or antibody detection kits

Journal of Microbiological Methods, ! 6 (1992) 231-237 ~ 1992 Elsevier Science Publishers B.V. All rights reserved 0167 - 701 '"'¢n~,./~,,~/.~v_,.uu""

231

MIMET 00531

Rapid diagnosis of systemic candidiasis using commercial antigen and/or antibody detection kits Kevin K. Smith and S.M. Hussain Qadri King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia (Received 12 February 1992; revision received 10 June 1992; accepted 12 June 1992)

Summary Systemic candidiasis is a life-threatening disease seen with increasing frequency in hospitalized patients. There has been considerable interest in rapid serological diagnosis, including assays for metabolites and circulating antigens and antibodies. We evaluated a commercially available antigen detection kit (Ramco Labs, Houston, TX) in combination with a commercially obtained antibody test (Allergy Immuno Technologies, Newport Beach, CA). Using the combination for the diagnosis of systemic candidiasis, 81% of patients gave serological indication of infection with one or both methods, confirmed by culture. The antigen titer on its o,,~.a had a positive and negative predictive value of 80 and 91% using a titer of t> 1:8 for a positive value and ~<1:2 for a negative value. Use of high IgM, high lgG and a positive antigen titer >t 1:8 gave a positive predictive value (PPV) of 99% with a sensitivity of 14% and specificity of 99%. Colonised, patients with candidemia and culture-negative patients were positive in 15, 32 and 27% of cases, respectively. Combined rapid antigen and antibody testing had a negative predictive value of 88%.

Key words: Candida spp.; Candidiasis detection; Serodiagnosis; Antibody to Candida albicans

Introduction

Candida species are commensals in the gastro-intestinal tract and normal resident flora of the skin, oral cavity and female genitalia. They are opportunistic organisms, capable of invading the body via trauma or persorption [1,2]. The incidence of systemic candidiasis has increased with the use of broad-spectrum antibiotics, immunosuppression, intravenous drug abuse, invasive techniques such as catheterization, and trauma due to burns and surgcry [3,4]. The diagnosis of deep seated candidiasis is difficult and often depends primarily on the clinical picture, with definitive diagnosis established by histopathology of visceral tissue [4]. Cultures for Candida must be interpreted with caution in order to avoid confusion between colonization, transient candidemia and systemic candidiasis. Blood cultures have Correspondence to: S.M. Hussain Qadri, Microbiology Labs., King Faisal Specialist Hospital and Research Centre, MBC-10, P.O. Box 3354, Riyadh 11211, Saudi ~,rabia.

232 been shown to be negative in 40-80% of patients with systemic candidiasis [5-7]; positive cultures could result from transient candidemia commonly associated with colonized indwelling intravenous catheters. However, immunosuppression or a high level of circulating yeast cells in a patient can lead to systemic infection [3]. Early diagnosis and treatment with antifungal drugs is necessary to reduce the incidence of mortality because delays can result in poor prognosis [8,9]. Early diagnosis to predict the absence of disease also reduces unnecessary antifungal therapy and the sideeffects from drug toxicity and synergistic toxicity in transplant patients receiving cyclosporin A [4]. In the present study, we evaluated a combination of two commercially available systems for the detection of systemic candidiasis. They consist of the Cand-Tec Candida antigen latex agglutination and Candiquant antibody ELISA for IgG and IgM classes against a purified cytoplasmic extract of C. albicans. Materials and Methods

Patients and sera Patient sera were collected over a 2-year period in suspected cases of candidiasis. Sera were immediately tested for antigens and then stored at - 2 0 ° C for ~uture antibody testing in batches. Patients were considered to have systemic candidiasis based on positive blood cultures over multiple days without evidence of contaminated catheters, culture-positive with positive histopathology, or a single C. tropicalis blood culture associated with positive surveillance cultures in neutropenic patients. Candidemia was diagnosed as either a single positive blood culture or multiple cultures positive due to colonization of an indwelling catheter. Colonization with Candida was determined from surveillance cultures of respiratory, fecal, urine, surface skin, or genital sites, or by the determination of a non-invasive infection of peritoneal or ascitic'fluids. 20 patients with respiratory candidiasis and 10 with vaginal candidiasis were also included in the study. Control subjects were patients at risk of fungal infection due to immunosuppression or treatment with broad-spectrum antibiotics without clinical evidence of infection and negative surveillance cultures; 15 normal healthy laboratory, workers were also included as controls. Antigen assay The presence of a heat-labile glycoprotein associated with Candida infection was detected using the Cand-Tec (Ramco Labs, Houston, TX) latex agglutinatiou assay. In the procedure, 20 pl of 1:2 diluted sera and 20/~1 of latex reagent were mixed on glass slides, and rotated for 10 min at 140 rpm. Results were read macroscopically, and positive sera were serially diluted to determine the endpoint. Antibody assay The presence of Candida antibodies was determined using ELISA kits from Allergy Immuno Technologies (Newport Beach, CA) which uses a purified cytoplasmic extract of C. albicans immobilized onto microwells. In the ELISA test, 100 pl of serially diluted positive control, negative control or specimen diluted 1:200 with sample diluent were added to each well of a microtiter plated with antigen. After incubation for 1 h at room temperature, the plates were washed five times with an

233 automatic plate washer. 100 pl of enzyme conjugate was added to all wells and incubated for 1 h at room temperature and washed five times. Then i00 /ai of substrate was added, incubated at room temperature for 30 min and finally 50/~1 of stop solution added. Absorbance was read at 405 nm. Calculations were performed by dividing the results of patient or positive control by the negative control. Resulting patient ratio values were compared with the positive control ratios to semi-quantitate.

Cultures Sterile fluids, blood and tissues were cultured using Bactec 6A bottles and growth was monitored on Bactec NR660 (Becton-Dickinson, Towson, MD). Other sterile sources were cultured using a combination of solid and liquid media including Sabouraud and Mycosel agar. All isolates were presumptively identified using the germ tube test and further ~peciated using the AutoMicrobic System yeast cards (Vitek Systems, Inc., Hazelwood, MO). Statistical analysis Sensitivity, specificity and predictive values were calculated as previously described [10]. Results

To evaluate the cross-reactivity between different Candida species and the C. albicans used for antigen and antibody preparation in the kits, we evaluated the mean titers between species causing systemic candidiasis. The mean titers in the CandTec test for C. albicans were 1:6.6, for C. tropicalis 1:5.2 and for C. parapsilosis 1:16, indicating that the Cand-Tec antigen was common for these species. Two of the 14 C. albicans and three of nine C. tropicalis patients gave high IgG and high IgM response. One of the C. parapsilosis p~ti,~nt~w ~1~,~~"i'4'~'~A~ hioh~,..Tgr,. ... and IgM .r~'~pnn~e-~ Of_. . .the._ . illllllllll,,I.lll~,.#li~l~llrllll.,.tl'll..#lllll,,,~lli~li.,i

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albicans showed a high IgG and IgM antibody response. Excluding immunocompromised patients, 22°/'0 of C. albicans, 43% C. tropicalis and 50% of C. parapsilosis patients had levels of antibody significant for diagnosis of systemic candidiasis, demonstrating the cross-reactivity between Candida species and the Candiquant antigen. A breakdown of antigen and antibody results for each classification of Candida exposure is shown in Table 1. The Cand-Tec result was a good indicator for differentiating systemic candidiasis from candidemia at a titer of 1:4 where 82% of systemic candidiasis patients were positive, but 93% of candidemia patients were negative. Diagnosis was more difficult when blood cultures were negative, but systemic candidiasis could not be ruled out without visceral organ biopsies. High IgM levels also appeared to be a good indicator for differentiating systemic candidiasis from candidemia, where the combination of a high IgM with a positive blood culture had a PPV of 99%. The sensitivities, specificities and PPV's for various antigen and antibody combinations are shown in Table 2. Of the four patients that were culture-negative and had Cand-Tec titers of 1:8, none had high anti-Candida IgG and IgM level, all were considered immunocompetent and only one was positive

_.~4 TABLE ! Antigen and antibody results Candida infection

Antibody level

Cand-Tec titer Neg.

1:2

i:4

>/1:8

IgM

IgG Neg

Lo

Hi

Neg

Lo

Hi

Systemic (n = 28)

3

2

7

16

I!

9

8

i!

7

10

Candidemia

I0

4

0

1

5

8

2

9

6

0

19

9

3

3

8

20

6

15

13

6

9

8

7

4a

17

7

4

16

8

4

15

0

0

0

7

5

3

5

7

3

(n = 15) Colonization (n = 34) No Candida isolated

(n = 28) Healthy lab workers 01 = 15) ~ All four had titers of 1:8. One patient had a non-viable yeast on sputum G r a m stain, one patient expired prior to further studies, one patient was receiving amphotericin B and the last patient had an overgrowth of Staplo,lococcus aureus on all blood cultures; systemic candidiasis was not suspected.

for rheumatoid factor by enzyme immunoassay. Overall, C. albicans accounted for 50% of systemic candidiasis, 57% of candidemia and 91% of the colonized patients as shown in Table 3. C. tropicalis was the second most frequent isolate being found in 32% of systemic candidiasis and 33% of e~n,-lide,,.,ia " +,', p u r u p--;, . .one. eacn . . of t~. . . . . . . . . . . . . . . . .~+h~.,. organisms ,e,..., .u.~ ua :_~i..a_a ,n~,uucu ..... two w. s u o .s t s ,. and glabrata, C. guilliermondii and C. humicola. Discussion

Sandford et al. [11] showed that fungal surveillance cultures were good indicators for predicting C. tropicalis disease but gave no good predictive values for C. albicans because the number of patients colonized far exceeded the number with clinical disease. Unavailability of deep organs for culture or histology coupled with prolonged time for culture to become positive has led many laboratories to explore rapid methods for diagnosing systemic candidiasis. Such tests include monitoring antibody levels, antigen detection using cell wall mannan, a heat labile glycoprotein and metabolite detection using gas liquid chromatography [1,3,12-21]. Unfortunately, many small laboratories do not have the ability to perform some of these tests due to lack of specialized equipment and expertise required for methods like gas-liquidchromatography, preparation of in-house reagents, etc. Previous investigators, using a titer of 1:4, found sensitivities of 45-91% for the Cand-Tec antigen and specificities of 90-100% [1,12,16]. PPV's ranged from 17 ~.o

235 TABLE 2 Efficiency of antigen and antibody results for systemic candidiasis Antigen and antibody combination

Sensitivity

Specificity

Positive predictive value

IgM high positive Cand-Tec /> 1:2

99.9

92.9

71.4

lgM high positive Cand-Tec >! !:4

80.0

95.1

66.7

IgM high positive Cand-Tec >i 1:8

60.0

98.7

85.7

IgM high positive IgG high positive Cand-Tec 1>i:4

i8.5

96.3

62.5

IgM high positive IgG high positive Cand-Tec >t 1:8

14.0

99.9

99.9

igM high positive IgG high Positive

22.2

96.3

66.7

IgM high positive

5;5.7

90.1

50.0

Cand-Tec >t 1:4

81.5

80.4

59.5

Cand-Tec f> 1:8

59.3

92.9

80.0

81% although m a n y workers used specific patient populations such as neutropenic or i m m u n o c o m p r o m i s e d subjects [13,17,21,22]. In a general hospital setting, including all patients, we found a PPV of 59.5%. Using a cut-off titer of 1:8 other investigators reported sensitivities of 4 5 - 7 1 % , specificities of 3 4 - 9 8 % and PPV o f 16-79% [13,16,21]. According to the m a n u f a c t u r e r of EIA high IgG and IgM level indicated TABLE 3 Incidence of C a n d i d a species cultured Candida

species cultured

C. a l b i c a n s

Patient grouping Systemic candidiasis

Candidemia

Colonization 31

14

8

C. t r o p i c a l i s

9

5

1

C. p a r a p s i l o s i s

2

-

-

C. g l a b r a t a

1

-

-

C. guillie, m o n d i i

-

1

-

C. h u m i c o l a C a n d i d a species not a l b i c a n s

i 1

1

3

236 an active Candida infection, whereas we found a specificity of 96.3%, a sensitivity of 22.2% but a PPV of only 66.7%. Ramco Labs indicates that a Cand-Tec titer of >t 1:4 is suggestive of systemic candidiasis while we found that with this titer the sensitivity was 81.5%, specificity 80.4% and PPV 59.5%. Increasing the cut-off titer to >~ 1:8 increased the PPV to 80%, specificity to 92.9% but lowered the sensitivity to 59.3%. The only acceptable PPV we found for the diagnosis of systemic candidiasis without clinical signs and symptoms was the combination of a high lgG and IgM level with an antigen titer of t> 1:8. However, a high lgM or associated with a positive Cand-Tec test or a Cand-Tec titer of >t 1:8, gave acceptable results for diagnostic use when clinical features of the patient were also taken in consideration. The results of antibody tests or Cand-Tec results ~< 1:4 alone were not useful for diagnostic purposes.

Acknowledgements The authors wish to thank Ms. Amy Pering for secretarial assistance and Mrs. Marilyn Smith for literature search.

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