iha aspergillus fermigatus

Acta path. microbiol. scand. Sect. B, 82: 871-878, 1974

INDIRECT HAEMAGGLUTINATION FOR DEMONSTRATION OF ANTIBODIES TO

ASPERGILLUS FUMIGATUS OLAV T~NDER and MARIANNE R~DSETIIER

The University of Bergen, School of Medicine, The Gade Institute, Department of Microbiology ; The Broegelmann Research Laboratory for Microbiology,

Bergen, Norway

Subhaemolytic amounts of saline extracts of homogenized, disrupted A. fumigatus sensitized tanned erythrocytes for agglutination to high titres (8,192) by an antiserum to A. fumigatus. Extracts of 5 different strains of A. funigatus were equally active. Erythrocytes sensitized by similarly prepared extracts of P. notaturn and an unclassified Aspergillus strain gave low titres (64 and 128), while extracts of some other fungi and bacteria had no sensitizing activity in tests with the antiserum. Results of absorption and inhibition experiments using the various preparations indicated that the sensitizing antigen(s) in extracts of A. fumigatus may be species specific. Results obtained with 441 human sera showed good correlation between high titres and presence of aspergillosis. Titres above 128 are highly indicative of active disease.

The diagnosis of human aspergillosis is based on clinical investigations, X-rays, repeated demonstrations of Aspergillus (A.) in pathological material, and serological and skin tests (18) with various antigens. How- ever, repeated isolations of fungus from sputum is not in itself proof that aspergillosis is present (14). Even antibodies in serum can be of doubtful diagnostic value, the most important clue is increase or decrease in titres.

Various serological tests have therefore been used: Complement fixation ( 1, 13, 21, 22) , double diffusion in agar (2, 3, 4, 7, 19), latex agglutination ( 10, 20), collodium agglutination (20) and immuno-electrophore- sis (1, 11, 12, 21). Because extracts of A .

Received 5.vii.74 Accepted 5.vii.74

fumigatus haemolyse erythrocytes, it has been claimed that indirect haemagglutination could not be used (9, 20). Complement fixation and latex agglutination give too low titres to be of diagnostic value (16, 20, 21, 22) and double diffusion in agar has therefore been most widely employed. The interpretation of results can be confusing, however, as cross-reactions with similarly prepared antigens from other fungi are frequent

Since clinical and radiological diagnosis of aspergillosis often encounters difficulties, a sensitive and specific serological technique would be an important tool. Efforts were therefore made to elaborate a haemagglutination test which allows an easv semiquanti- tation of the antibodies. The results are described in the present paper.

(1) .

87 1

M A ' I ' E R I A 1 . S A N D M E T H O D S

Aspergillus Strains Most of the experiments were carried out with

a strain of A. funzigatus isolated in the Department of Microbiology (later called Af). Four strains were obtained from Commenwealth Mycological Institute, Kew, Surrey, England: A . furnigatus I M I 16030, 48338, 131705, and 92877. A strain of Aspergillus isolated in this department and which could not be species determined was included (Aspergillus 3 ) . A . pseudoglaucus and A . niger were kindly provided by the Institute of General Microbiology, University of Bergen, and the State Institute of Public Health, Oslo, respectively.

Other Fungi and Bacteria The following species were included: P . notaturn,

C . albicans, 7'. rubrutn, 7'. schoenleinii, S. aureus, Micrococcus strains and E. coli.

Erythrocytes Whole blood from sheep, rabbit, guinea pig and

chicken were collected in equal amounts of Alse- ver's solution, and human blood in acid-citrate- dextrose solution. l 'he blood was kept a t 4" C. Before use the erythrocytes were washed 3 times in phosphate buffered saline, p H 7.2 (PBS) with centrifugation at 800 x g for 5 min and final packing at 1,000 x g for 10 min.

Formalin treated erythrocytes were prepared as described by Daniel et al. ( 5 ) .

Sera Four groups of human sera were selected as

follows: 1 ) randomly selected sera from blood donors ( 131), 2 ) sera from patients with various respiratory diseases (44), 3) sera from patients with various diseases ( 2 3 1 ) , and 4) sera from patients with definite or possible aspergillosis (35 ) .

The patients of the 3 control groups (1--3) were selected to give an age distribution similar to that in the patient group. They were not mat- ched according to sex.

Most of the sera from patients with respiratory diseases were received from the Department of 1,ting Diseases, Bergen University Hospital.

An antiserum to A . furnigatus Af was raised in a rabbit by immunization with a lyophilized washed culture suspended in PBS. The schedule consisted of weekly injections of 2 ml, the first week given subcutaneously and containing 1.6 mgJml, followed by intramtisciilar injections, and increasing the dose to 20 mg/ml. 'I'he rabbit received 6 injections and then a booster-dose 3 weeks later. Small bleedings were taken before each injection, and larger

bleedings 1 week after the 6th and the 7th injections.

Before use all sera were heated at 56" C for 30 min (inactivated sera).

Cult u res Surface culture: From pure cultures on Sabour-

aud's medium (Difco) the fungi were transferred to Roux bottles containing yeast extract medium (15) . 'l'he bottles were incubated at 37" C until a continuous mat of growth was established on the surface. The necessary incubation time varied from 3 days to 5 weeks, most of the Aspergillus strains had the shortest, and T . schoenleinii the longest time. C . albicans was only grown on Sabotiraud agar plates and the bacteria on nutrient agar p 1 ate s .

Suspension culture: Fungi were transferred to 1,000 ml Erlenmeyer flasks containing 30 ml yeast extract medium. 'I'he flasks were inciihated at 37" C on a shaker for 3 days. Material from such cultures contained very few conidia, as shown by Fukui & Yasuda ( 8 ) .

Harvesting was performed under a hood. 'I'he medium in the Roux bottles was poured off and the harvest shaken carefully with 1,000 ml of distilled water, the mycelium was transferred to a Buchner funnel and washed with 1,000 ml of distilled water.

Harvesting of the suspension culture was performed by separating the mycel clumps from the medium by filtration through filter paper, trans- fer to a Buchner funnel and washing with 2,000 ml of distilled water.

The harvests were then homogenized for 4 min at 180,000 rev/min in an Omni Mixer (Sonal l ) in ice water-bath with a small amount of PBS, and the suspension was transferred to a bacterial press (X-press, AB Biox, Stockholm ) . After free- zing at -25" C, the material was pressed 3 times. After lyophilization the material was kept in small screw-cap bottles at room temperature.

Preparation of Extracts 1 he lyophilized material was resuspended in

PBS and left at room temperature for 20 min. Unsoluble material was spun down at 1,000 x g for 5 min, and the supernatant (extract) was saved. 'I'he concentrations of the extracts will be given as mg lyophilized material per ml of PBS.

~.

Titrat ion of Haetriolytic Ac t i r i t y of the Extracts To twofold dilutions of extracts in 0.2 ml of

PBS was added 0.2 ml of a 2 per cent suspension of erythrocytes . After incubation a t 37" C for

30 min followed by 90 rnin at 20" C, the degree of lysis was recorded macroscopically: 100 per cent: 3 + , 50 per cent: 2 + , 25 per cent 1+, 0 per cent: -.

Indirect Haenlagglutination ( I H A ) Equal volumes of 2.5 per cent sheep erythrocytes

and tannic acid (E. Merck, Darmstadt, W-Ger- many) diluted 1 in 80,000 (wJv) were incubated at 37" C for 10 min. The tanned erythrocytes were washed thrice in PBS and packed. To 10 ml of a 0.5 per cent suspension of tanned erythrocytes was added 0.5 ml of extract a t proper concentrations. The mixtures were incubated at 37" C for 60 min. The sensitized erythrocytes (SSB) were washed thrice in PBS containing 2 per cent normal rabbit serum (diluent) and resuspended to 0.5 per cent in diluent.

All sera to be tested were absorbed with sheep erythrocytes by mixing equal amounts of serum diluted 1 in 4 and packed erythrocytes. After 15 min at room temperature, the erythrocytes were sedimented by centrifugation and the supernatant (absorbed serum) was saved.

The haemagglutination test was performed with microequipment (Cooke Engn. Co., Medical Re- search Division, 900 Slates Lane, Alexandria, Va., USA). To twofold dilutions of absorbed serum in 0.025 ml diluent was added 0.025 of SSB. The plates were shaken, incubated at 37" C for 30 min and then overnight a t 4" C. The haemagglutination was graded as 3 + , 2 +, 1 + and -, according to the appearance of the pattern of sedimented cells. The titre is given as the reciprocal of the highest serum dilution which gave agglutination.

Inhibition of I H A T o 0.025 ml of extracts of various concentrations

was added 0.025 ml of antiserum to A . furnigatus Af diluted in diluent. The mixtures were left a t room temperature for 45 min upon which 0.025 ml of SSB was added. Patterns were recorded as for IHA.

Absorption of Sera T o 0.5 ml of the antiserum to Aspergillus were

added 30 mg of lyophilized fungus material. The mixture was left a t room temperature for 60 rnin and then centrifuged at 1,000 x g for 10 min. The supernatant (absorbed serum) was tested in IHA.

R E S U L T S

Haernolytic Activity of Extracts Since it was known from earlier (15,

20) that saline extracts of Aspergillus haemolysed erythrocytes, we tested extracts of A . furnigatus Af against various species erythrocytes to find which erythrocytes were liable to lysis (Table 1). Erythrocytes from man and guinea pig were most sensitive, those from rabbit and chicken were least sensitive, while sheep erythrocytes showed medium sensitivity. Since rabbit and chicken erythrocytes are not well suited for indirect haemagglutination with human sera, we selected sheep erythrocytes for further experi-

TABLE 1. Haeniolysis of Different Species Erythrocytes by Extracts of A. fumigatus at Various Con- centrations

Amount of material extracted (mgJml) 60 30 15 7.5 3.75

Erythrocytes from

Man Sheep Rabbit Guinea pig Chicken Man (F) Sheep (F) Rabbit (F ) Man (T) Sheep (Tj Rabbit (T)

3 + 3 + 3 + 3 + 3 + - - - 3 + 3 + 3 +

3 + 3 + 3 + 3 + 3 + - - - 3 + 3 + 3 +

F: Formalinized. T: Tanned.

57 Arta path. niirtobiol. rcaiid. Sect. B, 82, 6 873

TABLE 2. Extracts of Cultured Material f r o m the Various Aspergillus Strains 7’ested for Haemolysis of Sheep Erythrocytes

Extract of Amount of material extracted (mg/ml)

120 60 3 0 15 7.5 Strains

A . fumigatus Af (Sf) ? + .I+ 9 + 2 t Af (SP) 16030 (Sf) 3 + 3 + 3 + 2 t 16030 (Sp) - 131705 (Sf) I + 48338 (Sf) -

92877 (Sf) 3 + 2 t A . pseudoglaucus 3 + .I+ 1 + A . niger Aspergillus 3

Sf: Surface culture. Sp: Suspension culture.

ments. I t should be noted that tanned erythrocytes showed unchanged sensitivity to the haemolysin, while forrnalinized erythrocytes were resistant.

Extracts of the various Aspergillus cultures were then tested for haemolytic activity against sheep erythrocytes. Table 2 shows that the activity varied between strains, and also within strains according to type of culture used. Extracts of material obtained from suspension cultures gave no lysis.

Sensitization of Erythrocytes for I H A Sheep erythrocytes were treated with ex-

tracts at the concentration of 1 /2 of the haemolysing amount or at a concentration of 120 mg/ml of non-haemolytic extracts. After

1’ABLE 3. Titres in Indirect Haemagglutination w i th Rabbit Antiserum to A. fumigatus Tes ted Against Sheep Erythrocytes Sensitized by Extracts

of the Various Fungi

Extracts of Titres

A . fumigatus ( 5 strains) 4096-16384 Aspergillus 3 128 P. notatum 64 A . niger and A . pseudoglaucus < I C . albicans < 1 T . rubruni < 1 T . schoenleinii < 1

washing and resuspension the erythrocytes were added to dilutions of the immune serum. No agglutination occurred, indicating that the extracts did not sensitize the erythrocytes.

Tanned erythrocytes were then treated similarly and tested against the antiserum to Aspergillus (Table 3 ) . Extracts of all 5 strains of A . fumigatus sensitized erythrocytes for agglutination to high titres, while extracts of Aspergillus 3 and P . notatuni gave very low titres. Extracts of the other fungi did not sensitize the erythrocytes. Ery- throcytes sensitized by extracts of material from suspension cultures gave usually titres lower by two steps, even with a concentration of 120 rng/ml, and the results were inconsistent. Further experiments were therefore performed with extracts of the surface cultures.

Lyophilized preparations kept at room temperature for 12 months showed no decrease in sensitizing activity. However, extracts of fresh, homogenized material which had not been disrupted in the bacterial press did not sensitize the erythrocytes.

Specificity of the Sensitizing Substanc 1’s

The specificities involved in agglutination of erythrocytes sensitized with A . funiigatus

874

TABLE 4. Indirect Haemagglutination with Antiserum Tested Against Erythrocytes Sensitized by Ex- tracts of A. fumigatus Af, Aspergillus 3 or P. notatum. Effect of Absorption of Serum with Cultured

Material of Each Fungus

8192

2048

512

128

32

8 -

Erythrocytes Antiserum to Serum diluted 1 in sensitized with A . fumigatus Af 4 16 64 256 1024 4096 16384

- -

-

-

-

A . fumigatus Af Unabs. 3 + 3 + 3 + 2 + I + I + - A . fumigatus Af - - - - - - - Aspergillus 3 3 + 3 + 3 + 2 + I + I + - P . notatum 3 + 3 + 3 + 1 + I + 1 +

Abs. with

Aspergillus 3

P . notatum

- - - - Unabs. I + 1+ I + Abs. with

- - A . fumigatus Af 1 + 1 + 1 + - Aspergillus 3 - - - - .-

P . notatum 1 + 1 + I + - - - - - -

- ~ Unabs. 1 + I + 1 + - - Abs. with

- - .~ A . fumigatus Af 1 + 1+ I + Aspergillus 3 I + I + 1 + P . notatum -

- - - -

- - - - - -

extracts were then investigated in inhibition studies using extracts of all fungi and bacteria mentioned (see Materials) against 4 agglutinating units of the antiserum. The results were clear-cut: Extracts of all 5 strains of A . fumigatus (from 1.25 to 10 mg/ml) inhibited the agglutination of erythrocytes sensitized with extracts of the A . fumigatus strains, while concentrations up to 120 mg/ml of the other organisms had no inhibiting effect.

Results of absorption experiments were in perfect accordance with the results of inhibition. Antiserum was absorbed with lyophilized material from various cultures including the 3 species of bacteria, and absorbed and unabsorbed serum samples were tested against erythrocytes sensitized with A . fumigatus Af. The material from all 5 A . fumigatus strains absorbed the agglutinins, while none of the other fungal or bacterial preparations had any effect. Particularly, Aspergillus 3 and P . notatum which sensitized erythrocytes for agglutination with antiserum, did not reduce the titre.

The latter results were further supported by the following observations. Samples of

57'

antiserum were absorbed with lyophilized material from A . fumigatus, Aspergillus 3 and P. notatum, respectively. The results presented in Table 4 show that each strain of fungi only absorbed the corresponding antibodies. This indicates that the concentration of the cross-reacting antigens are relatively

Titre

I I I I I I

0.625 1.25 2.5 5 10 20 m g A. fumigotus Aflml

Fig. 1 . Titres in indirect haemagglutination of rabbit antiserum to A . fumigatus Af tested against erythrocytes sensitized with various amounts of Aspergillus extract.

875

TAB1.E 5. Indirect Haemagglutination. Human Serum 7 e s t e d Against Erythrocytes Sensitized w i th Extract of A. fumigatus Af. Distribution of Number of Sera According t o 7’itres

Serum from l‘itres

<8 8 16 32 64 128 256 512 1024 2048 4006 81Ilfi No.

Blood donors 131 122 6 (94) (4)

lung diseases 44 (87) ( 7 )

(82) (2)

(40)

Patients with 38 3

various diseases 231 189 4

obs. aspergillosis 35 14

Figures in parentheses: Per cent.

low in A . fumigatus, and that the sensitizing antigen is fairly specific for A . fumigatus.

The antiserum was titrated against sheep erythrocytes sensitized with A . fumigatus Af extract at various concentrations prepared as twofold dilutions from an extract of 20 mg/ml (checkerboard titration). The results are shown in Fig. 1 . Extract at a concentration of 10 mg/ml sensitized the erythrocytes optimally. This sensitization was therefore employed in further experiments. (Higher concentrations lysed the erythrocytes as shown above).

All bleedings from the immunized rabbits were tested in IHA. The titres rose from <2 in the pre-immune samples to 4,096 after the 3rd injection and to 8,192 one week after the booster dose. This indicates that the technique will easily detect changes in titres during immunizations.

Haemagglutinins in Human Sera The 4 groups of human sera were tested in

IHA as developed. Each serum was tested a t least twice, and for each test the following control sera were included: 1 ) the antiserum, 2 ) a high titred serum (titre >512), 3 ) a medium titred serum (titre 128), and 4) a low titred serum (titre 16). Maximum variation of these control sera was I+ 1 dilution. The results are shown in Table 5 which is compiled on the basis of the mode titres

876

for each serum. Among the blood donor sera, as many as 94 per cent had titres <8, and none had titres >16. Among the 275 sera from patients with various lung diseases and other diseases, only one had a titre > 128. This was a patient with upper respiratory allergy, but the aetiology could not be established. Among the sera obtained from patients under observation for possible aspergillosis, 32 per cent had titres >128. It should be noted that, as regards the 17 patients whose sera had titres <32, the diagnosis aspergillosis was ruled out after the observation period. On the other hand, the 4 sera with titres >1,024 were from patients who after evaluation had definite aspergilloma or aspergillosis, while the 6 sera with titres 64 to 1,024 were from patients with either definite or possible aspergillosis or aspergilloma. Serum from a patient who underwent surgery for aspergilloma 6 months earlier, had a titre of 512.

Sequential sera were obtained during treat- ment of two patients with definite aspergillosis (asthma and bronchitis). The sera were kept frozen and tested on the same day. Sera from both patients showed a 4- to %fold decrease in titres after 3-4 weeks.

D I S C U S S I O N

In accord with earlier reports (15, 20) , we also found that extracts of surface cultures

of Aspergillus were haemolytic for erythrocytes. The activity varied somewhat with species of erythrocytes, but all were lysed. Since sheep erythrocytes showed medium lysis, and are widely used in routine serology, we selected these for our experiments.

Untreated erythrocytes were not sensitized by sub-haemolytic amounts of the extracts for agglutination by antiserum to Aspergillus. Rut tanned erythrocytes were not lysed to a higher degree, and sensitization could therefore be performed with a concentration of extracts which was close to the haemolytic amount. The titres obtained with antiserum tested against such sensitized erythrocytes were in a high range; i.e. the test showed a high sensitivity. This was also clearly de- monstrated by the sequential sera from the immunized rabbit. The titre increased from <2 to 4,098 in 3 weeks.

The results obtained by comparing the sensitizing activity of haemolytic and non-haemolytic extracts of A . fumigatus (extracts of surface and suspension cultures, respectively) showed that the sensitivity could not be increased by using concentrated non-haemolytic extracts. These results indicated that the sensitizing activity of each extract reached an optimal level of concentration at subhaemolytic amounts. By checkerboard titra- tions of antigen preparations and antisera, or titration of antigen in inhibition tests, the optimal concentration of sensitizing antigen can easily be determined employing another A . funiigatus antigen preparation.

A possibility which we did not explore further in this series of experiments, was to use formalinized erythrocytes as recently re- ported by Senet & Brisset ( 17) . Such erythrocytes were not lysed by the extracts, but in our preliminary experiments they gave inconsistent results in IHA.

The immune serum produced against A . jurnigatus Af agglutinated erythrocytes sensitized by any of the 5 A . fumigatus extracts to similar titres, while erythrocytes sensitized by most of the extracts of other fungi, or bacteria, including A . niger and pseudoglaucus, were not agglutinated. Erythrocytes sensitized

with extracts from P . notatum and Aspergil- lus 3 gave low titres. Apparently, the sensitizing antigen in extracts of A . fumigatus is species specific. The results of absorption experiments using the lyophilized cultured material of the various organisms supported this observation: Only A . fumigatus material re- duced the titres of the immune serum tested against A . fumigatus antigen in IHA. How- ever, experiments with antisera produced against other Aspergillus species and other fungi, together with extensive absorption experiments, are needed if the specificity of the described reactions is to be definitely established.

A technical point which should be stressed is the importance of complete disruption of the fungus material before preparation of the extract. No sensitizing activity was traced in extracts of material which had not been disrupted in the bacterial press.

The results obtained with the 441 human sera showed good correlation between high titres and presence of aspergillosis, and none of the sera with low titres were from patients with the disease. However, we could not establish a definite lower limit suggestive of specific disease. On the basis of the titres recorded we used the following groupings until a larger series of patients and controls had been investigated: Titres 2 3 2 : “normal”, titres 64-128: doubtful increase, and titres F256 : pathologically increased. I t is highly probable that patients whose sera fall within the last group have aspergillosis. The test may therefore be useful since A . fumigatus is responsible for 90 per cent of re- ported cases ( 6 ) .

The technique presented is simple to per- form. The sensitivity of the technique makes it easy to detect increases or decreases in titres while patients are under observation, particularly since between 80 and 90 per cent of the sera in the 3 control groups had no de- monstrable antibodies ( titre < 8 ) . Results from this study indicate that the antigen is connected with the mycelium, since conidia- free material contained it. Results of experiments designed to characterize the antigen,

877

and to investigate the relationship between entific Publications, Oxford, Edinburgh 1968, haemagglutinating and precipitating anti- pp. 78-79.

10. Hipp, S. s., Rerns, D . s., Tornpkins, V . & Buckley, H . R . : Latex slide agglutination test bodies to A . fumigatus, will be presented in a

forthcoming paper. for Aspergillus antibodies. Sabouraudia 8:

1.

2.

3.

4.

5.

6.

7.

8.

9.

R E F E R E N C E S

Biguet, J., Fruit, J., Vernes, A . & Capron, A . : La &action de fixation du complement et I’imrnunoClectrophoRse appliquhes au diagnostic immunologique de I’aspergillose pulmo- naire. Rev. Immunol. (Paris) 34: 193-203, 1970. Bronnestam, R . & Hallberg, T.: Precipitins against an antigen extract of Aspergillus fumigatus in patients with aspergillosis or other pulmonary diseases. Acta med. scand. 177: 385-392, 1965. Campell, M . J . & Clayton, Y . M.: Broncho- pulmonary aspergillosis. A correlation of the clinical and laboratory findings in 272 patients investigated for bronchopulmonary aspergillosis. Amer. Rev. resp. Dis. 89: 186-196, 1964. Coleman, R . M . & Kaufman, L . : Use of the immunodiffusion test in the serodiagnosis of aspergillosis. Appl. Microbiol. 23: 301-308, 1972. Daniel, T. M., Weyand, J . G . , J r . & Stavit- sky, A . B.: Micromethods for the study of pro- teins and antibodies. IV. Factors involved in the preparation and use of a stable preparation of formalinized, tannic acid-treated, pro- tein-sensitized erythrocytes for detection of antigen and antibody. J. Immunol. 90: 741.- 750, 1963. Davis, R. D . , Dulbecco, R. , Eisen, H . N . , Ginsberg, H . S . & Wood, W . R., Jr.: Microbio- logy. Harper & Row, New York, Evanston, London, and John Weatherhill Inc., Tokyo 1969, pp. 997-998. English, M . P. & Henderson, A . H . : Signifi- cance and interpretation of laboratory tests in pulmonary aspergillosis. J. clin. Path. 20: 832-834, 1967. Fukui, M . & Yasuda, 1.: Serological studies on Aspergillus fumigatus. Mycopathologia (Den Haag) 14: 39-54, 1961. Gell, P . G . H . & Coombs, R . R . A . : Clinical aspects of immunology. 2. ed. Blackwell Sci-

11 .

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

237-241, 1970. Longbottom, J . L. & Pepys, 1.: Pirlmonary aspergillosis. Diagnostic and immunological significance of antigens and C-substance in Aspergillus fumigatus. J. Path. Bact. 88: 141- 151, 1964. Longbottom, J . L., Pepys, J . & Clire, F . T.: Diagnostic precipitin tests in Aspergillus pulmonary mycetoma. Lancet i : 588--589, 1964. Parker, J . D . , Sarosi, G . A , , Doto, I . L. & Tosh, F . E.: Pulmonary aspergillosis in sana- toriums in the south central United States. Amer. Rev. resp. Dis. 101: 551-557, 1970. Pepys, J . , Riddell, R . W . , Citron, K . M., Clay- ton, Y . M . & Short, E . I .: Clinical and im- munologic significance of Aspergillus fumigatus in the sputum. Amer. Rev. resp. Dis. 80: 167- 180, 1959. Rutquist, Z,.: Studies on Aspergillus fumigatus; stability of haemolysin and toxin in crude filtrate. Acta vet. scand. 9: 350 -363, 1968. S a l r h , S. R.: Serologic relationship of fungus antigens. J. I.ab. clin. Med. 34: 1096 1104, 1949. Senet, J . M . & Brirset, C . : The diagnosis of aspergillosis by passive hemagglutination. Bio- medicine 19: 365-368, 1973. Stanley, N . F.: Biological properties of poly- saccharide and lipoid fractions from a patho- genic strain of Aspergillus fumigatus. Airst. J. exp. Biol. med. Sci. 28: 99-108, 1950. Stallybrass, F. C . : The precipitin test in human systemic aspergillosis. Mycopathologia (Den Haag) 21: 272- 278, 1963. Tomiikova‘, A . , Sach, J . , H o f e j f i , M . , Mal+, V. & Novcic‘kovci, D . : Antikorper gegen Peni- cillia iind Aspergilli. Z. 1mmirn.-Forsch. 120: 40-52, 1960. Walther, J . E. & Jones, R . D . : Serologic tests in diagnosis of aspergillosis. Dis. Chest. 53: 729-735, 1968. Weill, C. P., Pesle, G . D . & Mehl, P . : Diag- nostic des affections B Aspergillus par dCvia- tion du complement. Ann. Biol. clin. 27: 87.- 91, 1969.

878

iha aspergillus fermigatus

Documents