Evaluation o f a c a n d i d a antigen d e t e c t i o n m e t h o d (Cand-Tec): Experience from a university t e a c h i n g hospital

. Evaluation of a candida antigen detection method (Cand-Tec): Experience from a

a I: 16 .Q ua n d un litre s u peri eur ou ega! a l :4 scrva it de seuil compa tib le a vec le fe rm e diagn osUc de candi dose.Ia sens ibili tc et s pecifl cite globales atteig na ie nt 7 7 % e t 6 9 % respecti vem en t. landis que Ia valeur de prevision d 'u n test posiUf etail d e 48 % e l cell e cl\111 test negalif.de 89 % .Quancl les cas de p roba bili te elevee cl'infeclion ont ete indus cla ns !"a na lyse avec un litre s eu il s uperi eur ou ega! a 1:4 .Ia sens ibili te et specifl c ite globa les Ctaient de 70 % et 69 % respeclivem ent.La nd is que le pourcenta ge d e p revi s ion d 'un tes t posilif a tteigna il 68% et cclui cl'un test n ega lif.7 1 %.L'uli li te clu test Cand -Tec ne s•es l a melioree pour a uc un d es so us -groupes d e pa li c nls (n i po ur ceux q ui receva ienl d es s oi ns in len s ifs n i en s ui vant d es Utres seri es chez des pa Uen ls ind ividu c ls). Da ns Ia presenle experi ence.Je lest Ca n d -Tec n•a pas foumi assez de d onn ees s upp le me nla ircs po ur pc rmc llrc cl'inclu re ou d 'exclu re un cl iagnosUc fo rme! de canclidose syslemiqu c cl a ns cetle popu la tion ge ne ra te de pa Uenls a ha u ti•isq u e .S YSTEMI C CANDIDIASIS REMAI S A SI GNIFICANT CAUSE OF hospital mor tality particula rly among high ris k gro u ps.ll is prevalent in in ten s ive care unit.patien ts and in patien ts wiU1 variou s t.ypes of imm unosu ppress ion (1.2) .Deep-sealed candida!infection s a re diffi c ul t. to d iagn ose accu rately .res ulting in s ignillcant.delays in in itiation of appropriate an Ufungal U1erapy .Definitiv e d iagnosis based upon a •gold s ta n dard' definiti on of invasive candidiasis can on ly be made by histopa th ological demonstration of Candida species invasion of tissue(s) -seldom achieved in c ri tically ill patien ts .Instead.patients a r e most.often st.art.ed on empiric antifu ngal treatm en t when a probable diagn osis of invas ive candida!infection is made.Most.often, pa tients s u spected of h aving invasive infection h ave s ignifican t clini cal risk fac tors for fungal infection .a nd Candida species is s ubsequ ently isola ted from one or m o re impo rtant.body s ites, eg, blood .urine or spu tum (3).
Th e diffic ulties in making a d efinitive d iagn osis o f invasive candida!infec ti on h ave en cou raged th e deve lopm ent and evalua tion of immunological diagn ostic methods (4)(5)(6).Th e inse n s itivi ty of antibody detection meth od s in cri tically ill .immunocomprom ised pati en ts h as led to the developmen t of serodiagn os ti c tests fo r circ ulating can dida a ntigen s .The only comme rcially a vaila ble Candida s pecies de tection system (Can d-Tec: Ramco Labo ra to ri es.Texas) u ses uniforms ized latex particles coa ted wiU1 a n a nticandi da a ntibody to detect the p resen ce of candida a ntigen s in th e serum of patien ts by an agglutina ti on reaction (7).However .U1e a n tigen detected by tl1is system has n ot been purifi ed and characterized .so it.is n ot certain wh at. is being detected by the Cand -Tec lest.
At. present, U1e clinical u sefu ln ess of the Cand -Tec lest.remain s controve rsial.Severa l studies h ave reported •widely varying val u es for U1 e sen s itivi ty a nd specificity of the test in differe ntia ting be tween Candida species colonization a nd infection .Severa l s tud ies ha ve reported U1e Cand -Tec test.to be u seful for the detection of invasive can didiasis (8)(9)(10)(11)(12).However, oU1e r studies h ave not con firmed these results (1 3 -16).Most previou s studies h ave either fo cu sed on h igh ly selected pa tien t. popula tion s or small numbers of pa tien ts in whom a definite diagn osis of inva sive candidia sis h a d already been m ade by the •gold standa rd ' t echniques of pos itive fu n gal c ul tures for Candida species from deep tissu e bi ops ies, or histopath ological eviden ce of fungal infection from post mortem tissu e examin ation .Therefore. it was of interest to evaluate the Cand -Tec tes t re tros pectively in a gen eral h ospital popula tion a t ris k for invasive candida!infec tion.The m a in purpose of U1is s tudy was to decide if U1e test s hould b e offered by U1e clin ical microbiology la bora tory a nd.if so, to establish clear guidelines for its u se in diagn osing invas ive candidi asis.

Patient populatio n:
Foothil ls Hospi tal is a large univers ity Leaching instit ution providing a wide range of tertia ry care s ervices including in ten s ive care (traumas urgical.coron a ry , n eurological a nd bum units) to pa tie nts throughou t so uth ern Alberta .Ove r a one year pe riod , the candida antigen de tection system (Canci -Tec) was ret rospectively eva lu ate d in this gen eral patient.popula tion.Prior to U1e introduc tion of U1e Cand -Tec test by the clinical microbiology la boratory.a n edu ca ti on a l m e mora ndum was circ ula ted t.o all physicians at the h ospital outlining the principles of U1e tes t. and its u sefulness based upon previous s tudies .
During a on e year s tudy period .Cand -Tec tests were o rdered m ainly by specia li st physicia ns providing prim ary or intensive care to patients with ris k fa ctors for developing invasive candida!infec tion .The infecti ou s diseas es service cla rified the reasons for Cand-Tec tes ting in all cas es in whic h U1e cl inical informa tion provided t.o th e la bora tory was in a d equate or unclea r.No restriction s were initi a lly placed on the number of Cand-Tec tests th at could be don e per pa tient.Howeve r, three mont11s after introducti on, becau se of cost con straints a nd the practi ce of som e physicia n s in orde ring d aily tes ts .U1e la borat01y restricted the freq u en cy of testing t o no more th a n tlvo Cand -Tec determina tion s per week pe r pa ti ent.A cu t-off tib•e of ;::: 1:4 was con s idered to be s ignificant a nd poss ibly diagnostic of invasive candidias is .b ased upon previou s ly reported s tudy resu lts (7).All mi crobiology re ports read: "Cand -Tec titre ;::: 1: 14 which m ay be diagnostic of invas ive candidiasis .Please correlate wiU1 clinical pictu re." All p a tien ts who h a d Cand -Tec tests al so h a d extens ive s urveillance cultures for yeast take n from seve ral peripheral s ites includin g throat blood.s pu t um, stool and urine.
Data collection: All eligible patients in whom the Cand-Tec test was clone on one or more occasions were retrospectively reviewed.Between October 1, 1987 and October l. 1988 a total of 82 patients (43 females, 39 males) and 151 individual san1ples were evaluated.The flnal analyses included 79 patients (43 females, 36 males) and 125 individual samples.Three patients with rheumatoid arthritis were excluded because rheumatoid factor may interfere with the Cand-Tec test.Also, Cand-Tec tests clone on patients after amphotericin B treatment was started were not included for analysis.
Chart reviews recorded the following information: patient's age.sex.length of stay in hospital.diagnoses (including unde rlying inmmnosuppressive factors).recent surgery.total parenteral nutrition, admission to the intensive care unit.antibiotic thera py, results from fungal surveillance cultures including throat, sputum, and serial blood , urine and stool cultures, biochemical tests including peak serum creatinine levels, and all available biopsy and/or autopsy results.
Patients were classified into four groups based upon the following clinical and laboratory criteria.'Definite infection• -patients with definite evidence of disseminated candida infection demonstrated by histological evidence of tissue invasion and/or positive tissue fungal cultures and/or positive blood cultures on more than one occasion.'Probable infection' -patients with multiple clinical risk factors for invasive candidiasis (broad spectrum antibiotic use at the time of test and/or within two weeks preceding test.recent abdominal surgery.total parenteral nutrition, immunosuppression from any cause.prolonged hospital stay, or admission to an intensive care unit).as well as two or more significant peripheral sites culture positive for Candida species (eg.sputum.wound), and/or persistent candiduria (positive urine fungal culture for Candida species on more than one occasion) .'Colonization• -patients v.rith less than two of the clinical1isk factors for invasive cand idiasis outlined above ('probable infection• group) and a positive fungal culture from a single peripheral site other than blood.'No infection'patients with no clinical or fungal culture evidence of invasive candidal infection.Statistical analysis: All analyses were done using the highest titres for each patient.The sensitivity, specificity.and positive and negative predictive values for diagnosis of invasive candidiasis were calculated using cut-off Cand-Tec titres of ~1:4.~1:8 and ~1:16.All patients in the 'definite infection' group were included as disease positive.Results from patients in the •no infection• group were used as negative disease.The sensitivity.specificity, and positive and negative predictive values to support a suspected diagnosis of invasive candidiasis were calculated in a similar manner, except that patients in the •probable infection' group were also included as having positive disease.Microbiological methods: Culture of blood specimens was performed using a standard broth system (Bactec; Becton-Dickinson).All blood cultures requesting fungal isolation were plated to prima1y fungal media including inhibitory mold and chocolate agars.All blood fungal cultures were kept and analyzed for 21 clays.Routine bacteriological or fungal cu ltu res which isolated yeast(s) were identified as C aLbicans versus other Candida species by demonstration of germ t ube production.and by assin1ilation profiles with the API-20C (API Analytab Products, New York).Candida antigen detection: A commercially available latex agglutination system (Cand-Tec: Ramco Laboratories.Texas) was used to detect candida antigen.Samples were tested as previously described (7).If agglutination occurred at a ~1:2 dilution, then serial twofold dilutions (up to ~1 : 64) were done.The endpoint was the highest dilution at which agglu tination occurred.Rheumatoid factor was assayed in all patients with a positive titre.

RESULTS
Of the 79 patients, 13 had definite invasive candidiasis and were included in tl1e 'definite infection' group, 20 had probable infection.and 46 were only colonized with Candida species or had no evidence of infection (Table 1).The mean age of the study population was 52 years (range 0 to 81) and the mean lengt11 of stay in hospital was 4 7 clays (range one to 200).The majo1ity of patients studied were critically ill: 57% were situated in the in tens ive care unit, and 30% subsequently died (Table 1).The highest mortality rate occurred in the group with definite infection (44 .5%).Patients in each diagnostic group had pro longed intensive care unit stays, indicating tl1e overall severity of illness in tl1e present study population.Not surprisingly.patients in all diagnostic categories admitted to the intensive care unit had much longer hospital stays tl1an those who did not requ ire this type of care.
Table 1 also gives patient profiles for otl1er associated variables which are known to increase the risk of invasive candidiasis.The use of broad spectrum antibiotics and central total parenteral nutrition were common in tl1e 'definite' and 'probable infection• groups.Up to one-third of patients in each diagnostic patient group were imn1unocompromised for various reasons (Table 1).Most patients in the 'definite infection• group had renal insufficiency (62%).compared witl1 40% of patients with probable infection and on ly 13% of patients in tl1e •colonization/no infection' groups.Some patients with positive Cand-Tec titres had normal renal function at tl1e time of their tests.while oU1ers had mild to severe renal insufficiency.Amphotericin B treatment was common in patients witl1 definite or probable invasive candidal infection (Table 1).Most fungal cultures from patients in all groups isolated C a Lbicans .Candida tropicaLis was isolated from a protected brush specimen from one  titres prior to tl1e initiation of therapy.Most patients (77%) in the definite infection group had Cand-Tec titres :::: l :4.On average, Cand-Tec tests were done five days prior to definitive diagnosis of fungal infection and initiation of amphotericin B therapy.Of the six patients who died in tl1e definite infection group.three had post mortem examinations (Table 1).One patient had a Cand-Tec titre of :::: 1:32 five days prior to death and was confirmed at autopsy to have invasive candidiasis .The two otl1er autopsied patients had been on amphotericin B prior to death and showed no evidence of invasive candidiasis at post mortem examination.Both patients had been fungemic prior to death -the patient witll C a lbicans infection had serial Cand-Tec titres of 2' : 1:2 (five and nine days prior to death).while the patient with C parapsilosis infection had serial Cand-Tec titres of 2' : 1:8 and 2' : 1:16.
Most patients in the 'probable infection' group also had mu ltiple Cand-Tec titres done (Table 2).The highest Cand-Tec titre for most patients in this group was ~1:4 (70%).On average, Cand-Tec tests were done 12 days after diagnosis of fungal infection by e:11..1:ensive peripheral s ite cultures .As shown by the la rge standard deviation (Table 2), Cand-Tec tests were done in several patients several weeks after infection had been confirmed by peripheral body s ite fungal cult ures.
Most patients in the 'colonization' and 'no infection' groups h a d only single Cand-Tec titres done (Table 2).Most of the time, Cand-Tec tests were ordered randomly in these patient groups, and testing bore no relationship to cultures.The highest Cand-Tec titres in these patients were often :2: 1:4 (51 %), and the majority had titres 5. 1:8 (83%).
Table 3 outlines the ability of the Cand-Tec test to diagnose invasive candidiasis in the definite infection group for various cut-offtitres between :2: 1:4 and :::: 1:16.The Cand-Tec test was neither adequately sensitive or specific at a cut-off titre of :::: 1:4.Test sensitivity, specificity, and positive and negative predictive values were 77%, 61 % , 36% and 90%, respectively.The specificity could be improved by increasing the cut-off titre to :::: 1:8 (78% ) or :::: 1: 16 (96%).Sensitivity of the test substantially decreased as the cut-off titre was moved up to :::: 1:16 (30%).Similar changes occurred in the positive and negative predictive values for the test as the cut-off titre was increased.
Table 4 outlines the ability of the Cand-Tec test to support a clinical diagnosis of probable invasive candida!infection for various cut-off titres between :2: 1:4 and :::: 1: 16.Including patients in the •probable infection' group in the analyses did not appreciably change the ability of the Cand-Tec tests to diagnose invasive candidiasis accurately at a cut-off titre of :::: 1:4.Test sensitivity, specificity, and positive and negative predictive values were 70%, 61%.56% and 74%, respectively.Again , specificity could be improved by increasing the cut-off titre to :::: 1:8 (78%) or :::: 1:16 (96%).However, sensitivity of the test substantially decreased as the cut-off titre was moved up to :2: 1:16 (21 %).
Increasing the cut-off titre above :::: 1:4 did not significantly alter the positive and negative predictive values of the test.
Restricting the analyses further to patients who were critically ill and in the intensive care unit did not enhance the diagnostic ability of this test considered alone or in combination with culture data in definite exclusion of candida!infection.

DISCUSSION
Invasive candidiasis remains a serious and difficult infection to diagnose and treat in the authors' i.nstitution , particularly in critically ill patients.Over a one year period in a general hospital setting, tl1e authors evaluated 33 patients with a definite or probable diag-    4) demonstrated that the Cand-Tec test was less than optimal in h elping to confirm or rule out a diagnosis of invasive candidiasis.Not surprisingly , there was a trade-off between tl1e level of sensitivity and specificity for tl1e test depending upon the Cand-Tec titre used as tl1e cut-off for diagnosis of invasive infection.Even when the 'probable infection' group patients were excluded from analysis (Table 3), the maximal sensitivity (77%) occurred at a cut-off titre of :::: 1:4 when tl1e specificity of the test was only 69%.Similarly, positive and negative predictive values for the test were poor regardless of the cut-off Cand-Tec titre used (Tables 3,4).The variation between Tables 3 and 4 in the positive and negative predictive values observed for different cut-off levels occurred mainly because the prevalence of disease differed in the populations being considered in t11e analyses .To carry this further, if the test was to be used in a less selected population with lower disease prevalence than the present one, t11e positive predictive values would fall even further.The present results are comparable with t11e larger study of Cabezubo et al (12) who also demonstrated poor diagnostic ability of the Cand-Tec test in a general hospital popul a tion.However.Cabezubo el a l (12) suggested that the d etermina tion of seria l tit res might b e h e lpful in early diagnosis of invasive infection.Although the role of serial titres was not formally evaluated in th e present p a tient population, no predictabl e trend was found in patien ts who had multiple tilres done.Des pite no treatment.several patients demonstrated marked fluctuations in their titres from one determination to the n ext.whi ch furth e r co nfuse d a difficult clinical s ituation .
Other published data regarding the usefulness of the Cand-Tec test are contradictory.likely because highly p re-selected patient population s were studied.The initial study of Gentry et al (7) reported a sensitivity of 9 1% in 33 patients with confirmed invasive candidiasis (positive blood or organ tissue c ulture for C albican s plus fever) .In a controlled follow-up study evaluating tl1e Cand-Tec test in oilier high risk patient popu lations , Price and Gentry (9) demonstrated variable sensitivity but a universally high specificity (97%) for all patient groups using a cut-off titre of ;:o: 1:4.Subsequent studies by oilier groups have s hown the Cand-Tec test to have variable but not nearly as high sen sitivity and specificity in highly selected patient popula tions (8, 1 0.11.[14][15][16]. The present study and others (14)(15)(16) demonstrate that t11 e Cand-Tec test does not a ppreciably e nha nce physici a ns• ability to diagnose invasive candida!infection beyond standard histopathological and microbiologica l methods.In addition , ilie test did not d e monstra te sufficie nt specificity or n egative predictive value in tl1e present patient population to rule out abso lu tely invasive infection in patie nts colonized with Candida species.Therefore.it is not likely t11at set clinical and laboratory criteria would enhance t11e performan ce of the Cand-Tec test in a general hospital setting.In addition, false positive Cand-Tee results wiiliout careful clinical and la boratory follow-up of the patient may lead to t h e inappropriate initia tion of potentially toxic antifungal therapy.During ilie study.several patients wiili persistently e levated Cand-Tec ti t.res a nd oilienvise limite d evidence of invasive fun gal infection were empirically treated witl1 prolonged courses of an1photericin B.
At tl1e a uiliors' institution.ilie diagnosis of invasive candidiasis in high risk patients continues to rest witll the astute clinician.The Cand-Tec test is no longer p e rformed on a routine b asis by ilie hospital's clinical mic robiology laboratory .
CAN J INFECT DIS VOL 3 No 4 JULY/AUGUST 1992 Cand-Tec evaluation

TABLE 2 Characteristics of Cand-Tee testing versus candida! infection status
Candida species other than C albicans demonstrated s ignificant titres in the Cand-Tec test.Table2illustrates the characteristics of Cand-Tec testing versus the patient's designated candidal infection status.Most patients in the 'definite infection' group had multiple Cand-Tec titres done.Despite tissue culture evidence of invasive candidal infection, most patients did not demonstrate rising Cand-Tec

TABLE 3
Analysis of the Cand-Tec test's ability to diagnose invasive candidiasis in high risk patients*