Nonculture Methods for Diagnosis of Disseminated Candidiasis · 312 REISS ANDMORRISON In onestudy, 30%of58 nononcologicsurgical patients who developedDCwerereceiving systemiccorticosteroids

CLINICAL MICROBIOLOGY REVIEWS, OCt. 1993, p. 311-323 Vol. 6, No. 40893-8512/93/040311-13$02.00/0Copyright © 1993, American Society for Microbiology

Nonculture Methods for Diagnosis of Disseminated CandidiasisERROL REISS* AND CHRISTINE J. MORRISON

Molecular Mycology Section, Mycotic Diseases Branch, Division ofBacterial and Mycotic Diseases, NationalCenter for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333

INTRODUCTION ...................................................................... 311Types of Disseminated Candidiasis...................................................................... 311

SIGNIFICANCE OF CANDIDEMIA...................................................................... 312Primary Source for DC ...................................................................... 312Frequency of Positive Blood Cultures ...................................................................... 312Receptor-Ligand Clearance of C. albicans from the Bloodstream ..................................................313Candida Fungemia: Primary Disease and Predisposing Factors ....................................................313Summary ...................................................................... 313

MEASUREMENT OF SERUM D-ARABINITOL, A CANDIDA SPECIES METABOLITE ...................313Enzymatic-Fluorometric Determination of Serum D-Arabinitol .....................................................314Clinical Evaluation of Serum D-Arabinitol ...................................................................... 314Summary ...................................................................... 314

AMPLIFICATION OF DNA FROM C. ALBICANS IN BLOOD AND URINE ...................................314Application of PCR to Clinical Specimens ...................................................................... 315Prospects for PCR Detection of Candida spp. in Blood ...............................................................316Summary ...................................................................... 316

IMMUNOASSAY DETECTION OF MARKER ANTIGENS ..........................................................316Enolase (EC 4.2.1.11) ...................................................................... 317

Enolase activity in the 48-kDa antigen ...................................................................... 317Liposomal rhodamine immunoassay for serum C. albicans enolase.............................................317Summary ...................................................................... 317

Secreted Aspartyl Proteinase (EC 3.4.23.6) ....................................................................... 318Role of AP in DC ...................................................................... 318Proteinase antigenuria EIA in models of colonization and DC...................................................318Summary ...................................................................... 318

Immunoblot Detection of Candida Antigenemia/Antigenuria ........................................................318Summary ...................................................................... 318

CWMP Antigenemia ...................................................................... 318Definition and general plan of CWMP ...................................................................... 319Development of immunoassays to detect serum mannan ..........................................................319Sandwich EIA ...................................................................... 319RPLA tests ....................................................................... 320Summary ...................................................................... 320

SUMMARY AND CONCLUSIONS...................................................................... 320REFERENCES ...................................................................... 321

INTRODUCTION

The design of diagnostic laboratory methods for life-threatening infections with Candida species should take intoaccount the range of underlying host conditions and thechanging spectrum of emerging Candida species, which maybe linked to trends in antifungal therapy. Because mostpatients with disseminated candidiasis (DC) are immuno-compromised, the premise of the diagnostic methods to bediscussed is that the results they give are independent of afunctioning immune system. Residual innate and adaptiveimmunity in such patients can interfere with detection of theanalyte being measured by causing it to be complexed toantibodies or removed from circulation by nonantibodyreceptor-ligand interactions and then degraded by plasmaenzyme systems or lysosomal hydrolases. The methodsdevised can be categorized into those that detect D-arabini-

* Corresponding author.

311

tol, a low-molecular-weight metabolite, by enzymaticmeans; DNA after its amplification from Candida spp. inclinical specimens; and marker antigens by immunoassays.These methods use serum, whole blood, or urine specimensto detect Candida spp. or products. Antibody tests to detectDC will not be discussed here, and other reviews should beconsulted for current methods (14, 15, 32).

Types of Disseminated Candidiasis

DC is most often a nosocomial infection affecting twomajor groups of susceptible hosts: severely immunocompro-mised patients, i.e., those with a hematologic malignancy or

solid tumors, who are persistently granulocytopenic fromcytotoxic drug therapy (mean of 11.6 days of granulocyto-penia until the occurrence of fungemia) (29), and nononco-

logic patients, especially those who have undergone abdom-inal or cardiac surgery. Although these patients are notgranulocytopenic, their immune responses may be de-pressed by debilitation and systemic corticosteroid therapy.

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312 REISS AND MORRISON

In one study, 30% of 58 nononcologic surgical patients whodeveloped DC were receiving systemic corticosteroids (41).

Risk factors common to both groups of susceptible hostsinclude prolonged hospital stays (median of 48 days in onestudy [98]), prolonged and multiple antibiotic therapy, andindwelling catheters for hyperalimentation therapy. An un-desirable effect of multiple antibiotic therapy for fever or forprophylaxis is the stimulation of an antibiotic-resistant intes-tinal replacement flora, including Candida species. Surveil-lance cultures of the gastrointestinal and/or gynecologicaltracts often become positive for yeasts after 1 week or moreof broad-spectrum antibiotic therapy (2). Indigenous bacte-ria normally decrease the number of Candida spp. in the gutand inhibit adherence of Candida organisms to the mucosaeby forming a bacterial barrier layer in the mucus gel (38).DC can be either acute or chronic. Acute DC can develop

in both the oncologic and nononcologic risk groups. Theinfection occurs abruptly, with fever, fungemia, and inva-sion of visceral organs from mucosal foci in the gastrointes-tinal tract. Less frequently, skin lesions occur, and rarely,shock is present. High-grade candiduria in patients who donot have an indwelling urinary tract catheter indicates he-matogenous spread of Candida organisms to the kidney (18).Prompt antifungal therapy is essential for acute DC; in one

study, no patient with a hematologic malignancy who devel-oped Candida fungemia survived without therapy (41).

Chronic DC, or hepatosplenic candidiasis, occurs mostoften in leukemia patients at a point in their therapy whentheir granulocyte count returns toward normal. Persistentfever results from lesions that progress slowly over weeks or

months in the liver, spleen, kidney, and lungs (36, 91).Hepatosplenic lesions can be imaged by computed tomogra-phy, ultrasonography, and magnetic resonance imaging (91).Although initiation of antifungal therapy may not be asurgent as for acute DC, the chronic form is difficult to treat;46% or more of such patients studied did not respond toamphotericin B treatment (36, 91).

SIGNIFICANCE OF CANDIDEMUI

Nosocomial candidemia, reported through the NationalNosocomial Infection Surveillance System during the period1986 to 1989, accounted for 8% of all hospital-acquiredbloodstream infections and was the fourth most commoncause of septicemia (77). In the setting of serious immunesystem compromise, mortality rates among fungemia pa-tients have been estimated at between 50 and 80%, even

after treatment (2). In another study of nosocomial candi-demia, 88 (57%) of 154 patients with fungemia died, and ofthat number, 46% died within 1 week of the onset offungemia (98). For a high proportion (74%) of candidemicpatients on whom an autopsy was performed, candidiasiswas found to be the cause of death. Meunier et al. (54) foundthat 7 (41%) of 17 cancer patients from whom only one bloodculture was positive for Candida spp. died within 1 monthafter the fungemia occurred. Isolation of Candida speciesfrom the bloodstream of a patient with persistent, profoundgranulocytopenia (<100 polymorphonuclear leukocytes per

,ul for >7 days) is usually indicative of disseminated infec-tion (1).

Primary Source for DC

Transient fungemia may result from colonization of cen-

tral venous catheters by Candida species. Improved cathetermaintenance, i.e., frequent changing of solutions and atten-

tion to sterile technique, may aid in preventing suchfungemia. In a patient with a central venous catheter,candidemia may arise from contamination of the catheterfrom an outside (skin) source or from extrinsically andintrinsically contaminated intravenous fluids. Also, the cath-eter may function as a foreign body and be seeded via thebloodstream from a remote source of infection in the patient.Candidemia resulting from contamination of the catheterfrom the skin of the patient or a health care worker is oftenindistinguishable from fungemia arising from gastrointestinalcolonization. When 172 episodes of candidemia among 169hospitalized patients were studied, not stratified according tothe degree of immunosuppression, a contaminated intravas-cular device was the source of infection in 67 episodes(40%), the source was extravascular in 73 episodes (43%),two patients had endocarditis, and the source could not beidentified in 30 episodes (17%) (90). The importance ofremoving the central venous catheter from a patient withcandidemia was shown by a study in which antifungaltreatment alone, without removal of central venous cathe-ters, resulted in either death or recurrence of fungemia orfever in 9 (82%) of 11 granulocytopenic cancer patients (46).However, for severely granulocytopenic patients, the pri-mary concern is always restoration of the normal number ofcirculating leukocytes, and until that occurs, the risk of DCremains high even when central venous catheters are re-moved (1).The gastrointestinal tract is the most likely primary source

of infection in the granulocytopenic cancer patient. Cyto-toxic therapy causes ulcerations along the gastrointestinaltract. Antibiotics that suppress gram-negative anaerobespromote a resistant microflora that includes Candida spe-cies. Candida organisms may pass through intact mucosaeby persorption through the intestinal wall (42) or by pene-trative growth, perhaps aided by secreted pathogenic factorssuch as aspartyl proteinase (11) and phospholipase (4).Disruption of the integrity of the gastrointestinal tract, withresulting fungemia, may also occur in surgical patients (1).These factors set the stage for DC.

Frequency of Positive Blood Cultures

Routine blood cultures are relatively insensitive and maytake several days to become positive, but advances in bloodculture methods have improved the detection of fungemia.One promising commercial method is the lysis-centrifugationIsolator system, which releases fungi from leukocytes, pro-ducing a sediment that is plated onto five different agarmedia (89). The Isolator has an advantage over other meth-ods in reducing the time between inoculation and detectionof growth (32, 89). Among a well-defined population ofhigh-risk patients, 73% of those with histologically provencases of DC had positive blood cultures with the Isolatorsystem, and the mean time to recovery of Candida spp. was2.1 days (89).Depending on the series of patients, the methods used to

isolate and culture Candida spp., and the frequency ofsampling, between 25 and 82% of blood cultures fromleukemia patients with DC have been positive, as reviewedby Jones (32). Chronic DC patients with hepatospleniclesions have a low frequency of positive blood cultures.Thaler et al. (91) found only five patients with positive bloodcultures among 60 cases of hepatosplenic candidiasisgleaned from the world literature. Of the eight hepatospleniccandidiasis patients treated at the National Cancer Institute

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NONCULTURE METHODS FOR CANDIDIASIS DIAGNOSIS 313

between 1982 and 1985 (91), blood cultures were positive inonly two cases (25%).

Receptor-Ligand Clearance of C. albicans fromthe Bloodstream

C. albicans contains surface adhesins as well as ligandsthat interact with host receptors. These receptor-ligandinteractions probably contribute to the clearance of Candidaspp. from blood by entrapment in the sinusoidal endotheliumof the liver. Among these receptors is a C. albicans surfaceprotein that displays lectin-like binding to fucosyl residueson mammalian cells (10). Another mechanism of adherenceis thought to operate via binding of the mannose receptorprotein on Kupffer cells to the mannan moiety of C. albicans(85). Evidence is accumulating that C. albicans containsintegrin-like proteins in the outer cell wall that bind toArg-Gly-Asp (RGD) sequences on fibronectin, laminin, fi-brinogen, and human endothelium (76). Sawyer et al. (76)proposed that RGD sequences in soluble fibronectin couldfunction as opsonic bridges between C. albicans and Kupffercells, aiding in the clearance of Candida organisms from theblood.

Candida Fungemia: Primary Disease andPredisposing Factors

Since therapy may differ for different Candida species, animportant condition for those designing specific laboratorydiagnostic tests is the need to detect many different potentialfungal pathogens. Central venous catheters for hyperalimen-tation therapy have been associated with fungemia causedby, in descending order of relative frequency, C. albicans,C. tropicalis, C. parapsilosis, and C. glabrata (29, 41). Thecause of fungemia following abdominal surgery was C.albicans, C. tropicalis, or C. glabrata, also in descendingorder (29, 67). C. albicans was found more frequently inopen heart surgery-related fungemias than C. tropicalis (95).C. parapsilosis occurred more often in late-onset recurrentendocarditis (31).The attack rate for Candida fungemia in leukemia patients

at one cancer center over a 4-year period was 11.5% (41).Candida species isolated from the bloodstream of leukemiapatients include C. tropicalis and C. albicans (29), but withthe advent of prophylactic fluconazole therapy for autolo-gous marrow transplant recipients, there has been a shiftaway from susceptible C. albicans towards the more resis-tant C. krusei, C. glabrata, and C. parapsilosis as the causeof infection (100). Fungemia in patients with solid tumors isdue most frequently to C. albicans and C. tropicalis, but C.glabrata is also notably present (29).The proportion of Candida species causing fungemia in

one cancer center was: C. albicans, 51%; C. tropicalis, 25%;C. parapsilosis, 12%; C. glabrata, 9%; and other species,3% (41). The same relative prevalence of these species hasbeen observed in other cancer centers (29). In their series ofcancer patients, Meunier et al. (54) observed fewer C.tropicalis blood isolates and relatively more C. glabrataisolates, which may reflect a higher proportion of patientswith solid tumors than leukemia. Similarly, of yeasts cul-tured from blood over a recent 7-year period at the MayoClinic, the proportion of C. albicans and C. glabratafungemia was 55 and 12%, respectively (89). C. glabratafungemia is associated with a high mortality rate. In one

large series of 135 cases of fungemia, including 12 patientswith C. glabrata fungemia, the mortality rate from this

TABLE 1. Chronology of events in the detection of D-arabinitolin the serum of patients with DC

Observation Yr Reference

Serum arabinitol detection by GLC 1979 40Arabinitol production by C. albicans, 1981 5

C. tropicalis, other Candida spp.Ara/cr ratio compensates for renal 1982 101

dysfunctionC. albicans arabinitol is the 1985 6D-enantiomer

D-Arabinitol DH used in fluorometric 1985 82measurement of serum D-arabinitol

D-Arabinitol DH purified from K 1985 102pneumoniae to homogeneity

D/L-Arabinitol ratios computed by 1992 71GLC-negative chemical ionizationmass spectrometry

Enzymatic-fluorometric assay kit for 1992 21, 92serum D-arabinitol evaluated

species was 83% (41). C. parapsilosis is recognized for itsassociation with invasive vascular devices, including bloodpressure transducers and cutaneous catheters for adminis-tration of parenteral nutrition (96). C. krusei is notableamong the less frequent but emerging species for its relativeresistance to fluconazole. Colonization with C. krusei wasfound in 40% of leukemia patients receiving bone marrowtransplants and prophylactic fluconazole (100).

Summary

Clearly, the challenge for innovative laboratory methodsis to provide evidence of infection earlier than is possiblewith blood cultures, or to complement blood culture resultsby giving a positive result when blood cultures are negative.Early initiation of antifungal drug therapy is critical toreduce mortality in DC patients (1, 29, 41). The changingspectrum of clinically significant Candida species should beconsidered when designing alternatives to blood culture.

MEASUREMENT OF SERUM D-ARABIN1TOL, ACANDIDA SPECIES METABOLITE

Arabinitol was first discovered in the serum of DC patientsduring a test for the presence of serum mannose (40). Themethods for detecting serum D-arabinitol have been modifiedto correct for renal dysfunction, to differentiate host frommicrobial arabinitol, and to find a simpler, more rapid, andmore accessible clinical method than gas-liquid chromatog-raphy (GLC) (Table 1). Yeast pathogens producing arabini-tol include C. albicans, C. tropicalis, C. parapsilosis, and C.pseudotropicalis (C. kefyr), but not C krusei, C. glabrata, orCryptococcus neoformans (5). Natural host serum arabinitolaccumulates during renal insufficiency, which can be com-pensated for by determining the arabinitol/creatinine (ara/cr)ratio (101). C. albicans produces the D-enantiomer of arab-initol. It has proven possible, using GLC-negative chemicalionization mass spectrometry, to compute serum D/L-arab-initol ratios (71). Arabinitol also accumulates in urine duringDC, and almost half of the arabinitol in normal human urineis the D-enantiomer (6).

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Enzymatic-Fluorometric Determination ofSerum D-Arabinitol

Enzymatic determination of serum D-arabinitol is a rapid,quantitative test. Klebsiella pneumoniae D-arabinitol dehy-drogenase (DH) oxidizes serum D-arabinitol. In the enzy-matic-fluorometric assay alternative to GLC (82, 83), therate of NAD reduction catalyzed by D-arabinitol DH isdetected by fluorometry. The increase in fluorescence withreazurin as the fluorochrome is measured, and the D-arab-initol concentration is interpolated from a standard curve ofD-arabinitol standards in serum (82, 83). The utility of thetest may be limited because host serum D-mannitol is alsoattacked by D-arabinitol DH, but at a slower rate. Both hostD-mannitol and host D,L-arabinitol may be elevated duringrenal failure. Slightly higher values were obtained for theenzymatic determination than for the GLC determination,but the differences were not significant (84). D-Arabinitol DHwas previously prepared from sonic extracts of K. pneumo-niae by a classical method of differential salt precipitation,which resulted in impure reagents that could potentiallycompromise test reliability. Recently, anion-exchange andgel permeation chromatography were used to produce D-ar-abinitol DH that is homogeneous, as determined by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (102). The purified enzyme still oxidized D-mannitolbut at two-thirds the rate obtained with D-arabinitol.

Clinical Evaluation of Serum D-Arabinitol

Wong and colleagues (101-103) reported the ara/cr ratio asmicrograms of arabinitol per microgram of creatinine x 10,whereas Soyama and Ono (82, 83) and others (21, 92) usedthe ratio micromoles of arabinitol per milligram of creatine.The average D-ara/cr concentration in healthy adults, ac-cording to Soyama and Ono (82), was 0.6 (range, 0 to 1.7),umol/mg. Hospitalized cancer patients without evidence ofcandidiasis have a higher mean ara/cr ratio (0.069 ,g/,ug x10) than normal controls, reported as 0.024 (102) and 0.041(26). About one-third of infected patients have serum ara/crratios within 2 standard deviations of the mean value fornoninfected controls (26). Although human serum and urinecontain both D- and L-arabinitol, the elevated arabinitol inDC is considered to be the D-enantiomer because thatenantiomer is produced by C. albicans (6). Elevated D-ara-binitol in hospitalized patients receiving broad-spectrumantibiotics may also result from dietary D-arabinitol that isnot metabolized because of the suppression of normallyinactivating intestinal microflora (103).An alternative to measuring the ara/cr ratio was intro-

duced by Roboz and Katz (71), who used GLC-negativechemical ionization mass spectrometry to compute the D/L-arabinitol ratio. The mean serum D/L-arabinitol ratio for 29healthy control subjects was 1.76 + 0.47 (range, 0.77 to2.75). When the D/L-arabinitol ratio was 3 standard devia-tions greater than the mean of the normal ratio (that is,exceeded 3.18), the sensitivity for a positive test among 16patients with proven DC was 94% (D/L-arabinitol ratio range,3.2 to 50.1). Renal insufficiency did not appear to interferewith the diagnostic specificity of the D/L-arabinitol ratios.Soyama and Ono (82) reported two cases of autopsy-

confirmed DC in which the ara/cr ratios were 3.3 and 42.7,mol/mg (range in normal controls, 0 to 1.7 pmol/mg). Fujitaet al. (22) described a patient with C. albicans endocarditiswho tested positive over a 9-week period, with serum ara/crvalues peaking at 9.5 ,umol/mg. The range of serum D-ara/cr

ratios for seven patients with DC was between 4 and 7,umol/mg (84). Wong and Brauer (102) coupled the D-arab-initol DH reaction with capillary GLC to determine the meanara/cr ratio for 27 normal adults and 4 patients who wereknown from previous analyses to have had either fungemiaor invasive candidiasis and elevated serum arabinitol levels.The mean ara/cr ratio for six positive serum samples fromfour patients was 0.86 ,ug/,ug x 10; range, 0.11 to 2.4), andthe ara/cr ratio for healthy controls was 0.024.

Kits for the enzymatic-fluorometric determination of se-rum D-arabinitol are produced by the Nacalai Tesque Co.,Ltd., Kyoto, Japan, and have undergone preliminary evalu-ations. The first such study (92) was conducted with serumfrom febrile and afebrile patients with candiduria. The meanara/cr ratio in febrile candiduria patients (2.9 pLmol/mg) wassignificantly higher than in other patients who had candiduriabut were afebrile (0.8 ,umol/mg) or in control patients with-out candiduria (0.6 ,mol/mg). Moreover, the correlationcoefficient between the GLC and enzymatic fluorometricdeterminations (r = 0.943 [92]) was similar to that deter-mined earlier in a comparison of these two methods (84). Amore comprehensive analysis of the enzymatic-fluorometrickit method for serum arabinitol measurement drew upon aseries of 16 patients with histologic evidence of DC and 42patients with two or more positive blood cultures obtainedduring the same week (21). An ara/cr ratio 3 standarddeviations above the mean for controls (1.5 ,mol/mg) wasconsidered a positive result. By this criterion, 29 patientswith DC had positive tests. The sensitivity and specificitywere 50 and 91%, respectively. Fujita and Hashimoto (21)also observed elevated serum ara/cr ratios in 10% of high-risk control patients with cancer and/or bacterial sepsis butwithout evidence of candidiasis. This elevation was ob-served previously by Wong and Brauer (102). Fujita andHashimoto (21) attributed this rise to gastrointestinal colo-nization with Candida spp. or possibly to interference frommannitol with the enzymatic-fluorometric assay.

SummarySignificantly elevated serum D-ara/cr ratios are found for

approximately two-thirds of DC patients. Further prospec-tive evaluations are needed to decide the exact value of thistest and whether its application may lead to improved patientoutcome. The availability of a kit to detect D-arabinitol by anenzymatic-fluorometric method will facilitate longitudinalmonitoring of patients at risk for developing DC. Potentialinterference with D-arabinitol measurements by D-mannitolis amenable to further study and may be eliminated withadditional modifications.

AMPLIFICATION OF DNA FROM C. ALBICANS INBLOOD AND URINE

Depending upon the blood culture system used, the fre-quency of sampling, and the patient population under study,blood cultures can fail to detect as many as 75% of DC cases(32). Other shortcomings of blood culture techniques includethe variable 2- to 5-day interval necessary for detectinggrowth of Candida spp. (32) and the evidence that withouttreatment, immunosuppressed patients may die within 7days of the first positive blood culture (98). DNA probes,especially with the advent of the polymerase chain reaction(PCR), will likely facilitate earlier diagnosis. The scarceliterature to date describing DNA detection-based diagnosisof DC indicates a belief that detection of Candida spp. in



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blood may be limited by the rapid clearance of organismsfrom the venous circulation or that the technical difficultiesencountered when PCR is applied to fungal pathogens retardits application to clinical materials, especially blood. A smallnumber of reports have appeared that predict that PCRamplification methods for Candida spp. in clinical materialsare feasible, which should stimulate efforts to develop aclinically useful test.

Application of PCR to Clinical Specimens

One study of six surgical patients showed that C. albicansDNA was amplified by PCR from clinical specimens, includ-ing urine, blood, and sputum (9). PCR primers were derivedfrom the single-copy gene target sequence encoding cyto-chrome P-450 A14 a-lanosterol demethylase. This enzyme isin the pathway for ergosterol biosynthesis by Candidaspecies and is absent in bacteria, making the gene a selectivetarget.A multistep procedure for isolating Candida DNA was

described previously (9) and consisted of lysing leukocytesand erythrocytes with detergent, digesting human and bac-terial DNA with DNase, lysing Candida cell walls withzymolyase in the presence of reducing and chelating agents,solubilizing membranes with detergent, and digesting pro-teins with proteinase K. After phenol-chloroform extractionand ethanol precipitation, the DNA was sufficiently pure forTaq polymerase-generated PCR. PCR products were thendetected by ethidium bromide staining after agarose gelelectrophoresis. Quantitative recovery was estimated byseeding blood with known numbers of C. albicans blasto-conidia prior to DNA extraction procedures and PCR am-plification. The detection limit was 120 CFU/ml. A 244-bpamplicon was generated from the blood of three surgicalpatients; two of the three patients had blood cultures posi-tive for a yeast strain. No further information was providedabout the extent of candidiasis in these patients. PCRproducts derived from using the same primer pair sequencesgave amplicons of different molecular masses for one isolateeach of C. krusei, C. glabrata, and C. pseudotropicalis (C.kefyr). This heterogeneity of PCR products indicates that themethods used in this study could provide a tool for identify-ing different Candida species, a finding which would behelpful in guiding therapy. However, since as many as 50%of DC cases have fewer than 100 recoverable CFU/ml, thesensitivity of this method would need to be improved inorder to maximize its clinical utility.The approach taken by Miyakawa et al. (55) is potentially

more powerful because it combines a multicopy DNA targetsequence with Southern blotting to improve detection of theamplicon. The target sequence for PCR was a 2-kbp mito-chondrial DNA fragment, E03, cloned from C. albicans.These authors used a simpler method than that of Buchmanet al. (9) to extract Candida DNA. Spheroplast formationwas induced with zymolyase and 2-mercaptoethanol in sor-bitol-EDTA buffer, after which the sample was suspended inPCR buffer, Nonidet P-40 detergent, and proteinase K. Afterincubation, the sample was heated to inactivate the protein-ase K and then PCR amplified. Primers were 20-mers thatwere 1.8 kbp apart. The detection limit for the PCR productsafter agarose gel electrophoresis was 2 to 10 CFU/ml insaline and 100 CFU/ml in urine. Detection in urine wasenhanced to 10 CFU/ml by Southern blotting with cloned,32P-labeled E03 DNA as the probe. Southern blot hybrid-ization showed that the PCR product was specific for C.albicans and did not cross-react with seven other Candida

species (C. tropicalis, C. guilliernondii, C. krusei, C. parap-silosis, C. glabrata, C. lusitaniae, and C. kefyr). Thismethod has not been adapted to amplify DNA from Candidaspp. in blood, nor has it as yet been applied to clinicalspecimens.

Further simplification of sample preparation and adapta-tion of universal fungal primers to the detection of fungi inclinical specimens, but not in blood, was achieved by Hopferet al. (28). The clinical specimens (urine and tracheal aspi-rate) and controls (fungal saline suspensions) were diluted10-fold in Tris-buffered Triton X-100 detergent, boiled for 15min, and then shaken for 30 min with glass beads. The fungaloligonucleotide primers NS-4 and NS-5, spanning a 310-bpregion in the 18s rRNA gene complex, were used for PCRamplification. Ethidium bromide-stained agarose gel electro-phoresis permitted the detection of 10 to 100 CFU/ml.Candiduria was detected in three patients, and a trachealaspirate that was culture positive for C. lipolytica was alsopositive by PCR. Theoretically, all fungal DNA should beamplified by the primers used in this study. Therefore,HaeIII restriction enzyme digestion of the amplicons wasused to differentiate PCR products into five broad taxonomicgroups: Candida spp. and related yeasts; Cryptococcus andTnchosporon spp.; Aspergillus spp. and other septate fungalpathogens; zygomycetes; and systemic dimorphic fungalpathogens.The attractive features of this approach (28) are the broad

specificity and the possibility that genus- and species-spe-cific probes could be used to replace the need for identifica-tion via restriction enzyme digestion (62). In other respects,the sensitivity obtainable with clinical specimens was notdetermined, and the detection method was comparable toprevious efforts. It is doubtful that the method used toprepare the urine and tracheal aspirate samples (i.e., boilingand then shaking with glass beads) will be successful withblood specimens (personal observations).The actin gene of C albicans was successfully amplified

by PCR in serum samples from infected mice and from six ofeight human patients with candidemia but not in sera fromhealthy controls (35). The amplicon was detected by hybrid-ization to a 32P-labeled oligonucleotide probe. Blood cul-tures from the infected mice yielded 5 to 120 CFU/ml. ThePCR could detect as little as 50 fg of C. albicans genomicDNA spiked into serum. Whether naked DNA can remainundigested or uncleared from serum for a sufficient intervalto allow detection in clinical specimens needs further explo-ration.

In a preliminary study, Jordan (33) used the buffy coatfraction of whole blood from patients and controls. DNAwas released by zymolyase, and after phenol extraction andethanol precipitation, DNA encoding the chitin synthetasegene was amplified by PCR. The amplicons differed in sizeon ethidium bromide-stained agarose gels according to theCandida sp. tested. Chemiluminescent probes specific for C.albicans, C. glabrata, C. parapsilosis, and C tropicaliswere used in Southern blots to identify amplicons to thespecies level. When used for 15 patients with culture-provenDC, PCR and blood culture methods gave identical resultsfor 24 of 25 blood samples.A nested PCR with primers derived from the 18s rRNA

gene subunit of C. albicans was able to detect C. albicansDNA in blood samples from eight patients either on the sameday or 1 to 3 days after the blood cultures became positive(66). The 417-bp amplicon was detected by ethidium bromidestaining after agarose gel electrophoresis. The primers se-lected also amplified a 430- to 440-bp region in DNA from

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TABLE 2. Multicopy DNA sequences in C. albicans

Sie Copies/!efrNomenclature (Skibp) haploid Specificity Refer-genome

CARE-1 >0.47 2-12 C. albicans 45CARE-2 1.06 12-14 C. albicans 44Ca7a 1.1 30 C. albicans 75Ca3b 11 15-25 C. albicans + 75

C. tropicalis27ab 15 10 C. albicans 78MspI fragmentc 2.9 NDd C. albicans + 12

C. stellatoideaE03 mtDNAC 2 30-40 C. albicans 55mtDNA 41 30-40 C. albicans 99rDNAe 15 50-130 All Candida spp. 30

a Sixteen copies of a 23-bp telomeric repeat.b May be related mid-repetitive elements.c May be related mitochondrial DNA (mtDNA) sequences.d ND, not determined.e Gene cluster of 15 kbp repeat units on chromosome 2.

human leukocytes, tempering enthusiasm for this set ofconditions.

Prospects for PCR Detection of Candida spp. in Blood

Further evaluation of the optimal conditions for extractionof DNA from small numbers of Candida spp. in the blood isnecessary. Quantitative yeast cultures obtained from blooddrawn through indwelling catheters help to estimate thesensitivity that is likely to be needed (39). Eighteen percentof blood samples that grew yeast contained < 1 CFU/ml. Anadditional 35% contained between 1 and 100 CFU/ml. Thus,a sensitivity range of 1 to 100 CFU/ml is a minimum goal toduplicate the diagnostic ability of conventional blood cul-tures. Because blood cultures fail to detect about 25% of DCcases even with the best available method (lysis centrifuga-tion) (89), it is reasonable to expect the PCR method todetect 1 to 10 CFU/ml of blood if it is to be a significantimprovement. The extraction and purification proceduresneed to be simplified and made less labor-intensive. Whetherthe goals of increased sensitivity of detection and simplifi-cation are achievable remains to be demonstrated. PCRmethods for detecting Candida spp. in blood samples with asensitivity that exceeds that of conventional blood cultureswill likely require both multicopy gene target sequences andimproved sensitivity of detecting PCR products.Multicopy gene target sequences are more likely to suc-

cessfully detect small numbers of Candida spp. in the bloodthan are single-copy sequences. Several candidates are listedin Table 2, including four families of C. albicans mid-repetitive elements: CARE-1, CARE-2, Ca3, and Ca7. Themitochondrial DNA target sequence E03 was discussedabove. These sequences have the shortcoming of being C.albicans specific and consequently not able to detectfungemia resulting from other Candida species. Primersderived from conserved DNA sequences flanking the inter-nal transcribed spacer regions between the 5.8s and 18s or5.8s and 28s rRNA genes will amplify all fungal DNAs, butthe amplicon has the potential to be species specific. Zakroffet al. (104) have demonstrated that the internal transcribedspacer region can be used to amplify DNA from C. albicansartificially introduced into blood and that the labeled PCRproduct from this region can be used as a species-specificprobe. This possibility is attractive in providing presumptive

evidence of fungemia, with further identification to specieslevel facilitated either by nested PCR or by use of species-specific probes. Bowman (8) summarized the factors affect-ing PCR amplification of the 18s rRNA gene subunit forsystemic fungal pathogens, including stringent washing anddetection of the amplicon by dot blotting.Dot blots, or microtitration plate capture assays, are

attractive alternatives to ethidium bromide-stained agarosegels and should provide rapid presumptive results and fur-ther increase sensitivity (63). The possibility that the ampl-icons may vary in molecular size according to Candidaspecies (9) justifies the use of agarose gel electrophoresis andSouthern blotting for confirmation. Nonradioactive probeslabeled, for example, with digoxigenin will be more accept-able for clinical use than 32P-labeled probes. Intrinsic label-ing of the amplicons with biotinylated primers affords theprospect of further simplification.

SummaryPCR amplification of small numbers of Candida organisms

in the blood or other clinical specimens is a promisingapproach to overcome the limits of currently availableculture methods. Other obstacles will have to be surmountedbefore mycology laboratories can fully use PCR for diagno-sis (9, 63). The steps required to isolate target DNA prepa-ratory to PCR are too cumbersome and time-consuming.Further experimentation is needed to find the best way toconcentrate Candida cells from blood samples and to re-move interfering host substances. The possibility exists thatnaked fungal DNA persists in serum from some patients,reducing the amount of sample preparation. Finally, theoptimal conditions for the release of Candida DNA in a formsuitable to act as a template for Taq polymerase have yet tobe determined.

Multicopy gene targets are more likely to detect limitingnumbers of C. albicans than single-copy DNA sequences,and these should be compared to select the target thatprovides the greatest sensitivity. Intrinsic labeling of PCRproducts with biotinylated primers or labeling of the probewith biotin or other nonradioactive ligands will spare therequirement for radioactive detection. Coverage to detect allmedically important Candida species has been achieved, butspecies-specific probes are needed. Agarose gel electro-phoresis of PCR products is a cumbersome prototype thatshould be replaced by rapid, presumptive dot blot or microti-tration capture assay methods.

IMMUNOASSAY DETECTION OF MARKER ANTIGENS

The search for methods to detect traces of soluble prod-ucts of Candida species in the body fluids of hosts who lacka functioning immune system has led to various approachesto detect antigenemia and antigenuria. These tests have beenthe subject of recent reviews (14, 15, 32). The ability of evenseverely immunosuppressed patients to produce humoralimmune responses complicates the detection of circulatingCandida antigens because the formation of immune com-plexes will mask antigenic sites and blunt test sensitivity.Provisions for dissociating immune complexes or circum-venting them by detecting antigenuria rather than antigene-mia are designed into some of the immunoassays describedbelow. The status of efforts to evaluate four different markerantigens is discussed. Further evaluations are needed todetermine which, if any, of these tests will become availableto clinical laboratories and which will remain research tools



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or be available only in select reference laboratories. Most ofthe marker antigens discussed below are well-characterizedproteins or cell wall mannoprotein (CWMP). One antigene-mia test for candidiasis that detects a 56°C-labile antigen byreverse passive latex agglutination (RPLA) is the Cand-Tectest, marketed by Ramco Laboratories, Houston, Tex. (25).The Cand-Tec test has been the subject of several evalua-tions. The identity of the antigen being measured is un-

known, and the test has not been modified since previousreviews discussed its clinical utility in detail (15, 32).

Enolase (EC 4.2.1.11)

In a study by Matthews et al. (51), Western blot (immu-noblot) analysis with homogenate-supernatants of C. albi-cans revealed a multiplicity of antigens that reacted withsera from patients with DC, including an immunodominant47-kDa antigen. Independently, Strockbine et al. (88) pas-

saged C. albicans cytoplasmic solubles through concanava-

lin A-agarose to remove CWMP and other mannoproteinsand isolated a 48-kDa protein antigen. The 48-kDa proteindid not bind to concanavalin A and was purified by anion-exchange chromatography. Patients with DC had circulatingantibodies that immunoprecipitated the radioiodinated 48-kDa antigen. Monoclonal antibodies (MAbs) were preparedagainst this antigen and adapted to an enzyme immunoassay(EIA) to detect the 48-kDa protein in experimental murinecandidiasis (87, 93).

Enolase activity in the 48-kDa antigen. Enolase, an enzyme

of the oxidoreduction-phosphorylation stage of the glyco-lytic pathway, catalyzes the reversible dehydration of2-phospho-D-glycerate to high-energy phosphoenolpyru-vate. The Saccharomyces cerevisiae enzyme consists of a

dimer of two similar 40- to 50-kDa polypeptides. Polyclonalantibodies produced against purified S. cerevisiae enolaseprecipitated a C. albicans 48-kDa protein and reduced theenolase activity of the cytoplasmic supernatant (50). MAbsagainst the C. albicans antigen cross-reacted in Western blotanalysis with a -48-kDa protein in the S. cerevisiae homoge-nate. A 48-kDa protein was also translated in vitro from Calbicans mRNA in a rabbit reticulocyte expression system.The protein was then immunoprecipitated by anti-S. cerevi-siae enolase immunoglobulin G (IgG). Thus, there is evi-dence of immunologic cross-reactivity between enolase anda 48-kDa antigen in the serum of patients with DC.

Liposomal rhodamine immunoassay for serum C. albicansenolase. Qualitative detection of serum enolase was facili-tated by the development of a sandwich-type assay. Amurine IgA MAb was adsorbed to a nitrocellulose mem-

brane, and serum suspected of containing Candida enolasewas passaged through the membrane and captured. Poly-clonal rabbit anti-C. albicans enolase was applied and de-tected with a liposome containing rhodamine dye and coatedwith goat anti-rabbit IgG (94).

Patients from four oncology centers were screened pro-spectively over a 2-year period with a prototype liposomalimmunoassay from Becton Dickinson Co. (Philadelphia, Pa.)for detection of C. albicans enolase, developed in collabo-ration with Walsh and colleagues (94). Cancer patientsenrolled in the study had some or all of the following riskfactors for DC: cytotoxic therapy, long-term antibiotic ther-apy, central venous catheters, and persistent granulocytope-nia. A total of 24 patients with invasive candidiasis were

further classified with respect to whether only candidemiawas present or whether the diagnosis of DC was confirmedhistologically. Of 170 patients enrolled, 24 had invasive

TABLE 3. Enolase antigenemia in cancer patients with andwithout DC'

No. of patients-_Senitiity Positive

Group (n) Serum Serum ( predictiveenolase enolase value (%)positive negative

With DCTissue proven (13) 11 2 85 65Blood culture proven (11) 7 4 64 54Total (24) 18 6 75 75

No DC (146) 6 140

Total (170) 24 146

a Specificity for all patients was 96%. Negative predictive value was 96 to99%. Abstracted with permission from information appearing in reference 94.

candidiasis (Table 3). A total of 13 had tissue-proven DC,and 11 had Candida fungemia.When DC was confirmed by both positive blood culture

and deep-tissue biopsy, the sensitivity of the enolase detec-tion test was 75%, the positive predictive value was 75%,and the specificity was 96%. Multiple sampling of patientswas important because when sampling was done only once,test sensitivity was reduced to 54%. Positive blood culturescomplemented serum enolase detection, as shown for bothblood culture-positive and histology-positive groups. Ten of11 patients who were serum enolase positive had hepaticinvolvement. Also, for 5 of the 11 enolase-positive patients,the diagnosis was confirmed by histology alone. All 13 of thehistologically diagnosed DC patients had multiple negativeblood cultures: only 18 (13%) of 135 samples taken werepositive. The sensitivity of serum enolase detection in these13 histologically diagnosed cases, including all determina-tions, was 52%; 63 of 122 samples were positive. In compar-ison, 7 of 11 patients for whom the diagnosis was made onthe basis of fungemia were found to be serum enolasepositive. Among these 11 blood culture-diagnosed DC cases,cultures were positive for 36 of 46 samples (78%), whereasenolase was detected in 17 of 27 samples (63%).Summary. Enolase is produced by all Candida species,

and it is assumed that the capture MAb used in this test bindsenolases other than that of C. albicans. Direct proof of thisassumption is needed. Serum enolase is present in manycancer patients with DC. Multiple serum samples are neces-sary to maximize detection. The serum enolase immunoas-say complemented rather than replaced blood cultures.Approximately half of the cases were diagnosed by bothblood cultures and serum enolase determinations, and theother half were recognized by either positive serum enolasetests or positive blood cultures. Histologic proof was themajor diagnostic criterion in the blood culture-negativegroup. Serum enolase appears to be a marker of deep-tissueinvasion even in the absence of detectable fungemia. Afebrile cancer patient who does not have fungemia but whohas radiographic evidence of lesions consistent with he-patosplenic candidiasis may be spared a biopsy if the diag-nosis can be made by the enolase immunoassay. Since thereis no provision for dissociating soluble immune complexescontaining enolase, such immune complexes may blunt thetest's sensitivity.

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Secreted Aspartyl Proteinase (EC 3.4.23.6)

The production of an extracellular proteinase by C. albi-cans was observed by Staib (86) in 1965. Since then, it hasbeen suggested to be an important candidate virulence factorin candidiasis. C. albicans and C. tropicalis, which produceproteinases, are also the most pathogenic Candida species(7, 11, 43, 49, 73). Of the other less pathogenic Candidaspecies, C. parapsilosis secretes an antigenically distinctacid proteinase (13). C. albicans releases the aspartyl pro-teinase (AP) into the culture medium during growth on

proteinaceous substrates. The AP is active in the acidicmilieu (56), resulting from glucose metabolism, that sur-

rounds the blastoconidia.Role of AP in DC. A proteinase-deficient C. albicans

mutant had reduced mouse virulence and reverted to theproteinase-positive virulent form after mouse passage (43).The proteinases of C. albicans have been implicated as

causes of disseminated intravascular coagulation and vaso-

constriction (72). The enzyme was detected on the surface ofC. albicans colonizing a mouse kidney (49), and degradationof immunoglobulins, which occurs in the kidney duringinfection, may be the direct result of AP (73).

C. albicans AP also breaks down immunoglobulinspresent in saliva, including IgG, IgM, IgA2, and secretorycomponent (74). Proteinase has been localized by immuno-peroxidase on the surfaces of Candida blastoconidia phago-cytosed by murine peritoneal macrophages (7). As the blas-toconidia germinated at the lysosomal pH of 4.7 to 4.8,proteinase was demonstrable as a sheath on the pseudohy-phae. Proteinase production may aid C. albicans in escapingfrom the phagolysosome. The potential for AP to act as a

marker of disease activity was suggested by Neely andHolder (61), who showed, in a "1I-fibrinogen digestionassay, that the total serum proteolytic activity of skin-burned mice (12% of body surface, nonlethal) was higher ina group infected intravenously with C. albicans than in a

noninfected control group.

Proteinase antigenuria ETA in models of colonization andDC. Purification of AP and removal of mannan can beaccomplished by column chromatography with sequentialanion-exchange, gel permeation, and linear gradient anion-exchange steps (56). Purified AP contains negligible CWMPand does not stain on SDS-PAGE gels with silver stainsmodified for carbohydrate detection. The resulting purifiedenzyme preparation consists of a doublet of proteins (48 and49 kDa) having an identical N-terminal amino acid sequence

and a 41-kDa pepstatin A-binding protein with a uniqueN-terminal sequence (58).

Proteinase antigenuria was detected with a competitive-binding EIA in an immunosuppressed rabbit model of DC(57). The polyclonal antibodies used in the immunoassaywere prepared by immunizing mice with purified AP andthen conjugating the induced antibodies to horseradish per-

oxidase. Dialyzed urine from infected rabbits was combinedwith the diluted antiproteinase-horseradish peroxidase con-

jugate and incubated. This mixture was then transferred to

proteinase-coated microtitration wells, incubated again, and

washed. The color that developed upon addition of the

chromogen was read in a kinetic ETA reader, and the percent

inhibition was calculated with respect to normal rabbit urine,

used as the negative control.Rabbits immunosuppressed with cyclophosphamide and

cortisone were infected intravenously with 10 C. albicansblastoconidia. After 24 h, urine from eight rabbits demon-

strated significant inhibition (15 + 7%) in the competitive-

binding EIA. Inhibition increased daily, to a peak of 46% at3 days postinfection, in proportion to disease severity.Antigenuria persisted until the fifth day, when survivingrabbits were euthanized. Control experiments were con-ducted with rabbits mucosally colonized with C. albicans orinfected intravenously with Aspergillus fumigatus, Crypto-coccus neoformans, C. tropicalis, C. parapsilosis, or Ckrusei. No significant inhibition was detected in the antige-nuria test when specimens from control rabbits were used.Summary. AP is expressed by invading C. albicans strains

in DC and may play a role in allowing penetration ofanatomic barriers and escape from the phagolysosome.Results with the rabbit model suggest that the available EIAdetects AP antigenuria within 24 h of intravenous infectionand that it can discriminate between gastrointestinal C.albicans colonization and DC. Tests with urine from rabbitsinfected with A. fumigatus, C. neoformans, or other Can-dida species were negative. Further characterization of theantigen being detected is warranted; however, these prelim-inary results suggest that this test is a promising method toevaluate in humans with DC.

Immunoblot Detection of Candida Antigenemia/Antigenuria

Urine samples from 7 of 10 patients with DC (two or morepositive blood cultures) were positive in Western blot anal-ysis (19). The antiserum used to detect the antigen in urinewas produced in rabbits by immunizing them with a mannan-depleted cytoplasmic supernatant fraction of C. albicans. Allpositive antigenuria samples were found to contain a major47-kDa antigen. Urine antigens of 37 and 20 kDa were alsodetected in three and two samples, respectively. All urinesamples were culture negative for growth of C. albicans.Among patients who responded to 5 days of therapy withamphotericin B, the antigen disappeared from the urine. Fora patient with systemic lupus erythematosus who developedC. albicans osteomyelitis and circulating immune com-plexes, a 47-kDa Candida antigen was detectable afterprecipitation of the complexes with polyethylene glycol andWestern blotting (60).Summary. There are few reports on the detection of

antigenemia/antigenuria in DC by Western blot analysis.However, enough pilot studies have demonstrated the fea-sibility of this approach to encourage further development ofthis method.

CWMP Antigenemia

Among the antigens that circulate during infection with C.albicans or A. fumigatus are surface carbohydrates of thefungal cell wall, a family of homo- and heteromannans:galactomannan of A. fumigatus and CWMP of C. albicans(15, 23, 68). They exist as immune complexes that mustundergo dissociation before antigen detection. These anti-gens are stable and resist boiling, proteinase treatment, andacidic pH. They are labile to periodate oxidation and bind toconcanavalin A. Serum antigens are found in concentrationsin the low nanogram-per-milliliter range. Immunoassays forgalactomannan and CWMP are specific for invasive aspergil-losis and DC, respectively. Recent structural analysis haselucidated the unique epitopes of C. albicans CWMP, asdiscussed below. In an immunocompromised host with DC,CWMP-antibody complexes circulate in the blood (man-nanemia). Following dissociation of immune complexes, theantigen can be detected by either radioimmunoassay (RIA),EIA, or RPLA.



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TABLE 4. Detection of mannanemia in DC

Test No. of DC Sensitivity of Primary disease or condition Referencepatients detection (%) (no. of patients)

Competitive-binding RIA 11 46 Unspecified primary diseases 97Indirect-inhibition EIA 7 100 Leukemia/lymphoma 79Indirect-inhibition EIA 10 70 Leukemia 53

RPLA 18 72 Cancer, AIDS, abdominal surgery 323 78 Leukemia 346 0 Leukemia (2), lupus (2), diabetes, emphysema 245 0 Leukemia; postsurgery 6519 53 Cancer (8), postsurgery (4), other (7) 2758 38 Assorted, primary diseases unspecified 21a

Sandwich EIA 15 53 Cancer (8), other (7) 4813 54 Leukemia 1610 90 Cancer 2016 31 Cancer (6), marrow transplant (3), other (7) 4719 84 Hematology-oncology (13), other (6) 5914 86 Cancer (3), postsurgery (2), other (9) 6458 74 Assorted, primary diseases unspecified 21a

a In this study, RPLA and sandwich EIA tests were compared in parallel with the same antibody reagents and clinical specimens.

Definition and general plan of CWMP. The readily solubleouter wall layer of C. albicans and related Candida speciesis a homopolymer of D-mannose containing 3 to 5% covalentprotein and 1 to 2% phosphate, the latter as phosphodiester-linked mannan. CWMP is the immunodominant surfaceantigen of serotypes A and B. Sometimes it is called phos-phomannoprotein or simply mannan (23, 68, 80). The poly-saccharide moiety is linked by 0- and N-glycosidic bonds tostructural protein. An estimated 90% of the mannan isa-linked, but 10% is 3-mannan organized into a distinctdomain.Mannan contains an inner core linked by chitobiosyl-

aspartamido bonds to protein and an outer chain region thatinvolves the antigenic oligomannoside epitopes. Fourepitopes are known: two are serotype specific (A and Bserotypes), and two are common to both serotypes. Themost recent proposed structure for the serotype A-specificepitope is that of an unbranched mannohexaose composed of(1-*2)mannosyl residues, with three residues from thenonreducing end in the ,-configuration, the remainder beinga-linked (81). An important epitope common to both sero-types consists entirely of (1--2)-o-oligomannosides, with adegree of polymerization of between 2 and 6, and linkedthrough phosphodiester bonds to the a-mannan domain ofthe outer chain (81). Removal of the ,B-phosphomannan bydilute acid hydrolysis greatly reduces the serologic activityof both serotypes.Which of these epitopes is present in serum mannan

during infection? Evidence obtained with MAbs implicatesthe serotype A-specific epitope, which is shared with C.tropicalis (69). Other epitopes certainly are present in thecirculation, because antibodies produced against C. albicansB can detect mannanemia (70). Germ tube-specific antibod-ies are known to occur, because antibodies against germtubes, absorbed with blastoconidia, can still stain the germtubes during morphogenesis (68).Development of immunoassays to detect serum mannan.

The immunoassays designed to detect serum mannan aresummarized in Table 4. Heat-stable, pronase-resistant, pe-riodate-sensitive, concanavalin A-binding antigen compati-ble with CWMP has been detected in the serum of infectedanimals and humans (reviewed in references 15 and 17).

Serum mannan detection methods depend on dissociatingsoluble immune complexes in clinical specimens. This dis-sociation can be accomplished by proteinase digestion,exposure to extremes of pH, and/or heating to boiling. Anearly development in the detection of serum mannan in DCpatients was a competitive-binding RIA based on the obser-vation that 'I-mannan would not precipitate in half-satu-rated ammonium sulfate solutions but that the complexescould be precipitated in the presence of reference antibodies(97). This RIA detected antigenemia in 5 of 11 DC patients.Indirect EIA-inhibition tests soon appeared as alternativesto the RIA (53, 79). Segal et al. (79) detected significantantigenemia in all seven cancer patients they studied whohad autopsy-proven DC. Meckstroth et al. (53) used a similarETA to detect mannanemia in 7 of 10 DC patients studied.However, these indirect inhibition tests were technicallycumbersome. The test conditions made it difficult to com-pare percent inhibition results with a standard curve ofmannan plotted in nanograms per milliliter, complicating testinterpretation and standardization.Sandwich ETA. Lew et al. (48) demonstrated the feasibility

of detecting serum mannan in DC patients by a double-antibody sandwich ETA. This simplified format permitteddirect conversion of absorbance units into nanograms ofmannan per milliliter of serum. The method for a double-antibody sandwich ETA performed in microtitration plateswas described by Kaufman and Reiss (37). Depending on theprimary disease of the patient, source of reagents, andfrequency of sampling, the sensitivity for detecting serummannan by sandwich EIA has ranged from 31 to 90% (Table4). When present, serum mannan usually occurs in concen-trations in the low nanogram-per-milliliter range (1 to 10ng/ml), which is near the limit of test sensitivity. Tests forserum mannan have maintained a high degree of specificity,possibly aided by exposing serum samples to vigorous heattreatment before testing. Occasionally, infected patients willdemonstrate persistently elevated concentrations of serummannan, but such cases seem to be rare (52).

In one study that reported the highest test sensitivity,cancer patients were screened weekly (20). All DC patientshad at least one positive serum mannan test, and concentra-tions ranged from 2 to 48 ng/ml. A recent modification to the

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sandwich EIA has been to add antibodies against C. krusei tothe anti-C. albicans antibodies that are the most often used(21). Among a series of patients, including some with histo-logically proven DC and some who had two or more positiveblood cultures in 1 week, serum mannan concentrationsranged from 0.2 to 4.8 ng/ml; the sensitivity was 74%, andthe specificity was 100%. Antibodies against C. krusei failedto detect C. albicans or C. tropicalis antigens in vitro andwere less sensitive in detecting mannanemia in patientsinfected with either of the latter two species. However, inthe one documented case of DC caused by C. krusei,mannanemia was detected only by the homologous-antibodyreagent. Furthermore, in six cases of invasive candidiasisfrom which C. glabrata was isolated, no mannanemia couldbe detected by antibodies against C. albicans or C. krusei,indicating limitations of the currently available pool ofspecies-specific antibodies.The use of EIA to detect mannanemia was evaluated in

another series of 17 patients with and without cancer whohad proven or probable DC (47). Typically, one or twoserum samples from each patient were available to test.When the results obtained with serum samples drawn within1 week of a suspected fungal infection were considered, 5 of16 patients (31%) had serum mannan concentrations of >2ng/ml. Three cases of infections with C. krusei, C. paratro-picalis (C. kefrr), and C. parapsilosis went undetected,underlining the need for a blend of capture antibodies ofbroader specificity. When polyclonal antibodies against C.albicans serotype A were screened for their ability to detectmannans of other Candida species, it was clear that speciesother than C. albicans were detected less effectively and thatC. krusei mannan was not detectable (59). A commercialmannan detection sandwich EIA, the ICON Candida Assayrecently introduced by Hybritech Inc., Torrey Pines, Calif.,is conducted on a filter membrane in a cartridge device. TheICON Candida EIA demonstrated a high sensitivity fordetecting mannanemia in DC (86%), but antigenemia pre-ceded diagnosis by culture or biopsy for only 5 of the 14 DCpatients (64).RPLA tests. RPLA tests to detect serum antigen have

great appeal because of their simplicity. The cumulativeexperience with that format in detecting serum mannanduring DC has been variable. Among one series of well-studied patients for whom DC was established by biopsy,autopsy, or persistent candidemia during granulocytopenia,serum mannan was detected (positive RPLA titer of >1:4) in13 of 18 (72%) patients (3). Similarly, with their RPLA test,Kahn and Jones (34) found that serum mannan was detectedin 78% of leukemia patients with DC. The RPLA test is nearits sensitivity limit because antibody-coated latex particlestend to autoagglutinate and antigenemia occurs in concen-trations in the low nanogram-per-milliliter range (34). Bothof these RPLA tests were conducted with antibodies raisedagainst heat-killed blastoconidia of C. albicans. The reportof Fujita and Hashimoto (21) is noteworthy in that thecapture antibodies were adapted to both an RPLA test andthe sandwich EIA and the results were compared for thesame population of patients with DC. Their RPLA testsensitivity was 38%, whereas sensitivity with the sandwichEIA reached 74%. In two other reports (24, 65), a commer-cially available polyclonal anti-C. albicans antibody-coatedlatex reagent was used. The authors obtained uniformlynegative results. Not surprisingly, antigen detection is onlyas good as the capture antibodies employed.

Recently, Sanofi-Diagnostics Pasteur (Marnes-La-Co-quette, France) introduced an RPLA test in which latex

particles were coated with an antimannan MAb. This RPLAtest detected serum mannan in 10 of 19 (52.6%) immunosup-pressed and nonimmunosuppressed patients with DC provenby culture of blood samples or samples from other normallysterile sites (27). The monoclonal RPLA test for serummannan was recommended for immunosuppressed patientswho demonstrated weak antimannan antibody responses.Given the spectrum of Candida species capable of causingDC, it is likely that more than a single epitope specificity willbe needed to give maximal coverage. Clearly, RPLA testswith either high-titered polyclonal antibodies or MAbs ap-pear promising, especially as simple, presumptive test pro-cedures.Summary. The factors to be considered in evaluating the

results of published studies for the detection of serummannan in DC were delineated by de Repentigny (15).Briefly, they include the frequency of sampling, which isrelated to the proximity of sampling to the onset of DC; thedefinition of DC; and the spectrum of underlying diseases,including the degree of immunosuppression, the serotype ofC. albicans or the Candida species involved, the specificityand titer of the capture antibodies, and the method used toconduct the immunoassay. Mannanemia occurs in approxi-mately 31 to 90% of DC patients, depending on the above-mentioned factors. The highest test sensitivities have beenobtained for immunosuppressed leukemia patients. Resultsfrom most studies show that mannan concentrations occur inthe low nanogram-per-milliliter range, necessitating frequentsampling, especially during granulocytopenia. The availabil-ity of commercial tests for mannan detection is a recentdevelopment. Of the two RPLA tests that have come tomarket, one appears promising (27). However, in a singleside-by-side comparison of noncommercial sandwich EIAand RPLA tests, the latter was significantly less sensitive(21).A body of literature has accumulated from various labo-

ratories world-wide, suggesting that a positive serum man-nan test correlates well with DC and that there are fewfalse-positive reactions. To improve the mannan sandwichEIA for the diagnosis of DC, the sensitivity of the test mustbe increased into the picogram-per-milliliter range and cap-ture antibodies should include those against Candida speciesother than C. albicans. Also noteworthy for purposes ofpreparing capture antibodies is that germ tubes and hyphalforms may selectively express CWMP. Now that the natureof the epitopes is known, this information should be appliedto tailor MAbs to these epitopes. The use of MAbs mayimprove the test's insensitivity and narrow species spec-trum.

SUMMARY AND CONCLUSIONS

Two of the nonculture approaches to the diagnosis of DC,enzymatic-fluorometric determination of serum D-arabinitoland detection of marker antigens in antigenemia (enolase andCWMP), have been commercialized and have shown prom-ise in limited clinical trials. These approaches are not newbut are the culmination of efforts made over 10 or moreyears. Clearly, further fine-tuning of both metabolite andantigen detection is needed to simplify the methods and toimprove their sensitivity and specificity so that they will bevaluable in guiding clinical treatment decisions. An alterna-tive approach, detection of DC by DNA amplification meth-ods such as PCR, is a special case of a compelling technol-ogy and one that is capable of standardization acrossmicrobial genera. The availability of simplified PCR diagnos-



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tic methods for DC remains a tantalizing prospect. Never-theless, the development of methods to release DNA fromvery small numbers of Candida organisms in the blood in aform that is sufficiently free of inhibitors of PCR will requirefurther intensive effort. The maturation of these converginglaboratory approaches to nonculture diagnosis of DC leadsto more optimism about the eventual use of these methods inclinical laboratories.

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8. Bowman, B. H. 1992. Designing a PCR/probe detection systemfor pathogenic fungi. Clin. Immunol. Newsl. 12:65-69.

9. Buchman, T. G., M. Rossier, W. G. Merz, and P. Charache.1990. Detection of surgical pathogens by in vitro DNA ampli-fication. I. Rapid identification of Candida albicans by in vitroamplification of a fungus-specific gene. Surgery 108:338-347.

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