Zbl. Bakt. Hyg., I.Abt. Orig. A 247,537-542 (1980)

Department of Biology, Guru Nanak Dev University, Amritsar-143005, India

Efficacy of Counterimmunoelectrophoresis in the Detection of

Fungal Antibodies in Allergic Bronchopulmonary Mycoses!

Nutzen der Gegenstromimmunelektrophorese bei der Feststellung von Antikorpern bei allergischen bronchopulmonalen Mykosen

S.K. MEHTA and R.S. SANDHU

With 1 Figure· Received February 15, 1980

Abstract

The allergic bronchopulmonary mycoses (ABPM) are characterized by dual antibody response which includes immediate Type I and delayed Type III Arthus reactions to fungal antigens. In such cases the demonstration of precipitins is of diagnostic importance. In the present study the efficacy of counterimmunoelectrophoresis (CIE) has been compared with that of conventional agar gel double immunodiffusion technique (DID). Cases of allergic bronchopulmonary aspergillosis (ABP A) and candidiasis (ABPC) were diagnosed in a survey among patients with pulmonary diseases on the basis of positive clinical, radio­logical, mycological and immunological findings. Precipitating fungal antibodies were detected by CIE and DID techniques in 77 and 59 cases of ABPA and ABPC respectively. The former technique revealed double or multiple bands as against a single band observed in double immunodiffusion in a great majority of these cases. With CIE it was further possible to demonstrate the presence of precipitins in about 65 per cent of suspected cases of ABPM found otherwise negative in DID tests. The CIE has proved to be a more effica­cious serodiagnostic procedure in allergic bronchopulmonary mycoses in which antibody levels are known to vary a great deal.

Zusammenfassung

Allergische bronchopulmonale Mykosen zeigen charakteristischerweise eine zweifache Antikorperantwort einschlieBlich von Sofortreaktionen vom Typ lund verzogerten Reak­tionen vom Typ III nach Arthus auf Pilzantigene. In solchen Fallen ist der Nachweis von Priizipitinen von diagnostischer Bedeutung. In der vorliegenden Untersuchung wurde der Nutzen der Gegenstrom-Immunelektrophorese (CIE) dem der konventionellen doppelten Immundiffusionstechnik in Agargel (DID) gegeniibergestellt. Bei einer Erhebung unter

1 Paper presented at the First All India Joint Conference of Immunology and Allergy held at Srinagar (India), November 4-6,1979.

538 S. K. Mehta and R. S. Sandhu

Lungenpatienten wurden auf Grund positiver klinischer, radiologischer, mykologischer und immunologischer Befunde FaIle allergischer bronchopulmonaler Aspergillose (ABPA) und Candidiasis (ABPC) diagnostiziert.

Mit der CIE- und DID-Technik wurden bei 77 bzw. 59 Fallen von ABPA bzw. ABPC prazipitierende Antikorper festgestellt. Das erstgenannte Verfahren zeigte doppelte oder mehrfache Priizipitationslinien, wogegen die Doppel-Immundiffusion bei der Mehrzahl die­ser FaIle eine einzige Linie ergab. Weiterhin war es mittels CIE moglich, das Vorhandensein von Priizipitinen bei ca. 65% der ABPM-Verdachtsfalle nachzuweisen, die im DID-Test negativ waren. Die CIE erwies sich als effizienteres serodiagnostisches Verfahren bei aller­gischen bronchopulmonalen Mykosen, bei denen die Antikorperspiegel bekanntermagen erheblich schwanken.

Introduction

The detection of antibodies in fungal infections is achieved by employing various serological techniques. The double immunodiffusion (DID) is a more commonly used test in the diagnosis of allergic bronchopulmonary aspergillosis (ABPA) al­though in about 30 per cent of such cases the results remain negative primarily due to inadequate concentration of antibodies in the test sera. Bardana et al. (1), with the help of an extremely sensitive technique of primary binding, have shown a universal occurrence of fungal antibodies even in the sera of the normal subjects. Their method proved complementary to DID in ABPA (17). Recently, some scien­tists have obtained better results with counterimmunoelectrophoresis (CIE) in the serodiagnosis of fungal infections, particularly in the patients with comparatively low levels of specific precipitating antibodies (2-7,9,11-13,16,20,21). In this tech­nique favourable conditions are created for a manifest antigen-antibody reaction in the gel by unidirectional migration of antibodies under the influence of an electric field as against radial diffusion in DID. In the present study the comparative efficacy of CIE and DID has been evaluated in the serodiagnosis of allergic bronchopulmo­nary mycoses (ABPM) due to Aspergillus species and Candida albicans.

Materials and Methods

Sera: The study was made by examining the sera of patients with chronic chest diseases which included, bronchial asthma, tropical eosinophilia and pulmonary tuberculosis. The patients were registered at the V.P. Chest Institute and Rajan Babu T.B. Hospital, Delhi and T.B. and Chest Diseases Hospital, Amritsar. Sera taken from 120 healthy regular employees of Guru Nanak Dev University, Amritsar represented the control group. The sera were stored at - 18°C.

Antigens: Antigens were prepared from the culture filtrates of one strain each of Asper­gillus fumigatus, A. flavus, A. niger, A. nidulans and A. terreus, grown in glucose-aspara­gine broth as described earlier (8, 19). Candida albicans was grown in shake-culture in Sabouraud's broth at 26°C for one week. The culture filtrate served as the antigen source (10). The potency of each batch of antigen was tested against specific rabbit hyperimmune sera.

Double Immunodiffusion (DID): Six ml of 1 per cent Difco noble agar in McIlvaine citrate buffer, pH 7.3, was layered on a 75 x 50 mm glass slide (8, 14). One large central and six small peripheral wells were cut on the agar gel with a brass template (Fig. 1 a). The peripheral wells were filled with different antigens and the central well contained the

Counterimmunoelectrophoresis in ABPM 539

test serum. These plates were incubated in ~. moist chamber at 26 °C. The precipitin band usually appeared within 24-72 h. . Counterimmunoelectrophoresis (CIE ): It was carried out according to the method of Galussio et al. (4). A coat of 8 ml of one per cent Difco noble agar in veronal buffer, pH 8.2, was layered on a slide (75 x 50 mm). Six pairs of wells (3 mm in diameter) in two parallel rows 1.5 cm apart were cut. The wells near the cathode were filled with different antigens and those near the anode were filled with the test sera. The plates so prepared were placed in the electrophoresis tank kept in a refrigerator. Wicks of Whatman paper No.1 were used to bridge the gel with buffer. A current of 10 rnA per plate at 220 volts was allowed to pass for 90 min. The precipitin bands were stained with amido schwarz after thorough washings with distilled water and differentiated with 2 per cent acetic acid (Fig. 1b).

Criteria for the diagnosis : The diagnosis of allergic bronchopulmonary aspergillosis or allergic bronchopulmonary candidiasis (ABPC) was made in a patient if he /she had a history of asthma along with dual skin hypersensitivity to Aspergillus species and/or Can­dida albicans and gave evidence of: (1) pulmonary infiltrations with blood eosinophilia (2) positive sputum culture, and (3) demonstration of specific precipitins against Aspergillus species and/or C. albicans.

The cases in which one or more of the above mentioned three criteria were lacking, were labelled as suspected cases of ABP A or ABPC.

Results

The results obtained in the present study on 326 sera are summarized in T able 1. These serum samples were taken from 77 cases of ABPA, 42 of sus­pected ABP A, 59 of ABPC, 28 of suspected ABPC, and 120 normal subjects. The precipitins to Aspergillus fumigatus were demonstrated in all the sera (100%) from patients with ~J3PA by both the CIE and DID techniques. However, the precipitin bands were usually multiple in CIE as compared to single or occasional double hands in DID. Out of 42 cases of suspected ABPA, 28 (66.6%) showed precipitins against A. fumigatus in CIE alone and none in DID tests. Additional bands specific

Table 1. Detection of serum precipitins against Aspergillus fumigatus and Candida albicans in allergic bronchopulmonary aspergillosis (ABPA) and allergic bronchopulmonary candi-diasis (ABPC) by counterimmunoelectrophoresis (CIE) and double immunodiffusion (DID)

Sr. Clinical category Number Sera showing precipitin bands No. of cases CIE DID

Number % Number %

1. Allergic bronchopulmonary aspergillosis (ABP A) 77 77 (64)," 100 77 (12) 100

2. Suspected ABP A 42 28 (5) 66.6 0 a 3. Allergic bronchopulmonary

candidiasis (ABPC) 59 59 (48) 100 59 (0) 100

4. Suspected ABPC 28 18 (2) 64.3 0 0 5. Normal subjects 120 0 0 0 0

" Parentheses indicate the number of sera showing double or multiple bands

540 S. K. Mehta and R. S. Sandhu

Fig.lb

Fig. 1. Comparison of double immunodiffusion (a) and counterimmunoelectrophoresis (b) demonstrating double and multiple precipitin bands respectively against A. fumigatus with the serum of a patient with allergic bronchopulmonary aspergillosis. In CIE, bands were also present against A. niger. Wells labelled as S contained patient's serum and wells 1, 2, 3,4, 5,6 contained antigens of A. fumigatus, A. flavus, A. niger, A. nidulans, A. terreus and C. albicans respectively.

to other species of Aspergillus were observed in 5 cases against A. f!avus, in 3 cases each against A. niger and A. nidulans and in 4 cases against. A terreus. The CIE technique revealed multiple bands against A. fumigatus in as many as 64 out of 77 patients with ABP A, while sera of only 12 patients showed double bands in DID. Out of 77 ABPA cases, 9 gave 5 bands, 14 gave 4 bands, 25 gave 3 bands, 16 gave 2 bands and 13 gave a single band each in counterimmunoelectrophoresis.

The precipitins against C. albicans were observed in all the 59 cases of ABPC by both CIE and DID techniques. Invariably a solitary diffused (H-type) band was observed in double immunodiffusion test whereas counterimmunoelectrophoresis revealed additional sharp (R-type) bands (15) that is, 7 sera showed 4 bands, 13 showed 3 bands, 16 showed 2 bands and 12 showed one additional band each. Of the 28 suspected cases of ABPC 18 (64.3%) had precipitins (H-type band) against candidin in CIE but none in DID tests.

In none of the cases, the precipitins against Aspergillus species and Candida albicans were simultaneously present.

Counterimmunoelectrophoresis in ABPM 541

Discussion

The quantitative levels of fungal antibodies show great variation in allergic bron­chopulmonary mycoses. However, in view of the fact that even low levels of pre­cipitins have pathogenetic implications in such diseases (15), their detection by sensitive techniques assumes great diagnostic importance. In this context, the pre­sent study demonstrates that CIE is a more efficacious technique. Besides giving positive results in all the cases of allergic bronchopulmonary aspergillosis and allergic bronchopulmonary candidiasis it has succeeded in revealing precipitins in about 65 per cent of the suspected cases as well, which were otherwise uniformly negative by the DID method. The CIE is a more rapid method yielding results within 90 min as against 48 h required in DID procedure. The sensitivity of CIE is further reflected in the multiplicity of precipitin bands which were seen in 83.1 and 81.3 per cent of ABPA and ABPC cases, respectively. The explanation perhaps lies in the fact that antibodies in CIE are not diluted in all directions by radial dif­fusion as is the case in DID plates. They are directed towards the cathode and show manifest reaction with the antigen diffusing from the opposite direction. The tech­nique of CIE is also more convenient for investigating several serum samples simul­taneously on a single plate (11). In order to eliminate nonspecific reactions which involve the serum C-reactive proteins, it is recommended to wash the gels with 5% sodium citrate before staining (9). In the present study, nonspecific reactions were not seen. The CIE does not seem to be an over-sensitive method as may be said of the primary binding test in which significant levels of antibodies have to be determined in relation to the control for diagnostic purposes. The inability of CIE to reveal antibodies in about 35 per cent of the suspected cases of ABPM may be attributed to extremely poor levels of antibodies in such cases. Failure to detect antibodies may not necessarily preclude allergic bronchopulmonary disease due to fungi if other criteria are fairly suggestive (15).

Acknowledgements. A part of this work has been carried out at the Department of Medical Mycology, V.P.Chest Institute, Delhi. SKM is also thankful to the Council of Scientific and Industrial Research, New Delhi, for the award of Research Fellowship.

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Dr. R.S.Sandhu, Professor and Head, Department of Biology, Guru Nanak Dev Uni .. versity, Amritsar-143005, India