sensitivity to antifungal drugs [3]. Scedosporium inflatum and Lomentospora prolificans
FIG. 2. Genomic relationships within
Group 1
Group 2
Pseudallescheria boydiP
0-17%
0-6%
Scedosporium inflatum"~ CBS CBS IP IP IP IP
114.90 b 467.74 c 1236.80 1883.89 1912.90 1913.90
foot osteomyelitis green house soil foot osteoarthritis blood, medullar aplasia blood, acute leukaemia blood, myeloblastic leukaemia (96-100%)
aData adapted from [4]. ~Fype culture of Scedosporium inflatum. ~Type culture of Lomentospora prolificans.
Use of DNA probes for rapid identification of medically important fungi Genotypic methods can also be used for rapid, accurate species identification in the clinical laboratory. DNA probes specific for variable regions of rRNA have been developed and are now available commercially for the identification of Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis and Cryptococcus neoforroans (Murphy Clark et al., Abstract FT; Rubin et al., Abstract F6, 90th meeting of the American Society for Microbiology, Anaheim, California, May 13-17, 1990). Specific diagnosis of histoplasmosis, blastomycosis and coccidioidomycosis frequently requires obtaining tissue through invasive procedures with culture and/or histological confirmation. Recovery of these fungi may take 3 days to 6 weeks with an additional 3 days to 2 weeks for species identification. Therefore, the dimorphic fungi are the perfect organisms for the application of rapid identification systems. The DNA hybridization assay requires less than 1 h for identification from a single small colony from any medium, even if it is not a pure culture. In addition, either the yeast or filamentous forms of fungi are appropriate for testing with the probe assay system. The results of an evaluation of four probes for the identification of these important
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human pathogenic fungi are presented in Table 4 (Stockman et al., Abstract F5, 90th meeting of the American Society for Microbiology, Anaheim, California, May 13-17, 1990.) These assays provide an accuracy of identification comparable to that obtained with exoantigen testing; however, the probe assay results are available with 1 h compared with 72 h with the exoantigen tests. The application of the DNA probe assays to direct detection of fungal pathogens in tissue has not been accomplished but studies are currently underway. TABLE 4. Use of DNA probes for rapid identification of medically important fungia
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Fungal species tested
H. B. C. C.
capsulatum dermatitidis immitis neoformans
Sensitivity (isolates positive/isolates tested)
Specificity (non-target isolates/non-target isolates tested)
111/112 (99.1%) 88/89 (98.9%) 119/119 (100%) 103/103 (100%)
164/165 (99.4%) 228/228 (100%) 164/164 (100%) 237/237 (100%)
aData adapted from Stockman et al. Abstract F5, 90th Meeting of the American Society for Microbiology, Anaheim, California, May 13-17, 1990.
In conclusion, the methods described in this manuscript have the potential to dramatically improve the laboratory diagnosis of human mycoses. The rapid tests, for the detection of Aspergillus and Candida antigens, the detection of o-arabinitol produced by Candida species, and detection of specific DNA in clinical specimens, all provide results within hours. This dramatic reduction in time for positive results compared to culture techniques should permit earlier institution of specific antifungal chemotherapy and reduce the need for performing invasive procedures. Negative results, however, may not reduce the use of empirical antifungal therapy due to the possibility of false negatives with all of these tests. Importantly, these tests need to be performed on multiple specimens from each high risk patient and it must be emphasized that these tests augment but do not replace culture methods for the important opportunistic pathogens. Widespread use of all of these tests is not possible at the present time since many reagents are not universally available. Furthermore use of these tests is limited due to the requirement for specialized equipment not currently available in clinical laboratories. The methods for accurate species delineation and for rapid identification of medically important fungi will also aid in the diagnosis of human infections. The use of DNA/DNA or DNA/RNA hybridization assays can save days to weeks for accurate identification. The non-radiolabelled probes, with a reasonable shelf-life, will permit many laboratories to expand their ability to identify fungal pathogens. ACKNOWLED GEMENTS We wish to thank Mark Kielkucki for preparation of the manuscript and all the individuals who work in the laboratories of the authors who deserve special thanks.
CONTRIBUTORS The contributors to this symposium were: M. A. Pfaller, Laboratory aids in the diagnosis of invasive canclidiasis; T. R. Rogers, Diagnosis of pubnonary and dissemi-
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nated aspergillosis; G. D. Roberts, The application of nucleic acid probe testing in the clinical mycology laboratory; E. Gueho, Phylogenetic relationships among species of Cryptococcus and Trichosporon as determined by partial rRNA sequences. The coconvenors were W. Merz and C. De Vroey.
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1. BUCHMAN,T. G., ROSSIER,M., MERZ, W. G. & CHARACHE,P. 1990. Detection of surgical pathogens by in vitro DNA amplification. Part 1. Rapid identification of Candida albicans by in vitro amplification of a fungus-specific gene. Surgery, 108, 338-347. 2. CABEZUDO,I., PFALLER,M., GERARDEN,T., KOONTZ,F., WENZEL, R., GINGRICH,R., HECKMAN,K.
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& BURNS, C. P. 1989. Value of the Cand-Tec candida antigen assay in the diagnosis and therapy of systemic candidiasis in high-risk patients. European Journal of Clinical Microbiology and Infectious Diseases, 8, 770-777. DvpoNx, B., IMVROVISl,L. & RONIN, O. 1991. Aspects epidemiologiques et cliniques des infections ~. Scedosporium et Pseudallescheria. Journal de Mycologie M(dicale, 1, 33-42. GuEuo, E. & DE HOOO, G. S. 1991. Taxonomy of the medical species of PseudaUescheria and Scedosporium. Journal de Mycologie M3dicale, 1, 3-9. JONES, J. M. 1990. Laboratory diagnosis of invasive candidiasis. Clinical Microbiology Reviews, 3, 32-45. MURRAY, P. R. 1991. Comparison of the lysis-centrifugation and agitated biphasic blood culture systems for detection of fungemia. Journal of Clinical Microbiology, 29, 96-98. MURV.AY,P. R., SPmzo, A. W. & NILES, A. C. 1991. Clinical comparison of the recoveries of bloodstream pathogens in Septi-Chek brain heart infusion broth with saponin, Septi-Chek tryptic soy broth, and the isolator lysis-centrifugation system. Journal of Clinical Microbiology, 29, 901-905. NESS, M. J., VAUGHAN,W. P. & WOODS, G. L. 1989. Candida antigen latex test for detection of invasive candidiasis in immunocompromised patients. Journal oflnfectious Diseases, 159(3), 495-502. ROGERS, T. R., HAYNES, K. A. & BAaNES, R. A. 1990. Value of antigen detection in predicting invasive pulmonary aspergillosis. Lancet, 336, 1210-1213. SALmN, I. F., MCGINNIS,M. R., DYKSTV,A, M. J. & RINALDI, M. G. 1988. Scedosporium inflatum an emerging pathogen. Journal of Clinical Microbiology, 26, 498-503.
11. WALSH,T. J., HATHORN,J. W., SOBEL,J. D., MERZ, W. G., SANCHEZ,V., MARET, S. M., BUCKLEY, S. R., PFALLER,M. A., SCHAUFELE,R., SLIVA,C., NAVARRO,E., LECCIONES,J., CHANDRASEKAR,P., LEE, J. & Ptzzo, P. A. 1991. Detection of circulating caudida enolase by immunoassay in patients with cancer and invasive candidiasis. New England Journal of Medicine, 324, 1026-1031. 12. Pfaller et al. Abstract C25, 91st meeting of the American Society for Microbiology, Dallas TX, May 5-9, 1991.
