Mycopathologia 118: 153-162, 1992. 9 1992 Kluwer Academic Publishers. Printed in the Netherlands.
Diagnosis of disseminated candidiasis in hospitalized patients using the Cand-Tec latex agglutination assay Martha L. Sanchez 1, Michael A. Pfaller, 2 Ignacio Cabezudo, 1 Martha Bale * & Barry Buschelman 1
1Department of Pathology, University of Iowa College of Medicine, Iowa City, IA 52242; 2Department of Pathology, Oregon Health Sciences University, Portland, OR 97201-3098, USA Received 7 August 1991; accepted in revised form 15 December 1991
Key words: Candidiasis, antigen detection, diagnosis
Abstract
A total of 1,303 sera from 202 patients at risk for disseminated candidiasis were analyzed for the presence of Candida antigen using a commercially available latex agglutination test (Cand-Tec). Twentythree patients had disseminated candidiasis documented by positive blood cultures, deep biopsy culture and histopathology or autopsy. Six patients had transient candidemia, 15 patients had candiduria, 62 patients were not colonized yet treated empirically with amphotericin B, and 46 patients were not colonized and not treated with amphotericin B. The sensitivity and specificity of the Candida antigen test for the diagnosis of disseminated candidiasis was 87% and 36% (threshold titer of ~>1:2), 70% and 60% (I>1:4), and 30% and 85% (t>1:8), respectively. In contrast to previous studies we were unable to demonstrate a prognostic role for the Candida antigen test in patients with documented disseminated candidiasis. The lack of sensitivity and specificity of the Cand-Tec Candida antigen test precludes its use in the diagnosis of disseminated candidiasis.
Introduction
Candida species can be isolated as normal flora from the oral cavity, gastrointestinal tract and female genitalia of healthy humans [1-4]. Opportunistic infections with these organisms have occurred with increasing frequency over the past two decades [2, 5, 7, 9, 11]. Wey et al. [8, 12] described several important risk factors for candidemia including exposure to broad-spectrum antibiotics, colonization with Candida spp, and the presence of a Hickman catheter. Patients at increased risk for invasive candidiasis include neonates [1, 2] patients with
extensive burns [6] and those undergoing major surgery [1,2,8] and aggressive antineoplastic chemotherapy [5, 13, 14]. Usually these patients have one or more intravascular catheters and other medical devices such as a Foley catheter, artificial Ventilatory assistance, and Swan-Ganz catheters. In hospitalized patients one of the major problems in diagnosis of invasive or disseminated candidiasis is the paucity of clinical signs and symptoms [1, 5, 11, 12, 15]. Blood cultures are frequently positive late in the course of infection and may detect less than 50% of patients with disseminated candidiasis [9]. Cultures may be in-
154 adequate to distinguish colonization from infection and clinicians are frequently forced to administer antifungal agents empirically to febrile neutropenic patients [2, 5, 9, 11, 13, 16]. Given these problems, a different approach to the diagnosis of invasive candidiasis in hospitalized patients is warranted. As a result, several serologic techniques have been used to identify the presence of specific antibodies, circulating Candida antigens or metabolites in the serum of infected patients [4, 14, 17-21]. None of these approaches have been found to be uniformly successful [14, 16, 21-32]. Detection of antibodies has failed because those patients at greatest risk for disseminated candidiasis generally are incapable of producing specific antibodies. Similarly, certain Candida antigens are rapidly cleared from the circulation with the formation of immune complexes [21, 33, 34]. Consequently, adequate sensitivity for detection of disseminated disease has been difficult to achieve with assays for the presence of Candida antibodies or antigens in serum [14, 16, 18, 23, 26, 30, 32, 34]. The introduction of a simple latex agglutination assay for detection of heat-labile Candida antigen in serum (Cand-Tec, Ramco Laboratories, Houston, TX) has provided a rapid and inexpensive means of assessing the clinical usefulness of antigen detection in the diagnosis of disseminated candidiasis. We and others have evaluated this assay previously with mixed results [14, 21, 1232]. The purpose of the present study was to extend our previous observations on the usefulness of the Cand-Tec assay in the diagnosis and management of disseminated candidiasis in hospitalized patients.
Materials and methods
Patients and sera. A prospective study was con-
ducted on 202 patients hospitalized in the University of Iowa Hospital from 1987 to 1988. Sera for Candida antigen testing were obtained routinely from these patients on admission and weekly thereafter. Additional sera were obtained if
symptoms and clinical signs were suggestive of an infectious process. All sera were tested prospectively without knowledge of the patient's clinical condition. A total of 1,303 sera was collected from patients hospitalized on general and transplant surgery, hematology-oncology, bone marrow transplantation, and general medicine services, as well as burn and intensive care units. All patients had similar risk factors for disseminated candidiasis including broad-spectrum antibiotic therapy and central venous catheterization. Antibiotic therapy was defined when patients were given broad-spectrum antibacterial agents for at least one 24-hour period. Renal function was assessed by reviewing laboratory results (serum creatinine) for each patient. Renal failure was defined as a serum creatinine ~>2.0 mg/dl. Cultures. Fungal surveillance cultures were obtained on admission and weekly thereafter throughout hospitalization, and were processed as described previously in order to assess the colonization status of the patient [35]. Blood cultures were performed, when indicated clinically, by both a standard broth system (Bactec, Becton Dickenson, Towson, MD) and the Isolator lysiscentrifugation system (Wampole, USA) to maximize the chances of detection of candidemia [36]. Cultures of specimens from other sites were performed as indicated clinically. Diagnostic groups. The medical record of each
patient was reviewed by two of the authors (M.L.S. and I.C.) for evidence of disseminated infection, transient candidemia, localized infection or colonization. In addition, the outcome of hospitalization (survival or death) and the administration of systemic antifungal therapy were ascertained. The patients were stratified into six diagnostic groups (Table 1). Group 1 comprised 23 patients (176 sera) with disseminated candidiasis. Four patients (18%) were neutropenic (WBC < 500/mm3), and three had a hematologic malignancy. Four patients (18%) had a low neutrophil count (500-1000/mm 3) and 15 (64%) were
155
Table 1. Diagnostic groups, inclusion criteria, and crude mortality of 202 patients Diagnostic groups
Number of patients
Inclusioncriteria
Hematologic disorder* (%)
1. Disseminated candidiasis
23
Culture or histologic evidence from deep-organ biopsy and/or/>2 blood cultures obtained from separate sites at least 24 hours apart Single positive blood culture and no other evidence for disseminated infection. Urine culture containing >/1,000 C.F.U./ml in pure culture. Oral, gastrointestinal and/or respiratory colonization, but no documentation of disseminated disease. Negative surveillance cultures and no documentation of disseminated disease, but treated with antifungal agents. Negative surveillance cultures, no documented disseminated infection and not treated with antifungal agents.
13
70
48
50
67
33
60
60
20
89
79
24
92
100
32
2. Transient candidemia
6
3. Candiduria
15
4. Colonized
62
5. Non-colonized, treated
50
6. Non colonized non treated
46
Patients on amphotericin B (%)
Crude mortality (%)
28
* Includes leukemia lymphoma, aplastic anemia, and bone marrow transplantation.
non-neutropenic W B C > 1000/mm3). Of the 23 patients, 16 (70%) received amphotericin B and 20 (87%) received m o r e than four antibiotics per day. Seventeen patients (74%) had renal impairment. Eleven patients (48%) died, and in two patients autopsies were p e r f o r m e d which revealed disseminated candidiasis. Group 2 consisted of six patients (47 sera) classified as having transient candidemia. Three patients (50%) had an underlying hematologic malignancy and two (33%) were neutropenic ( W B C < 5 0 0 / m m 3 ) . Five patients (83%) received more than four antibiotics per day and four (67%) were treated with amphotericin B. Five patients (83%) had intravascular catheters and three (50%) had renal impairment. Two of the six patients (33%) died; however, autopsies were not performed. Group 3 consisted of 15 patients (101 sera) classified as having candiduria. Nine of these patients (60%) had an underlying hematologic malignancy and were neutropenic, Fourteen patients (93%) received four or m o r e antibiotics per day, 12 (80%) had an intravenous catheter, and nine (60%) re-
ceived amphotericin B. Seven patients (47%) had renal impairment. Three of the 15 patients died. No autopsies were performed. Group 4 consisted of 62 patients (531 sera) who had mucous m e m brane colonization alone documented by surveillance cultures. Fifty-five (89%) of these patients had hematologic malignancies and 49 (79%) were neutropenic. Forty-nine patients (79%) were treated empirically with amphotericin B for culturenegative fever which had not responded to therapy with broad-spectrum antibiotics. Fifty-seven patients (92%) had intravenous catheters and 35 (56%) had renal impairment. Autopsies of eight of 15 patients from this group who died did not show evidence of disseminated candidiasis. Group 5 comprised 50 non-colonized, treated patients (366 sera) who had negative surveillance cultures for Candida spp. and had no evidence of local or disseminated candidiasis. Forty-six patients (92%) had underlying hematologic disease and/or underwent bone m a r r o w transplantation and 42 (84%) were neutropenic. All patients in this group received amphotericin B empirically
156 for culture-negative fever which persisted despite therapy with broad-spectrum antibiotics. Fortyeight patients (98%) received more than four antibiotics per day, 46 (92%) had an intravascular device and 19 (35%) suffered renal impairment. Autopsies of five of 15 patients from this group who died did not show evidence of disseminated candidiasis. Group 6 comprised 46 non-colonized, non-treated patients (78 sera), who had negative surveillance cultures and no evidence of local or disseminated candidiasis. Thirteen of these patients (28%) had hematologic malignancies and 10 (22%) were neutropenic. None of the patients received amphotericin B, 12 (26%) received more than four antibiotics per day, 15 (33%) had an intravenous catheter and nine (20%) had renal impairment. Autopsy of one of four patients from this group who died did not show evidence of disseminated candidiasis.
Candida antigen assay. The Cand-Tec Candida detection assay (Ramco Laboratories, Houston, TX) detects poorly defined, heat-labile Candida antigens and was performed according to the manufacturer's instructions as described previously [28]. All sera were tested at an initial dilution of 1:2 in glycine-buffered saline (pH 8.4). The test was considered positive if the dilution showed clear agglutination and negative when there was no visible agglutination of the latex particles. Positive sera (agglutination at 1 :2) were serially diluted in glycine-buffered saline and tested with the Cand-Tec reagent. The final result was defined as that dilution of serum in buffer that still produced obvious agglutination. Positive and negative controls were included in every run. All sera with titers of 1:2 or greater were further tested for non-specific agglutination using a nonimmune antibody-coated latex control (Meridian Diagnostic, USA). The manufacturer recommends that a titer of/>1:4 be considered as positive evidence of disseminated candidiasis; however, for purposes of this study results were calculated using titers of >~1:2, I>1:4 and ~>1:8 as indicating a positive result.
Stat&tieal analys&. Statistical analysis was performed using either the chi-square test with Yate's correction or the two-tailed Fisher's exact test for comparison of proportions. Pearson's correlation coefficient was used to analyze the possible correlation between Candida antigen titers and serum creatinine. Values of p < 0.05 were considered significant. Sensitivity, specificity and predictive values were calculated by the method described by Galen and Gambino [37].
Results
Disseminated candidiasis (Group 1). Twentythree of 202 patients (11%) were diagnosed as having disseminated candidiasis (Table 2). Fifteen patients (65%) had positive blood cultures, seven patients had positive cultures of normally sterile body fluids (peritoneal fluid and pleural fluid) including two with positive blood cultures, six patients (26%) had positive deep tissue biopsies (blood cultures were also positive in four of these patients), and one patient was diagnosed at autopsy. In contrast with previous studies from this institution [28], only three patients (13%) had an underlying hematologic disorder, four (17%) had solid tumors and 16 (70%) had other underlying diseases (Table 2). Of the 23 patients with disseminated candidiasis, 18 (78%) were infected with a single species of Candida: 12 with C. albicans, four with C. glabrata, one with C. lusitaniae, and one with a Candida species detected by histopathology only. Five patients were infected with more than one species of Candida, including one with C. albicans and C. krusei, one with C. albicans and C. glabrata, one with C. albicans, C. glabrata, and C. parapsilosis, and one with C. albicans, C. parapsilosis, and C. tropical& (Table 2). We performed 176 Candida antigen determinations in this group (mean 7.6 tests per patient). Peak antigen titers were >~1:2 in 20 patients (87%), />1:4 in 16 patients (70%), and i>1:8 in seven patients (30%) (Tables 2 and 3). Five pa-
157 Table 2. Antigen titers determined with the Cand-Tec latex agglutination test in patients with disseminated candidiasis Patient number
Underlying disease
Evidence of infectiona
Candida species
Peak titer
Outcome
1 2 3 4 5 6 7 8 9
Hepatitis Acute abdomen Diabetes Cervical cancer Lung cancer Bowel obstruction Acute abdomen Lymphoma Ischemic bowel
SBF SBF BC BC SBF BC HP HP SBF
Died Survived Survived Survived Died Survived Survived Died Died
Renal transplant Multiple sclerosis Bowel obstruction Neuroblastoma Abdominal
HP BC BC BC BC, HP
NS c 1:4 1:16 Negative 1:2
Survived Survived Died Survived Survived
15
Neurosurgery
BC
1 :8
Died
16 17
Stroke Gastrointestinal Hemorrhage Short-gut syndrome Bacterial peritonitis Systemic lupus erythematosus Coronary heart disease
BC, SBF BC, HP
1:16 1:4
Died Died
BC, SBF SBF BC
glabrata albicans albicans glabrata glabrata albicans albicans species b albicans glabrata albicans lusitaniae albicins aIbicans aIbicans glabrata tropicalis albicans parapsilosis tropicalis albicans albicans krusei glabrata albicans albicans
1:8 1:4 1:8 1:4 1:4 1:4 1 :2 1:8 Negative
10 11 12 13 14
C. C. C. C. C. C. C. C. C. C. C. C. Cl C. C. C. C. C. C. C. C. C. C. C. C. C.
1:4 1:2 1:32
Survived Died Died Died
BC, HP BC
glabrata parapsilosis albicans albicans albicans
1:4
Leukemia Lymphoma
C. C. C. C. C.
1:2 1:32
Survived Survived
18 19 20 21
22 23
BC, HP
BC = blood culture, HP = histopathology, SBF = sterile body fluid other than blood. b Seen on HP only, not identified to species. c N S = non-specific agglutination.
Table 3. Candida antigen titers determined with the Cand-Tec test in 202 high risk patients Diagnostic groups
1. 2. 3. 4. 5. 6.
Disseminated candidiasis* Transient candidemia Candiduria Colonized Non-colonized, treated Non-colonized, non-treated*
No. of patients
23 6 15 62 50 46
* Two patients had non-specific agglutination in all sera.
Number of patients (%) with peak titer of />1:2
/>1:4
/>1:8
20(87) 5(83) 9(60) 44(71) 32(64) 24(52)
16(70) 2(34) 6(40) 26(42) 16(32) 6(13)
7(30) 1(17) 2(13) 9(15) 6(12) 2(4)
158 tients (21%) had one or two positive (>1:2) sera and 15 (65%) had three or more positive sera during the course of their hospitalization. One patient with positive blood cultures for both C. albicans and C. glabrata and one with C. albicans fungemia had negative titers. One additional patient with C. albicans fungemia showed nonspecific agglutination in all sera tested. Ten of the 17 patients (59%) with disseminated infection due to C. albicans had antigen titers >1:4 compared to six of six patients infected with other species of Candida (p > 0.05 for the comparison of C. albicans infected patients with all others). Nine (64%) of 14 patients with a peak titer of ~1:8
(p > 0.05). Twelve patients had more than two serum samples obtained during treatment for disseminated disease. Antigen titers increased at least twofold during the course of therapy in five patients and three (60%) of these patients died with disseminated infection. In contrast, antigen titers decreased by at least two-fold in seven patients during therapy and only two (29%) died of the infection. These results were not significantly different (p = 0.31). We determined the temporal relationship between positive antigen titers and positive culture results for Candida in the 20 antigen-positive patients with disseminated infection. Sera were positive for antigen at titers of >1:2 at least one day prior to diagnosis by culture or biopsy in seven (35%) and on the same day in four (20%) patients. Of the remaining nine patients (45%), five had positive titers between one and three days after positive cultures or biopsy and four had titers which were positive > 4 days after positive cultures. The relationship between renal function and Candida antigen titer was examined in Group 1 patients by matching the antigen titer with the corresponding serum creatinine determination. The Pearson's correlation coefficient failed to show a significant relationship between these values (r = 0.43, p = 0.15).
Table 4. Analysisof the Cand-Tec latex agglutinationtest as a predictor of disseminatedcandidiasis* Candida antigen threshold titer >1:2 Sensitivity (%) 87 Specificity(%) 36 Positive predictive value (%) 15 Negative predictive value (%) 96
>1:4 70 60 18 94
>1:8 30 85 21 90
* Prevalence of disseminatedcandidiasiswas 11%.
Disseminated candid&sis patients (group 1) in comparison with other diagnostic groups. Circulating antigen was also apparently detected in a number of patients without documented invasive infection (Groups 2 to 6, Table 3). Overall, 64% of patients in Groups 2-6 had titers >1:2, 31% had titers >1:4 and 11% had titers >1:8 (p < 0.05 for all comparisons versus Group 1). Analysis of the number of antigen-positive patients in Group 1 versus each diagnostic group at each of the threshold antigen titers (>1:2, >1:4, >1:8) revealed significant differences between Group 1 and 4 at >1:4, between Group 1 and 5 at >1:2 and >1:4, and between Group 1 and 6 at all three threshold antigen titers. There was no significant difference in antigen titers between Group 1 and Group 2 (transient candidemia) and between Group 1 and Group 3 (candiduria). Although a number of patients in Groups 2 to 5 received empiric amphotericin B therapy, disseminated candidiasis was not documented in any of them. Autopsies were performed in 13 of the 35 patients who died in Groups 2 to 5, and none demonstrated Candida invasion.
Predictive value of Candida antigen titers. The prevalence of documented disseminated candidiasis was 11% (23 of 202 patients). In analysis of the Cand-Tec latex agglutination test as a predictor of disseminated candidiasis (Table 4), the greatest sensitivity was obtained using a threshold titer of >1:2 (87%). This result was higher than that obtained with a threshold titer of either/> 1:4 (70%, p > 0.05) or >1:8 (30%, p < 0.05). The
159 specificity of the Cand-Tec test improved significantly using threshold titers of >~1:4 (60%) or I>1:8 (85%) compared to ~>1:2 (36%, p < 0.05). The positive predictive value was low (15-21%) for all threshold titers. The application of a higher threshold titer did not significantly improve the positive predictive value of the test relative to the low (~>1:2) threshold titer (p < 0.05 for all comparisons). The negative predictive value was significantly improved when the low (/>1:2) threshold titer was used (96%, p < 0.05) compared to either/>1:4 (94%) or ~>1:8 (90%).
Discussion
Given the problems associated with the clinical and microbiologic diagnosis of disseminated candidiasis it is reasonable to examine carefully the potential role of antigen detection as a means of early diagnosis of this important infection. The Cand-Tec latex agglutination assay for detection of circulating Candida antigen is a commercially available assay which is relatively inexpensive, technically simple, and less time consuming to perform than previously described antigen detection assays [4, 17, 19-21, 25]. We and other investigators have evaluated this test for the diagnosis of deep-seated candidiasis over the past seven years and the results have shown many discrepancies (Table 5). The variation in sensitivity and specificity obtained with the Cand-Tec assay in the different published studies is tremendous. The reported sensitivities range from 19% to 100% with specificities of 29% to 99% (Table 5). These differences are certainly due in large part to the differences in definition of disseminated candidiasis and the selection of cases and controls employed in the various studies. The diagnostic sensitivity of the assay may be impacted by the transient nature of the antigenemia which requires multiple sera to be collected serially in order to maximize detection of circulating antigen [14]. The apparent lack of specificity of the assay in certain studies has been more difficult to explain and has
been further impaired by the undefined nature of the heat-labile antigen. Ness et al. [26] reported the lowest specificity of 29% obtained in a highly selected population of bone marrow transplant patients. They found an apparent association of antigenemia with elevated serum creatinine. This observation suggests that antigens accumulate in the serum of patients with renal failure and may explain both the poor specificity in the study of Ness et al. [26] and the variable specificity observed by other investigators (Table 5). The present study was performed in an effort to extend the findings of our previous study [28] on the role of the Cand-Tec test in the diagnosis and therapy of candidiasis in hospitalized patients. In our previous study we found a high sensitivity (95%) and negative predictive value (98%) but a poor specificity (50%) and positive predictive value (31%) when a threshold titer of />1:2 was employed, suggesting that the test may be useful as a screening test in high risk patients. More importantly, we found that the determination of serial antigen titers during the course of antifungal therapy in patients with documented disseminated infection was useful in monitoring the effectiveness of treatment. In contrast, the sensitivity, specificity and positive predictive values obtained in the present study were all considerably lower than that observed previously regardless of the threshold titer employed (Tables 4 and 5). Furthermore, the present study failed to confirm the usefulness of serial antigen determinations in monitoring the effectiveness of antifungal therapy. The reasons for these differences are unclear but may be due to differences in the populations studied. Seventy-three percent of the patients with disseminated candidiasis (Group 1) in our previous study had an underlying hematologic malignancy or bone marrow transplantation and were neutropenic, whereas only 13% of Group 1 patients in the present study suffered from these underlying conditions. Thus, the clinical and antigenic response to antifungal chemotherapy may be considerably different in the predominantly neutropenic patients comprising Group 1 in the previous study versus the non-
160
Table .5. Summary of the results of published studies using the Cand-Tec test for the diagnosis of disseminated candidiasisa Number of patients References Kahn and Jones [14], Group B Bailey et al. [23] Escuro et al. [24] Kahn and Jones [14], Group A Bougnoux et al. [21] Ness et al. [26] Burnie and Williams [27] Lemieux et al. [25] Present study Cabezudo et al. [281 Piens et al. [29] Gentry et al. [30] Price and Gentry [31]u Fung et al. [32]
Disseminated infection
No infection
32
118
19
87
18 29 23
3i 113 74
28 38 48
97 77 89
22 11 30 28 23 37 9 33 11 6
142 41 481 184 179 158 95 154 117 18
50 55 67 68 70 73 78 91 100 100
92 29 94 92 60 72 91 99 51 88
Sensitifivity (%)
Specificity(%)
a Latex agglutination titer/>1:4 considered positive. b Includes only high-risk patient population.
neutropenic G r o u p 1 patients in the present study. The large n u m b e r of false-positive results obtained in this study is consistent with the findings of Cabezudo et al. [28], Ness et al. [26] and Price and G e n t r y [31] (Table 5). Most notable was the observation that 60-71% of patients with candiduria ( G r o u p 3) or mucosal colonization ( G r o u p 4) had sera with titers of i>1:2 (Table 3). This substantiates our previous observation that patients who are colonized or who have localized, but not disseminated, infections m a y have circulating antigen presumably due to defects in the oral, gastrointestinal or genitourinary mucosa [281. It has been suggested by Ness et al. [26] and Bougnoux et al. [21] that antigens detected by the Cand-Tec assay are accumulated in the serum of patients with renal failure and therefore titers are increased but meaningless. Analysis of the relationship between serum creatinine and antigenemia in b o t h the earlier study of Cabezudo et al. [28] and in the present study failed to support this contention. We did not observe a significant correlation between antigenemia and elevated
serum creatinine and thus cannot ascribe the false-positives observed in our patients to impaired renal function. In conclusion, we were unable to establish a firm basis for the clinical utility of the Cand-Tec Candida antigen test in the diagnosis and therapy of disseminated candidiasis. This is in contrast to our previous fndings [28] and in agreement with the conclusions of other investigators [14, 21, 2 3 26]. The m o d e r a t e sensitivity, p o o r specificity and positive predictive value coupled with a lack of prognostic utility suggests that the assay has little, if any, clinical utility and in fact m a y not be measuring a Candida-specific antigen. Additional efforts to develop rapid, culture-independent methods for the diagnosis of disseminated candidiasis should continue; however, any new products must be subjected to rigorous clinical trials prior to their application in hospitalized patients.
Acknowledgements We acknowledge the excellent assistance of the nurses, physicians assistants, and medical staff of
161
the various clinical services at the University of Iowa Hospitals and Clinics. The technical expertise and assistance of the medical technologists in the Special Microbiology Laboratory and the secretarial skills of Bea Dacy are also greatly appreciated.
17.
18. 19.
20.
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Address for correspondence: Michael A. Pfaller, MD, Department of Clinical Pathology, L471, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201-3098, USA. Tel: (503) 494-4105.
Diagnosis of disseminated candidiasis in hospitalized patients using the Cand-Tec latex agglutination assay Martha L. Sanchez 1, Michael A. Pfaller, 2 Ignacio Cabezudo, 1 Martha Bale * & Barry Buschelman 1
1Department of Pathology, University of Iowa College of Medicine, Iowa City, IA 52242; 2Department of Pathology, Oregon Health Sciences University, Portland, OR 97201-3098, USA Received 7 August 1991; accepted in revised form 15 December 1991
Key words: Candidiasis, antigen detection, diagnosis
Abstract
A total of 1,303 sera from 202 patients at risk for disseminated candidiasis were analyzed for the presence of Candida antigen using a commercially available latex agglutination test (Cand-Tec). Twentythree patients had disseminated candidiasis documented by positive blood cultures, deep biopsy culture and histopathology or autopsy. Six patients had transient candidemia, 15 patients had candiduria, 62 patients were not colonized yet treated empirically with amphotericin B, and 46 patients were not colonized and not treated with amphotericin B. The sensitivity and specificity of the Candida antigen test for the diagnosis of disseminated candidiasis was 87% and 36% (threshold titer of ~>1:2), 70% and 60% (I>1:4), and 30% and 85% (t>1:8), respectively. In contrast to previous studies we were unable to demonstrate a prognostic role for the Candida antigen test in patients with documented disseminated candidiasis. The lack of sensitivity and specificity of the Cand-Tec Candida antigen test precludes its use in the diagnosis of disseminated candidiasis.
Introduction
Candida species can be isolated as normal flora from the oral cavity, gastrointestinal tract and female genitalia of healthy humans [1-4]. Opportunistic infections with these organisms have occurred with increasing frequency over the past two decades [2, 5, 7, 9, 11]. Wey et al. [8, 12] described several important risk factors for candidemia including exposure to broad-spectrum antibiotics, colonization with Candida spp, and the presence of a Hickman catheter. Patients at increased risk for invasive candidiasis include neonates [1, 2] patients with
extensive burns [6] and those undergoing major surgery [1,2,8] and aggressive antineoplastic chemotherapy [5, 13, 14]. Usually these patients have one or more intravascular catheters and other medical devices such as a Foley catheter, artificial Ventilatory assistance, and Swan-Ganz catheters. In hospitalized patients one of the major problems in diagnosis of invasive or disseminated candidiasis is the paucity of clinical signs and symptoms [1, 5, 11, 12, 15]. Blood cultures are frequently positive late in the course of infection and may detect less than 50% of patients with disseminated candidiasis [9]. Cultures may be in-
154 adequate to distinguish colonization from infection and clinicians are frequently forced to administer antifungal agents empirically to febrile neutropenic patients [2, 5, 9, 11, 13, 16]. Given these problems, a different approach to the diagnosis of invasive candidiasis in hospitalized patients is warranted. As a result, several serologic techniques have been used to identify the presence of specific antibodies, circulating Candida antigens or metabolites in the serum of infected patients [4, 14, 17-21]. None of these approaches have been found to be uniformly successful [14, 16, 21-32]. Detection of antibodies has failed because those patients at greatest risk for disseminated candidiasis generally are incapable of producing specific antibodies. Similarly, certain Candida antigens are rapidly cleared from the circulation with the formation of immune complexes [21, 33, 34]. Consequently, adequate sensitivity for detection of disseminated disease has been difficult to achieve with assays for the presence of Candida antibodies or antigens in serum [14, 16, 18, 23, 26, 30, 32, 34]. The introduction of a simple latex agglutination assay for detection of heat-labile Candida antigen in serum (Cand-Tec, Ramco Laboratories, Houston, TX) has provided a rapid and inexpensive means of assessing the clinical usefulness of antigen detection in the diagnosis of disseminated candidiasis. We and others have evaluated this assay previously with mixed results [14, 21, 1232]. The purpose of the present study was to extend our previous observations on the usefulness of the Cand-Tec assay in the diagnosis and management of disseminated candidiasis in hospitalized patients.
Materials and methods
Patients and sera. A prospective study was con-
ducted on 202 patients hospitalized in the University of Iowa Hospital from 1987 to 1988. Sera for Candida antigen testing were obtained routinely from these patients on admission and weekly thereafter. Additional sera were obtained if
symptoms and clinical signs were suggestive of an infectious process. All sera were tested prospectively without knowledge of the patient's clinical condition. A total of 1,303 sera was collected from patients hospitalized on general and transplant surgery, hematology-oncology, bone marrow transplantation, and general medicine services, as well as burn and intensive care units. All patients had similar risk factors for disseminated candidiasis including broad-spectrum antibiotic therapy and central venous catheterization. Antibiotic therapy was defined when patients were given broad-spectrum antibacterial agents for at least one 24-hour period. Renal function was assessed by reviewing laboratory results (serum creatinine) for each patient. Renal failure was defined as a serum creatinine ~>2.0 mg/dl. Cultures. Fungal surveillance cultures were obtained on admission and weekly thereafter throughout hospitalization, and were processed as described previously in order to assess the colonization status of the patient [35]. Blood cultures were performed, when indicated clinically, by both a standard broth system (Bactec, Becton Dickenson, Towson, MD) and the Isolator lysiscentrifugation system (Wampole, USA) to maximize the chances of detection of candidemia [36]. Cultures of specimens from other sites were performed as indicated clinically. Diagnostic groups. The medical record of each
patient was reviewed by two of the authors (M.L.S. and I.C.) for evidence of disseminated infection, transient candidemia, localized infection or colonization. In addition, the outcome of hospitalization (survival or death) and the administration of systemic antifungal therapy were ascertained. The patients were stratified into six diagnostic groups (Table 1). Group 1 comprised 23 patients (176 sera) with disseminated candidiasis. Four patients (18%) were neutropenic (WBC < 500/mm3), and three had a hematologic malignancy. Four patients (18%) had a low neutrophil count (500-1000/mm 3) and 15 (64%) were
155
Table 1. Diagnostic groups, inclusion criteria, and crude mortality of 202 patients Diagnostic groups
Number of patients
Inclusioncriteria
Hematologic disorder* (%)
1. Disseminated candidiasis
23
Culture or histologic evidence from deep-organ biopsy and/or/>2 blood cultures obtained from separate sites at least 24 hours apart Single positive blood culture and no other evidence for disseminated infection. Urine culture containing >/1,000 C.F.U./ml in pure culture. Oral, gastrointestinal and/or respiratory colonization, but no documentation of disseminated disease. Negative surveillance cultures and no documentation of disseminated disease, but treated with antifungal agents. Negative surveillance cultures, no documented disseminated infection and not treated with antifungal agents.
13
70
48
50
67
33
60
60
20
89
79
24
92
100
32
2. Transient candidemia
6
3. Candiduria
15
4. Colonized
62
5. Non-colonized, treated
50
6. Non colonized non treated
46
Patients on amphotericin B (%)
Crude mortality (%)
28
* Includes leukemia lymphoma, aplastic anemia, and bone marrow transplantation.
non-neutropenic W B C > 1000/mm3). Of the 23 patients, 16 (70%) received amphotericin B and 20 (87%) received m o r e than four antibiotics per day. Seventeen patients (74%) had renal impairment. Eleven patients (48%) died, and in two patients autopsies were p e r f o r m e d which revealed disseminated candidiasis. Group 2 consisted of six patients (47 sera) classified as having transient candidemia. Three patients (50%) had an underlying hematologic malignancy and two (33%) were neutropenic ( W B C < 5 0 0 / m m 3 ) . Five patients (83%) received more than four antibiotics per day and four (67%) were treated with amphotericin B. Five patients (83%) had intravascular catheters and three (50%) had renal impairment. Two of the six patients (33%) died; however, autopsies were not performed. Group 3 consisted of 15 patients (101 sera) classified as having candiduria. Nine of these patients (60%) had an underlying hematologic malignancy and were neutropenic, Fourteen patients (93%) received four or m o r e antibiotics per day, 12 (80%) had an intravenous catheter, and nine (60%) re-
ceived amphotericin B. Seven patients (47%) had renal impairment. Three of the 15 patients died. No autopsies were performed. Group 4 consisted of 62 patients (531 sera) who had mucous m e m brane colonization alone documented by surveillance cultures. Fifty-five (89%) of these patients had hematologic malignancies and 49 (79%) were neutropenic. Forty-nine patients (79%) were treated empirically with amphotericin B for culturenegative fever which had not responded to therapy with broad-spectrum antibiotics. Fifty-seven patients (92%) had intravenous catheters and 35 (56%) had renal impairment. Autopsies of eight of 15 patients from this group who died did not show evidence of disseminated candidiasis. Group 5 comprised 50 non-colonized, treated patients (366 sera) who had negative surveillance cultures for Candida spp. and had no evidence of local or disseminated candidiasis. Forty-six patients (92%) had underlying hematologic disease and/or underwent bone m a r r o w transplantation and 42 (84%) were neutropenic. All patients in this group received amphotericin B empirically
156 for culture-negative fever which persisted despite therapy with broad-spectrum antibiotics. Fortyeight patients (98%) received more than four antibiotics per day, 46 (92%) had an intravascular device and 19 (35%) suffered renal impairment. Autopsies of five of 15 patients from this group who died did not show evidence of disseminated candidiasis. Group 6 comprised 46 non-colonized, non-treated patients (78 sera), who had negative surveillance cultures and no evidence of local or disseminated candidiasis. Thirteen of these patients (28%) had hematologic malignancies and 10 (22%) were neutropenic. None of the patients received amphotericin B, 12 (26%) received more than four antibiotics per day, 15 (33%) had an intravenous catheter and nine (20%) had renal impairment. Autopsy of one of four patients from this group who died did not show evidence of disseminated candidiasis.
Candida antigen assay. The Cand-Tec Candida detection assay (Ramco Laboratories, Houston, TX) detects poorly defined, heat-labile Candida antigens and was performed according to the manufacturer's instructions as described previously [28]. All sera were tested at an initial dilution of 1:2 in glycine-buffered saline (pH 8.4). The test was considered positive if the dilution showed clear agglutination and negative when there was no visible agglutination of the latex particles. Positive sera (agglutination at 1 :2) were serially diluted in glycine-buffered saline and tested with the Cand-Tec reagent. The final result was defined as that dilution of serum in buffer that still produced obvious agglutination. Positive and negative controls were included in every run. All sera with titers of 1:2 or greater were further tested for non-specific agglutination using a nonimmune antibody-coated latex control (Meridian Diagnostic, USA). The manufacturer recommends that a titer of/>1:4 be considered as positive evidence of disseminated candidiasis; however, for purposes of this study results were calculated using titers of >~1:2, I>1:4 and ~>1:8 as indicating a positive result.
Stat&tieal analys&. Statistical analysis was performed using either the chi-square test with Yate's correction or the two-tailed Fisher's exact test for comparison of proportions. Pearson's correlation coefficient was used to analyze the possible correlation between Candida antigen titers and serum creatinine. Values of p < 0.05 were considered significant. Sensitivity, specificity and predictive values were calculated by the method described by Galen and Gambino [37].
Results
Disseminated candidiasis (Group 1). Twentythree of 202 patients (11%) were diagnosed as having disseminated candidiasis (Table 2). Fifteen patients (65%) had positive blood cultures, seven patients had positive cultures of normally sterile body fluids (peritoneal fluid and pleural fluid) including two with positive blood cultures, six patients (26%) had positive deep tissue biopsies (blood cultures were also positive in four of these patients), and one patient was diagnosed at autopsy. In contrast with previous studies from this institution [28], only three patients (13%) had an underlying hematologic disorder, four (17%) had solid tumors and 16 (70%) had other underlying diseases (Table 2). Of the 23 patients with disseminated candidiasis, 18 (78%) were infected with a single species of Candida: 12 with C. albicans, four with C. glabrata, one with C. lusitaniae, and one with a Candida species detected by histopathology only. Five patients were infected with more than one species of Candida, including one with C. albicans and C. krusei, one with C. albicans and C. glabrata, one with C. albicans, C. glabrata, and C. parapsilosis, and one with C. albicans, C. parapsilosis, and C. tropical& (Table 2). We performed 176 Candida antigen determinations in this group (mean 7.6 tests per patient). Peak antigen titers were >~1:2 in 20 patients (87%), />1:4 in 16 patients (70%), and i>1:8 in seven patients (30%) (Tables 2 and 3). Five pa-
157 Table 2. Antigen titers determined with the Cand-Tec latex agglutination test in patients with disseminated candidiasis Patient number
Underlying disease
Evidence of infectiona
Candida species
Peak titer
Outcome
1 2 3 4 5 6 7 8 9
Hepatitis Acute abdomen Diabetes Cervical cancer Lung cancer Bowel obstruction Acute abdomen Lymphoma Ischemic bowel
SBF SBF BC BC SBF BC HP HP SBF
Died Survived Survived Survived Died Survived Survived Died Died
Renal transplant Multiple sclerosis Bowel obstruction Neuroblastoma Abdominal
HP BC BC BC BC, HP
NS c 1:4 1:16 Negative 1:2
Survived Survived Died Survived Survived
15
Neurosurgery
BC
1 :8
Died
16 17
Stroke Gastrointestinal Hemorrhage Short-gut syndrome Bacterial peritonitis Systemic lupus erythematosus Coronary heart disease
BC, SBF BC, HP
1:16 1:4
Died Died
BC, SBF SBF BC
glabrata albicans albicans glabrata glabrata albicans albicans species b albicans glabrata albicans lusitaniae albicins aIbicans aIbicans glabrata tropicalis albicans parapsilosis tropicalis albicans albicans krusei glabrata albicans albicans
1:8 1:4 1:8 1:4 1:4 1:4 1 :2 1:8 Negative
10 11 12 13 14
C. C. C. C. C. C. C. C. C. C. C. C. Cl C. C. C. C. C. C. C. C. C. C. C. C. C.
1:4 1:2 1:32
Survived Died Died Died
BC, HP BC
glabrata parapsilosis albicans albicans albicans
1:4
Leukemia Lymphoma
C. C. C. C. C.
1:2 1:32
Survived Survived
18 19 20 21
22 23
BC, HP
BC = blood culture, HP = histopathology, SBF = sterile body fluid other than blood. b Seen on HP only, not identified to species. c N S = non-specific agglutination.
Table 3. Candida antigen titers determined with the Cand-Tec test in 202 high risk patients Diagnostic groups
1. 2. 3. 4. 5. 6.
Disseminated candidiasis* Transient candidemia Candiduria Colonized Non-colonized, treated Non-colonized, non-treated*
No. of patients
23 6 15 62 50 46
* Two patients had non-specific agglutination in all sera.
Number of patients (%) with peak titer of />1:2
/>1:4
/>1:8
20(87) 5(83) 9(60) 44(71) 32(64) 24(52)
16(70) 2(34) 6(40) 26(42) 16(32) 6(13)
7(30) 1(17) 2(13) 9(15) 6(12) 2(4)
158 tients (21%) had one or two positive (>1:2) sera and 15 (65%) had three or more positive sera during the course of their hospitalization. One patient with positive blood cultures for both C. albicans and C. glabrata and one with C. albicans fungemia had negative titers. One additional patient with C. albicans fungemia showed nonspecific agglutination in all sera tested. Ten of the 17 patients (59%) with disseminated infection due to C. albicans had antigen titers >1:4 compared to six of six patients infected with other species of Candida (p > 0.05 for the comparison of C. albicans infected patients with all others). Nine (64%) of 14 patients with a peak titer of ~1:8
(p > 0.05). Twelve patients had more than two serum samples obtained during treatment for disseminated disease. Antigen titers increased at least twofold during the course of therapy in five patients and three (60%) of these patients died with disseminated infection. In contrast, antigen titers decreased by at least two-fold in seven patients during therapy and only two (29%) died of the infection. These results were not significantly different (p = 0.31). We determined the temporal relationship between positive antigen titers and positive culture results for Candida in the 20 antigen-positive patients with disseminated infection. Sera were positive for antigen at titers of >1:2 at least one day prior to diagnosis by culture or biopsy in seven (35%) and on the same day in four (20%) patients. Of the remaining nine patients (45%), five had positive titers between one and three days after positive cultures or biopsy and four had titers which were positive > 4 days after positive cultures. The relationship between renal function and Candida antigen titer was examined in Group 1 patients by matching the antigen titer with the corresponding serum creatinine determination. The Pearson's correlation coefficient failed to show a significant relationship between these values (r = 0.43, p = 0.15).
Table 4. Analysisof the Cand-Tec latex agglutinationtest as a predictor of disseminatedcandidiasis* Candida antigen threshold titer >1:2 Sensitivity (%) 87 Specificity(%) 36 Positive predictive value (%) 15 Negative predictive value (%) 96
>1:4 70 60 18 94
>1:8 30 85 21 90
* Prevalence of disseminatedcandidiasiswas 11%.
Disseminated candid&sis patients (group 1) in comparison with other diagnostic groups. Circulating antigen was also apparently detected in a number of patients without documented invasive infection (Groups 2 to 6, Table 3). Overall, 64% of patients in Groups 2-6 had titers >1:2, 31% had titers >1:4 and 11% had titers >1:8 (p < 0.05 for all comparisons versus Group 1). Analysis of the number of antigen-positive patients in Group 1 versus each diagnostic group at each of the threshold antigen titers (>1:2, >1:4, >1:8) revealed significant differences between Group 1 and 4 at >1:4, between Group 1 and 5 at >1:2 and >1:4, and between Group 1 and 6 at all three threshold antigen titers. There was no significant difference in antigen titers between Group 1 and Group 2 (transient candidemia) and between Group 1 and Group 3 (candiduria). Although a number of patients in Groups 2 to 5 received empiric amphotericin B therapy, disseminated candidiasis was not documented in any of them. Autopsies were performed in 13 of the 35 patients who died in Groups 2 to 5, and none demonstrated Candida invasion.
Predictive value of Candida antigen titers. The prevalence of documented disseminated candidiasis was 11% (23 of 202 patients). In analysis of the Cand-Tec latex agglutination test as a predictor of disseminated candidiasis (Table 4), the greatest sensitivity was obtained using a threshold titer of >1:2 (87%). This result was higher than that obtained with a threshold titer of either/> 1:4 (70%, p > 0.05) or >1:8 (30%, p < 0.05). The
159 specificity of the Cand-Tec test improved significantly using threshold titers of >~1:4 (60%) or I>1:8 (85%) compared to ~>1:2 (36%, p < 0.05). The positive predictive value was low (15-21%) for all threshold titers. The application of a higher threshold titer did not significantly improve the positive predictive value of the test relative to the low (~>1:2) threshold titer (p < 0.05 for all comparisons). The negative predictive value was significantly improved when the low (/>1:2) threshold titer was used (96%, p < 0.05) compared to either/>1:4 (94%) or ~>1:8 (90%).
Discussion
Given the problems associated with the clinical and microbiologic diagnosis of disseminated candidiasis it is reasonable to examine carefully the potential role of antigen detection as a means of early diagnosis of this important infection. The Cand-Tec latex agglutination assay for detection of circulating Candida antigen is a commercially available assay which is relatively inexpensive, technically simple, and less time consuming to perform than previously described antigen detection assays [4, 17, 19-21, 25]. We and other investigators have evaluated this test for the diagnosis of deep-seated candidiasis over the past seven years and the results have shown many discrepancies (Table 5). The variation in sensitivity and specificity obtained with the Cand-Tec assay in the different published studies is tremendous. The reported sensitivities range from 19% to 100% with specificities of 29% to 99% (Table 5). These differences are certainly due in large part to the differences in definition of disseminated candidiasis and the selection of cases and controls employed in the various studies. The diagnostic sensitivity of the assay may be impacted by the transient nature of the antigenemia which requires multiple sera to be collected serially in order to maximize detection of circulating antigen [14]. The apparent lack of specificity of the assay in certain studies has been more difficult to explain and has
been further impaired by the undefined nature of the heat-labile antigen. Ness et al. [26] reported the lowest specificity of 29% obtained in a highly selected population of bone marrow transplant patients. They found an apparent association of antigenemia with elevated serum creatinine. This observation suggests that antigens accumulate in the serum of patients with renal failure and may explain both the poor specificity in the study of Ness et al. [26] and the variable specificity observed by other investigators (Table 5). The present study was performed in an effort to extend the findings of our previous study [28] on the role of the Cand-Tec test in the diagnosis and therapy of candidiasis in hospitalized patients. In our previous study we found a high sensitivity (95%) and negative predictive value (98%) but a poor specificity (50%) and positive predictive value (31%) when a threshold titer of />1:2 was employed, suggesting that the test may be useful as a screening test in high risk patients. More importantly, we found that the determination of serial antigen titers during the course of antifungal therapy in patients with documented disseminated infection was useful in monitoring the effectiveness of treatment. In contrast, the sensitivity, specificity and positive predictive values obtained in the present study were all considerably lower than that observed previously regardless of the threshold titer employed (Tables 4 and 5). Furthermore, the present study failed to confirm the usefulness of serial antigen determinations in monitoring the effectiveness of antifungal therapy. The reasons for these differences are unclear but may be due to differences in the populations studied. Seventy-three percent of the patients with disseminated candidiasis (Group 1) in our previous study had an underlying hematologic malignancy or bone marrow transplantation and were neutropenic, whereas only 13% of Group 1 patients in the present study suffered from these underlying conditions. Thus, the clinical and antigenic response to antifungal chemotherapy may be considerably different in the predominantly neutropenic patients comprising Group 1 in the previous study versus the non-
160
Table .5. Summary of the results of published studies using the Cand-Tec test for the diagnosis of disseminated candidiasisa Number of patients References Kahn and Jones [14], Group B Bailey et al. [23] Escuro et al. [24] Kahn and Jones [14], Group A Bougnoux et al. [21] Ness et al. [26] Burnie and Williams [27] Lemieux et al. [25] Present study Cabezudo et al. [281 Piens et al. [29] Gentry et al. [30] Price and Gentry [31]u Fung et al. [32]
Disseminated infection
No infection
32
118
19
87
18 29 23
3i 113 74
28 38 48
97 77 89
22 11 30 28 23 37 9 33 11 6
142 41 481 184 179 158 95 154 117 18
50 55 67 68 70 73 78 91 100 100
92 29 94 92 60 72 91 99 51 88
Sensitifivity (%)
Specificity(%)
a Latex agglutination titer/>1:4 considered positive. b Includes only high-risk patient population.
neutropenic G r o u p 1 patients in the present study. The large n u m b e r of false-positive results obtained in this study is consistent with the findings of Cabezudo et al. [28], Ness et al. [26] and Price and G e n t r y [31] (Table 5). Most notable was the observation that 60-71% of patients with candiduria ( G r o u p 3) or mucosal colonization ( G r o u p 4) had sera with titers of i>1:2 (Table 3). This substantiates our previous observation that patients who are colonized or who have localized, but not disseminated, infections m a y have circulating antigen presumably due to defects in the oral, gastrointestinal or genitourinary mucosa [281. It has been suggested by Ness et al. [26] and Bougnoux et al. [21] that antigens detected by the Cand-Tec assay are accumulated in the serum of patients with renal failure and therefore titers are increased but meaningless. Analysis of the relationship between serum creatinine and antigenemia in b o t h the earlier study of Cabezudo et al. [28] and in the present study failed to support this contention. We did not observe a significant correlation between antigenemia and elevated
serum creatinine and thus cannot ascribe the false-positives observed in our patients to impaired renal function. In conclusion, we were unable to establish a firm basis for the clinical utility of the Cand-Tec Candida antigen test in the diagnosis and therapy of disseminated candidiasis. This is in contrast to our previous fndings [28] and in agreement with the conclusions of other investigators [14, 21, 2 3 26]. The m o d e r a t e sensitivity, p o o r specificity and positive predictive value coupled with a lack of prognostic utility suggests that the assay has little, if any, clinical utility and in fact m a y not be measuring a Candida-specific antigen. Additional efforts to develop rapid, culture-independent methods for the diagnosis of disseminated candidiasis should continue; however, any new products must be subjected to rigorous clinical trials prior to their application in hospitalized patients.
Acknowledgements We acknowledge the excellent assistance of the nurses, physicians assistants, and medical staff of
161
the various clinical services at the University of Iowa Hospitals and Clinics. The technical expertise and assistance of the medical technologists in the Special Microbiology Laboratory and the secretarial skills of Bea Dacy are also greatly appreciated.
17.
18. 19.
20.
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Address for correspondence: Michael A. Pfaller, MD, Department of Clinical Pathology, L471, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201-3098, USA. Tel: (503) 494-4105.
