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Candida: A Causative Agent of an Emerging Infection

2001; Elsevier BV; Volume: 6; Issue: 3 Linguagem: Inglês

10.1046/j.0022-202x.2001.00047.x

1529-1774

Mahmoud A. Ghannoum,

Pneumonia and Respiratory Infections

Incidences of infections due to Candida have increased over the last 15–20 y. This increase in the incidence and the high associated mortality rate despite therapy has focused the attention on this disease and prompted investigators to undertake research aimed at understanding the pathogenesis of this disease as well as methods to treat it. This paper discusses recent developments in the Candida field and the impact they have on patient management. Incidences of infections due to Candida have increased over the last 15–20 y. This increase in the incidence and the high associated mortality rate despite therapy has focused the attention on this disease and prompted investigators to undertake research aimed at understanding the pathogenesis of this disease as well as methods to treat it. This paper discusses recent developments in the Candida field and the impact they have on patient management. Emerging infectious diseases are diseases of infectious origin whose incidence in humans has increased within the past two decades or whose incidence threatens to increase in the near future. In this regard, Candida is among those diseases whose incidence has increased over the past 15–20 y.Wey et al., 1998Wey S.B. Mori M. Pfaller M.A. Woolson R.F. Wenzel R.P. Hospital-acquired candidemia. The attributable mortality and excess length of stay.Arch Intern Med. 1998; 148: 2642-2645Crossref Scopus (768) Google Scholar provided the first evidence that fungi had begun preying on the growing population of patients with impaired immune systems (e.g., AIDS, cancer patients). In their study, these authors showed that nearly 40% of all deaths from hospital-acquired infections were not due to bacteria or viruses, but to fungi, with the main culprit being Candida. Supporting evidence for the increase in the incidence of candidal infections could be derived from various databases, including the National Hospital Discharge Survey (NHDS), the Professional Activity Survey (PAS), and the McDonnell Douglas Automation Company Medical Records System (McAuto), which showed that in the 1980s the incidence of candidiasis in hospitalized patients has increased from <0.5 to about 2 per 1000 hospitalized admission (Fisher-Hoch and Hutwagner, 1995Fisher-Hoch S.P. Hutwagner L. Opportunistic Candidiasis. An epidemic of the 1980s.Clin Infect Dis. 1995; 21: 897-904Crossref PubMed Scopus (140) Google Scholar). Interestingly, age-specific rates of oropharyngeal candidiasis derived from NHDS data demonstrated clearly that the increase in this incidence was true irrespective of the age group (Fisher-Hoch and Hutwagner, 1995Fisher-Hoch S.P. Hutwagner L. Opportunistic Candidiasis. An epidemic of the 1980s.Clin Infect Dis. 1995; 21: 897-904Crossref PubMed Scopus (140) Google Scholar). Follow-up surveillance studies conducted in the 1990s demonstrated that the high incidence of candidal infections in hospitalized patients is still true.Edmond et al., 1999Edmond M.B. Wallace S.E. McClish D.K. Pfaller M.A. Jones R.N. Wenzel R.P. Nosocomial bloodstream infections in United States hospitals: a three-year analysis.Clin Infect Dis. 1999; 29: 239-244Crossref PubMed Scopus (1211) Google Scholar undertook a survey referred to as Scope Project (Surveillance and Control of Pathogens of Epidemiologic Importance) where they conducted concurrent surveillance for bloodstream infections at 49 hospitals over a 3 y period. In this study, more than 10 000 Gram-positive, Gram-negative, and yeast infections were monitored. Data analyzes showed that whereas Gram-positive and Gram-negative bacteria accounted for 64% and 27% of cases, respectively, yeast accounted for 8% of cases. Furthermore, Enterobacteria, Serratia, and coagulase (–) staphylococci and Candida were more likely to cause infection in the critical care setting. This study shows that Candida continues to be an important cause of nosocomial bloodstream infection. Importantly, similar to earlier findings, yeasts were associated with the highest crude mortality (40%). Unlike the situation with candidal bloodstream infections, which remain high, the incidence of esophageal candidiasis in the HIV/AIDS setting has decreased. This decrease could be attributed to the host immune reconstitution brought about by the new antiretroviral therapy (Highly Active Antiretroviral Therapy, HAART). The decrease in esophageal candidiasis in both adults and children is documented in the study ofKaplan et al., 2000Kaplan J.E. Hanson D. Dworkin M.S. et al.Epidemiology of human immunodeficiency virus-associated opportunistic infections in the United States in the era of highly active antiretroviral therapy.Clin Infect Dis. 2000; 30: S5-S14Crossref PubMed Scopus (583) Google Scholar, who investigated the epidemiology of HIV-associated opportunistic infections (OI) in the U.S.A. in the era of HAART (Kaplan et al., 2000Kaplan J.E. Hanson D. Dworkin M.S. et al.Epidemiology of human immunodeficiency virus-associated opportunistic infections in the United States in the era of highly active antiretroviral therapy.Clin Infect Dis. 2000; 30: S5-S14Crossref PubMed Scopus (583) Google Scholar). These authors showed that, among other OI, the incidence of esophageal candidiasis has dropped significantly between 1996 and 1997; however, a disturbing trend is seen between 1997 and 1998, where the incidence of esophageal candidiasis seems to be on the incline again. This observation indicates that it is prudent to continue following the epidemiology of this and other opportunistic infections, and that it is too early to draw firm conclusions regarding such trends. The decrease in the incidence of OI, including candidiasis, has been attributed to the restoration of specific immunity (Kelleher et al., 1996Kelleher A.D. Carr A. Zaunders J. Cooper D.A. Alterations in the immune response of HIV-infected subjects treated with HIB-specific protease inhibitor, ritonavir.J Infect Dis. 1996; 173: 321-329Crossref PubMed Scopus (310) Google Scholar;Autran et al., 1997Autran B. Carcelain G. Li T.S. et al.Positive effects of combined antiretroviral therapy on CD4 T cell homeostasis and function in advanced HIV disease.Science. 1997; 277: 112-116Crossref PubMed Scopus (1591) Google Scholar;Pakker et al., 1997Pakker N.G. Roos M.T. van Leeuwen R. et al.Patterns of T-cell repopulation, virus load reduction, and restoration of T-cell function in HIV-infected persons during therapy with different antiretroviral agents.J Acquir Immune Defic Syndr Hum Retrovitrol. 1997; 16: 318-326Crossref PubMed Scopus (85) Google Scholar;Angel et al., 1998Angel J.B. Kuman A. Parato K. et al.Improvement in cell-mediated immune functions during potent anti-human immunodeficiency virus therapy with ritonavir plus saquinavir.J Infect Dis. 1998; 177: 898-904Crossref PubMed Scopus (120) Google Scholar); however, protease inhibitors could also be exerting a direct effect on C. albicans secretory aspartyl proteases (Sap), which have been shown to be virulence factors for C. albicans (Hube, 1996Hube B. Candida albicans secreted aspartyl proteinases.Curr Top Med Mycol. 1996; 7: 55-69PubMed Google Scholar). In this regard, Cassone et al showed that the protease inhibitors indinavir and ritonavir inhibited Sap activity and production (Cassone et al., 1999Cassone A. DeBernardis F. Torosantucci A. Tacconelli E. Tumbarello M. Cauda R. In vitro and in vivo anticandidal activity of human immunodeficiency virus protease inhibitors.J Infect Dis. 1999; 180: 448-453Crossref PubMed Scopus (171) Google Scholar). Further studies are required to demonstrate whether Sap inhibition is implicated in the decrease of candidiasis in AIDS patients. It is critical to clarify that the observation that the incidence of OI, including candidiasis, is decreasing in the HIV-infected patients is limited to developed countries (mainly the U.S.A. and Western Europe). The incidence of these devastating diseases is still high in developing countries, however, particularly sub-Saharan Africa where between 20% and 50% (depending on the country or regions within a country) of the population are infected with this virus (Schooley, 2000Schooley R. HIV pathogenesis.Presented at the 49th Annual Montagna Symposium on the Biology of Skin. Snowmass, Colorado2000Google Scholar). The increase in the incidence of serious candidiasis and the high associated mortality rate, even with therapy (35%-50%), has focussed the attention on this disease and prompted investigations aimed at improving our understanding of the pathogenesis of this disease, and host–parasite interactions (Pittet et al., 1994Pittet D. Tarara D. Wenzel R.P. Nosocomial bloodstream infection in critically ill patients. excess length of stay, extra costs, and attributable mortality.JAMA. 1994; 271: 1598-1601Crossref PubMed Scopus (1253) Google Scholar). This paper will focus on recent developments in the Candida field that impact patient management. Candidiasis is an infection of the immunocompromised. This implies that for Candida to cause an infection, one or more risk factors should be present. A number of factors have been suggested, including underlying immune insufficiency, use of broad-spectrum antibiotics, cytotoxic agents (e.g., anticancer agents), immunosuppressive drugs, and central venous catheters. Although a number of Candida species exist, only 10 species have been associated with infection to any significant degree. These include: C. albicans, C. tropicalis, C. glabrata, C. krusei, C. lusitaniae, C. parapsilosis, C. guilliermondii, C. pseudotropicalis, C. rugosa, and C. stellatoidea. For a long time (up to early 1990s), C. albicans was the main etiologic agent of candidiasis, responsible for more than 70% of all Candida infections. With the introduction of the azoles (late 1980s, early 1990s), a shift in the candidal species causing infection started to occur with non-albicans becoming more important in the disease process. This point is clearly shown in the Edmond et al study that reported that the non-albicans species caused between 30% and 54% of the yeast infection (Edmond et al., 1999Edmond M.B. Wallace S.E. McClish D.K. Pfaller M.A. Jones R.N. Wenzel R.P. Nosocomial bloodstream infections in United States hospitals: a three-year analysis.Clin Infect Dis. 1999; 29: 239-244Crossref PubMed Scopus (1211) Google Scholar). Moreover, these authors showed that the extent of yeast infections caused by non-albicans Candida varied with the geographic region. According to their study, the north-east region of the U.S.A. had the highest percentage (54%) of nosocomial bloodstream infections due to non-albicans species, and the south-west had the least (30%). Candida glabrata and C. tropicalis were the second and third causative Candida, after C. albicans, respectively (Edmond et al., 1999Edmond M.B. Wallace S.E. McClish D.K. Pfaller M.A. Jones R.N. Wenzel R.P. Nosocomial bloodstream infections in United States hospitals: a three-year analysis.Clin Infect Dis. 1999; 29: 239-244Crossref PubMed Scopus (1211) Google Scholar). This change in the pattern of candidal species causing infections impacts the management of this disease. For example, C. glabrata tends to have higher minimum inhibitory concentrations (MIC) values to the currently used antifungals, particularly the azoles (MIC 16–32 µg per ml). Moreover, infections caused by this species are associated with poor prognosis (Nguyen et al., 1996Nguyen M.H. Peacock J.E. Morris A.J. et al.The changing face of candidemia: emergence of non-Candida albicans species and antifungal resistance.Am J Med. 1996; 100: 617-623Abstract Full Text PDF PubMed Scopus (702) Google Scholar). Thus, the use of high doses of fluconazole (800 mg daily) is recommended for the management of candidiasis caused by C. glabrata. Currently, bedside evaluation, blood cultures, diagnostic imaging, and biopsies are standard clinical and laboratory tools used in the diagnosis of invasive candidiasis. Unfortunately, these methods lack sensitivity in the early recognition of this infection and are imprecise as markers of complete eradication of infection. Many of the early attempts of nonculture diagnosis of invasive candidiasis lack sensitivity; however, advances in the identification and purification of potential antigens, monoclonal antibody production, epitope mapping, recombinant DNA techniques, and polymerase chain reaction (PCR) should contribute towards further development of diagnostic methods for fungi that are increased in both sensitivity and specificity. As these studies have been extensively reviewed (Jones, 1990Jones J.M. Laboratory diagnosis of invasive candidiasis.Clin Microbiol Rev. 1990; 3: 32-45PubMed Google Scholar;Walsh et al., 1991Walsh T.J. Hathorn J.W. Sobel J.D. et al.Detection of circulating Candida enolase by immunoassay in patients with cancer and invasive candidiasis.N Engl J Med. 1991; 324: 1026-1031Crossref PubMed Scopus (203) Google Scholar;de Repentigny, 1992de Repentigny L. Serodiagnosis of candidiasis, aspergillosis, and cryptococcosis.Clin Infect Dis. 1992; 14: S11-S22Crossref PubMed Google Scholar;Reiss and Morrison, 1993Reiss E. Morrison C.J. Nonculture methods for diagnosis of disseminated candidiasis.Clin Microbiol Rev. 1993; 6: 311-323PubMed Google Scholar;Verweij et al., 1998Verweij P.E. Poulain D. Obayashi T. Patterson T.F. Denning D.W. Ponton J. Current trends in the detection of antigenaemia, metabolites and cell wall markers for the diagnosis and therapeutic monitoring of fungal infections.Med Mycol. 1998; 36: 146-155PubMed Google Scholar), a brief description of the current status of research will be provided in this proposal. Detection of Candida spp. in the bloodstream is a key factor for establishing a diagnosis of invasive candidiasis. There have been numerous advances in blood culture methodology over the past 10–15 y that have contributed to improved detection of candidemia (Bougnoux et al., 1990Bougnoux M.E. Hill C. Moissenet D. et al.Comparison of antibody, antigen, and metabolite assays for hospitalized patients with disseminated or peripheral candidiasis.J Clin Microbiol. 1990; 28: 905-909PubMed Google Scholar;Kohno et al., 1993Kohno S. Mitsutake K. Maesaki S. et al.An evaluation of serodiagnostic tests in patients with candidemia; beta-glucan, mannan, Candida antigen by Candidiasis-Techniques and D-arabinitol.Microbiol Immunol. 1993; 37: 207-212Crossref PubMed Scopus (60) Google Scholar;Chen et al., 1997Chen Y.C. Chang S.C. Sun C.C. Yang L.S. Hsieh W.C. Luh K.T. Secular trends in the epidemiology of nosocomial fungal infections at a teaching hospital in Taiwan, 1981–93.Infect Control Hosp Epidemiol. 1997; 18: 369-375Crossref PubMed Google Scholar). Presently the lysis centrifugation and Bactec methods are the most superior of the systems developed. Although these methods are clearly a technologic advance these systems have a low sensitivity in the early detection of candidemia (between 25% and 82%) (Jones, 1990Jones J.M. Laboratory diagnosis of invasive candidiasis.Clin Microbiol Rev. 1990; 3: 32-45PubMed Google Scholar). Additionally, analysis of the available data indicates that maximum detection of fungemia is achieved when more than one blood culture system is employed (Pfaller and Wenzel, 1992Pfaller M.A. Wenzel R. Impact of the changing epidemiology of fungal infections in the 1990s.Eur J Clin Microbiol Infect Dis. 1992; 11: 287-291Crossref PubMed Scopus (221) Google Scholar). The length of time (minimum of 5 d) needed to recover Candida is another limitation with blood culture methods. These drawbacks emphasize the need for innovative nonculture methods that allow early detection of candidiasis, and/or complement blood culture. Molecular and biochemical factors involved in Candida infections have been investigated to develop nonculture diagnostic methods. These factors include (i) Candida antigens, (ii) antibodies against Candida antigens, (iii) Candida metabolites, and (iv) Candida DNA sequences. Several workers attempted to develop immunoassays for detecting C. albicans antigens in sera from infected patients (Bennett, 1987Bennett J.E. Rapid diagnosis of candidiasis and aspergillosis.Rev Infect Dis. 1987; 9: 398-402Crossref PubMed Scopus (43) Google Scholar;Jones, 1990Jones J.M. Laboratory diagnosis of invasive candidiasis.Clin Microbiol Rev. 1990; 3: 32-45PubMed Google Scholar;de Repentigny, 1992de Repentigny L. Serodiagnosis of candidiasis, aspergillosis, and cryptococcosis.Clin Infect Dis. 1992; 14: S11-S22Crossref PubMed Google Scholar;Reiss and Morrison, 1993Reiss E. Morrison C.J. Nonculture methods for diagnosis of disseminated candidiasis.Clin Microbiol Rev. 1993; 6: 311-323PubMed Google Scholar). These investigators employed radioimmunoassay (RIA), enzyme-linked immunoassay (ELISA), and latex agglutination to identify circulating and/or cytoplasmic antigens. The cell wall mannan (mannoprotein) of Candida has been studied extensively at the immunologic level. Thus, mannan extracts of varying degrees of purity have been used for serodiagnosis (Odds, 1988Odds F.C. Serodiagnosis of candidosis.in: Odds F.C. Candida and Candidosis: Review and Bibliography. London, Ballière-Tindall1988: 235-251Google Scholar). Cell wall mannan antigen is not usually detectable unless the immune and other circulating complexes are disrupted by hydrolysis (Walsh et al., 1995Walsh T.J. Lyman C.A. Pizzo P.A. Laboratory diagnosis of invasive fungal infections in patients with neoplastic diseases. Balliere's.Clin Infect Dis. 1995; 2: 25-70Google Scholar). Another limitation to using mannan is that it is cleared rapidly from the serum (i.e., transient). Consequently, repeated serum sampling is necessary (Jones, 1990Jones J.M. Laboratory diagnosis of invasive candidiasis.Clin Microbiol Rev. 1990; 3: 32-45PubMed Google Scholar;de Repentigny, 1992de Repentigny L. Serodiagnosis of candidiasis, aspergillosis, and cryptococcosis.Clin Infect Dis. 1992; 14: S11-S22Crossref PubMed Google Scholar). In spite of these limitations a number of commercial tests are available in Europe for mannan detection. Unfortunately the sensitivity of these tests is still a drawback (Fujita and Hashimoto, 1992Fujita S. Hashimoto T. Detection of serum Candida antigens by enzyme-linked immunosorbent assay and a latex agglutination test with anti-Candida albicans and anti-Candida krusei antibodies.J Clin Microbiol. 1992; 30: 3132-3137PubMed Google Scholar). Candida enolase, a 48 kDa cytoplasmic antigen, is recognized by human sera, and is associated with invasive candidiasis (Strockbine et al., 1984Strockbine N.A. Largen M.T. Zweibel S.M. Buckley H.R. Identification and molecular weight characterization of antigens from Candida albicans that are recognized by human sera.Infect Immun. 1984; 43: 715-721PubMed Google Scholar). A multicenter study of high-risk cancer patients with invasive candidiasis showed that the enolase-based method has a 54% sensitivity (Walsh et al., 1991Walsh T.J. Hathorn J.W. Sobel J.D. et al.Detection of circulating Candida enolase by immunoassay in patients with cancer and invasive candidiasis.N Engl J Med. 1991; 324: 1026-1031Crossref PubMed Scopus (203) Google Scholar). Multiple serum sampling improved sensitivity (up to 75%) and specificity (up to 96%). Although enolase showed a lot of initial promise, developing a commercial assay was not cost effective (Sendid et al., 1999Sendid B. Tabouret M. Poirot J.L. Mathieu D. Fruit J. Poulain D. New enzyme immunoassays for sensitive detection of circulating Candida albicans mannan and antimannan antibodies: useful combined test for diagnosis of systemic candidiasis.J Clin Microbiol. 1999; 37: 1510-1517PubMed Google Scholar). Furthermore, the need for multiple sampling to improve sensitivity indicates that enolase-based diagnostic assay can complement but not replace blood cultures in the diagnosis of invasive candidiasis (Walsh et al., 1991Walsh T.J. Hathorn J.W. Sobel J.D. et al.Detection of circulating Candida enolase by immunoassay in patients with cancer and invasive candidiasis.N Engl J Med. 1991; 324: 1026-1031Crossref PubMed Scopus (203) Google Scholar). Anti-Candida antibodies have been investigated as diagnostic markers of invasive candidiasis (Taschdjian et al., 1973Taschdjian C.L. Seelig M.S. Kozinn P.J. Serological diagnosis of candidal infections.CRC Crit Rev Clin Laboratory Sci. 1973; 4: 19-59Crossref PubMed Scopus (51) Google Scholar;Kozinn et al., 1978Kozinn P.J. Taschdjian C.L. Goldberg P.K. et al.Efficiency of serologic tests in the diagnosis of systemic candidiasis.Am J Clin Pathol. 1978; 70: 893-898PubMed Google Scholar;van Deventer et al., 1996van Deventer A.J. Goessens W.H. van Zeijl J.H. Mouton J.W. Michel M.F. Verbrugh H.A. Kinetics of anti-mannan antibodies useful in confirming invasive candidiasis in immunocompromised patients.Microbiol Immunol. 1996; 40: 125-131Crossref PubMed Scopus (18) Google Scholar). Anti-Candida-enolase antibody was detected in sera of less severely immunocompromised patients with invasive candidiasis (Walsh et al., 1991Walsh T.J. Hathorn J.W. Sobel J.D. et al.Detection of circulating Candida enolase by immunoassay in patients with cancer and invasive candidiasis.N Engl J Med. 1991; 324: 1026-1031Crossref PubMed Scopus (203) Google Scholar). Furthermore, antimannan antibody was detected in patients with mycologically and clinically proven candidiasis (Sendid et al., 1999Sendid B. Tabouret M. Poirot J.L. Mathieu D. Fruit J. Poulain D. New enzyme immunoassays for sensitive detection of circulating Candida albicans mannan and antimannan antibodies: useful combined test for diagnosis of systemic candidiasis.J Clin Microbiol. 1999; 37: 1510-1517PubMed Google Scholar). Unfortunately, the low, sometimes undetectable, levels of certain antibodies in immunocompromised patients limit their reliability as diagnostic markers. D-arabinitol, a sugar alcohol metabolite from C. albicans, has been used as a marker for invasive candidiasis (Christensson et al., 1997Christensson B. Wiebe T. Pehrson C. Larsson L. Diagnosis of invasive candidiasis in neutropenic children with cancer by determination of D-arabinitol/L-arabinitol ratios in urine.J Clin Microbiol. 1997; 35: 636-640PubMed Google Scholar). D-arabinitol is cleared by glomerular filtration in the body. Thus, impaired renal function may lead to elevated serum concentrations of D-arabinitol, which can be detected by gas-liquid chromatography (GLC) or by mass spectroscopy (MS) with selected ion monitoring. Elevated levels of D-arabinitol are observed in cases of invasive candidiasis, but not in infections due to C. glabrata and C. krusei (Lehtonen et al., 1996Lehtonen L. Anttila V.J. Ruutu T. Salonen J. Nikoskelainen J. Eerola E. Ruutu P. Diagnosis of disseminated candidiasis by measurement of urine of D-arabinitol/L-arabinitol ratio.J Clin Microbiol. 1996; 34: 2175-2179PubMed Google Scholar). Although elevated levels of D-arabinitol are specific to invasive candidiasis, the amount of time and cost involved and also the complexities of GLC and MS discourage the use of this method as a routine diagnostic tool (McSharry et al., 1993McSharry C. Lewis C. Cruickshank G. Richardson M.D. Measurement of serum arabinitol by gas-liquid chromatography: limitations for detection of systemic Candida infections.J Clin Pathol. 1993; 46: 475-476Crossref PubMed Scopus (4) Google Scholar). Nonetheless, an enzymatic-fluorometric method for determination of serum D-arabinitol has been developed using Klebsiella pneumoniae D-arabinitol dehydrogenase that oxidizes D-arabinitol (Walsh et al., 1994Walsh T.J. Lee J.W. Sien T. et al.Serum of D-arabinitol measured by automated quantitative enzymatic assay for detection and therapeutic monitoring of experimental disseminated candidiasis: correlation with tissue concentrations of Candida albicans.J Med Vet Mycol. 1994; 32: 205-215Crossref PubMed Scopus (22) Google Scholar). The utility of D-arabinitol is still under investigation. 1,3- β-D-glucan is a major structural component of the fungal cell wall that is released by several pathogenic fungi including C. albicans. Detection of 1,3- β-D-glucan is based on the coagulation system in the Limulus amoebocyte (Ikegami et al., 1988Ikegami K. Ikemura K. Shimazu T. Shibuya M. Sugimoto H. Yoshioka T. Sugimoto T. Early diagnosis of invasive candidiasis and rapid evaluation of antifungal therapy by combined use of conventional chromogenic limulus test and a newly developed endotoxin specific assay.J Trauma. 1988; 28: 1118-1126Crossref PubMed Scopus (29) Google Scholar;Obayashi et al., 1995Obayashi T. Yoshida M. Mori T. et al.Plasma (1→3)-beta-D-glucan measurement in diagnosis of invasive deep mycosis and fungal febrile episodes.Lancet. 1995; 345: 17-20Abstract PubMed Scopus (378) Google Scholar). Although 1,3- β-D-glucan has been detected in plasma from patients with systemic mycoses, concentration of this component was significantly higher in those with invasive candidiasis. Thus, this assay may have some clinical utility for diagnosis of invasive candidiasis; however, this approach is still under investigation. Several laboratories are actively pursuing the development of PCR-based tests for rapid diagnosis of invasive candidiasis. These assays have focussed on the use of universal fungal primers, multicopy gene targets, and species-specific DNA regions (Reiss et al., 1998Reiss E. Tanaka K. Bruker G. et al.Molecular diagnosis and epidemiology of fungal infections.Med Mycol. 1998; 36: 249-257PubMed Google Scholar). The universal fungal oligonucleotide primer pair ITS86 and ITS4, which are designed based on the conserved sequences of the 5.8S and 28S ribosomal DNA, respectively, amplifies a species-specific internal transcribed spacer, ITS2 region (Lott et al., 1998Lott T.F. Burns B.M. Zancope-Oliveira R. Elie C.M. Reiss E. Sequence analysis of the internal transcribed spacer 2 (ITS2) form yeast species within the genus Candida.Curr Microbiol. 1998; 36: 63-69Crossref PubMed Scopus (63) Google Scholar). The variability in length of this region between different fungal species is the basis for diagnosis. Similarly, sequences from the fungal lanosterol 14α-demethylase gene have been used in a PCR approach to develop a diagnostic test for invasive candidiasis (Buchman et al., 1990Buchman T. Rossier M. Merz W.G. Charache P. Detection of surgical pathogens by in vitro DNA amplification. Part I. Rapid identification of Candida albicans by in vitro amplification of a fungus-specific gene.Surgery. 1990; 108: 338-346PubMed Google Scholar;Morace et al., 1999Morace G. Pagano L. Sanguinetti M. et al.PCR-restriction enzyme analysis for detection of Candida DNA in blood from febrile patients with hematological malignancies.J Clin Microbiol. 1999; 37: 1871-1875PubMed Google Scholar). The family of secreted aspartic proteinase genes has also been the focus of PCR amplification (Flahaut et al., 1998Flahaut M. Sanglard D. Monod M. Bille J. Rossier M. Rapid detection of Candida albicans in clinical samples by DNA amplification of common regions from C. albicans-secreted aspartic proteinase genes.J Clin Microbiol. 1998; 36: 395-401PubMed Google Scholar). The use of PCR as a diagnostic technique for fungal infections has gained favor because it is rapid, sensitive, and specific; however, the high sensitivity can in fact be a potential disadvantage because detection of commensal C. albicans strains, in otherwise uninfected individuals, may produce false positive reactions (Flahaut et al., 1998Flahaut M. Sanglard D. Monod M. Bille J. Rossier M. Rapid detection of Candida albicans in clinical samples by DNA amplification of common regions from C. albicans-secreted aspartic proteinase genes.J Clin Microbiol. 1998; 36: 395-401PubMed Google Scholar). Likewise DNA sequences from dead or degrading fungal cells may also be amplified. Taken together, although extensive attempts at developing methods to diagnose fungal infections, including Candida, are currently being pursued, all of these methods are still investigational, making conclusions regarding their efficiency premature. To date, the contributions of antibody or antigen detection tests remain unsatisfactory. Studies using PCR to detect candidiasis in neutropenic patients have shown some promise; however, clinical trials are needed to evaluate their utility. The development of standardized antifungal susceptibility testing procedures was spearheaded by the National Committee for Clinical Laboratory Standards (NCCLS), Subcommittee for Antifungal Susceptibility Testing. This development passed through a number of phases: (i) Examination of the role of variables such as inoculum preparation, inoculum size, medium composition, incubation temperature, incubation time, and endpoint definition on interlaboratory variability (Guinet et al., 1988Guinet R. Nerson D. DeClosets F. et al.Collaborative evaluation in seven laboratories of a standardized micromethod for yeast susceptibility testing.J Clin Microbiol. 1988; 26: 2307-2312PubMed Google Scholar;Pfaller et al., 1988Pfaller M.A. Burmeister L. Bartlett M.S. Rinaldi M.G. Multicenter evaluation of four methods of yeast inoculum preparation.J Clin Microbiol. 1988; 26: 1437-1441PubMed Google Scholar,Pfaller et al., 1990Pfaller M.A. Rinaldi M.G. Galgiani J.N. et al.Collaborative investigation of variables in susceptibility testing of yeasts.Antimicrob Agents Chemother. 1990; 34: 1648-1654Crossref PubMed Scopus (132) Google Scholar;Espinel-Ingroff et al., 1991Espinel-Ingroff A. Kerkering T.M. Goldson P.R. 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