Allergic bronchopulmonary aspergillosis—A north American rarity

Allergic

Bronchopulmonary North

Aspergillosis-A

American

Clinical and Immunologic

Rarity* Characteristics

RAYMOND G. SLAVIN, M.D., DENNIS J. STANCZYK, M.D., ANDREW J. LONIGRO, M.D. and GORONWY

0.

BROUN,

SR.,M.D.

St. Louis, Missouri

Allergic bronchopulmonary aspergillosis has olten been reported from England, but it is exceedingly rare in the United States. .4 case is described which fulfills all the clinical and laboratory characteristics of the syndrome, including low grade fever and episodic wheezing, expectoration of brown plugs containing septate hyphae, transient pulmonary infiltrates, eosinophilia of blood and sputum, positive immediate skin tests and positive precipitating antibody. In addition, a positive intrabronchial challenge to Aspergillus fumigatus was demonstrated. Both skin sensitizing and precipitating antibody to A. fumigatus are thought to play a role in the pathogenesis of this disease. Cessation of symptoms followed successful theraov with intrabronchiallv administered amphotericin B and sodium iodide 1,

and orally administered Prednisone.

SPERGILLUSis a genus of mold that is hardy and highly ubiquitous. Its abundance in nature has been amply demonstrated by almost pure growths of this perennial fungus from such diverse sources as fertile soil, furs, compost piles, swimming pool water, flour, human hair and common foodstuffs [l-3]. Any species of Aspergillus may infect man, but the one most commonly seen is fumigatus. The respiratory tract is the most frequent site of infection, but the external auditory canal, skin and nails, eyes, sinuses, meninges and bone have also been invaded [4]. Because of the wide distribution and perennial presence of Aspergillus fumigatus, the fungus is probably inhaled and expectorated by the population at large [5]. Since it is a common contaminant of spmum and may be present at times in the respiratory tract without harmful effects, the mere culture of A. fumigatus from sputum cannot by itself be taken as proof of causal relation to clinical manifestations. Pulmonary disease due to A. fumigatus has

been recognized since the nineteenth century, when Sluyton [6] first reported an Aspergillus fungal mass in the lung cavity of a woman. Later, Redon [7] described pulmonary aspergillosis in wigmakers and pigeon breeders. Since then, pulmonary aspergillosis has been well established as a clinical entity. Various classifications of disease caused by Aspergillus have been offered [1,8], but the clearest seems to be that presented by Wahner et al. [S]. In group 1 of their classification, Aspergillus actually invades tissues. There is unequivocal evidence of pathogenicity with invasion of the bronchial walls producing a definite bronchitis. Pneumonia, abscess formation or chronic ganuloma may also occur. This form may become systemic and is seen most often in patients whose resistance is compromised either by severe underlying disease or by the use of steroids, antibiotics, marrow-depressing drugs or immunosuppressive agents. In group 2, the most common form, local growth of the organism occurs without inva-

A

* From the Section of Allergy and Irmnunology, Department of Internal Medicine, St. Louis University School of Medicine, St. Louis, Missouri 63104. This work was supported in part by U. S. Public Health Service Grant No. Al 07303.02. Requests for reprints should be addressed to Raymond G. Slavin M.D. Manuscript received August 28, 1968.

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sion of tissue. Mycelial growth may occur in bronchial lumens, bronchiectatic cavities or bronchogenic cysts. At most, there is a superficial invasion of the lining of a cavity. In this form aspergillosis occurs as a secondary complication to an anatomic abnormality such as a cavity or a cyst. A third group of pulmonary diseases due to Aspergillus was described in 1952 by Hinson, Moon and Plummer [IO] and designated allergic bronchopulmonary aspergillosis. The essential features of this syndrome were episodes of wheezing with low grade fever, expectoration of characteristic brown plugs, transient pulmonary infiltrates and eosinophilia of the blood and sputum. Since the initial description, reports of allergic aspergillosis associated with posifive immediate wheal and erythema skin reactions and precipitins to Aspergillus extracts have appeared principally from England [5,11,12]. Because of the evident extreme rarity of this condition in the United States, we are reporting a case which fulfills all the clinical and laboratory criteria of allergic aspergillosis. We hope that the immunologic survey described will aid in a better understanding of the pulmonary hypersensitivity syndromes in general. CASE REPORT

A thirty-one year old white male meat salesman (A.O.) entered the St. Louis University Hospital in January 1968 with a history of asthma since the age of five which was of moderate severity and frequency until the age of fifteen. Hyposensitization to mold and house dust brought about relief of symptoms. In the summer of 1965 he was repeatedly exposed to a neighbor’s compost pile. In July 1966 he was admitted to a hospital with pneumonitis in the lower lobe of the left lung, unresponsive to penicillin therapy; his temperature was 99%. The white blood cell count was 12,000 per cu. mm. with ‘21 per cent eosinophils. Sputum grew out Aspergillus, and scalene node and bronchial biopsy specimens revealed nonspecific inflammatory response. ACTH treatment was instituted with clearing of the infiltrate and clinical improvement. In July 1967 the patient was readmitted with a three month history of cough ansd fatigue and several days of chills and fever. The white blood cell count was 15,000 per cu. mm. with 24 per cent eosinophils. A chest roentgenogram showed an infiltrate in the upper lobe of the left lung. FolIowing tetracycline therapy the patient’s condition improved and there was marked clearing of the pulmonary infiltrate. Four months later he reentered VOL.

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the hospital with increasing cough, dyspnea, wheezing and atelectasis in the lower lobe of the left lung. Temperature was 101.3’~. The white blood cell count was 16,000 per cu. mm. with 18 per cent eosinophils. Aspergillus was grown from sputum, and thick tenacious mucous was aspirated from the upper and lower lobes of the left lung on bronchoscopy. The patient was started on a regimen of nystatin, 50 mg. four times a day, and intense therapy with saturated solution of potassium iodide. Because of failure to improve on this program, he was readmitted in January 1968. On this admission it was noted that he was wheezing and expectorating brown plugs in his sputum which contained mycelia on microscopic examination. White blood cell count was 12,000 per cu. mm. with 21 per cent eosinophils. Serum immunoglobulin levels were normal. Sputum culture was positive for A. fumigatus on three occasions, and sputum smear revealed many eosinophils and septate hyphae on numerous occasions. Chest roentgenograms revealed an infiltrate in the upper lobe of the right lung. Bronchograms showed bronchiectasis of upper and middle lobes of the right lung and the upper and lower lobes of the left lung. Peripheral blood eosinophilia remained between 18 and 31 per cent. Aerosol nebulization of amphotericin B, 5 mg. per cc. twice a day was initiated, alternating with a 10 per cent solution of sodium iodide four times a day. Hyposensitization to mixed molds, including A. fumigatus was begun. Four weeks later there was a marked decrease in clinical wheezing and rales with a decline of peripheral eosinophilia to 3 per cent. Treatment with intrabronchially administered amphotericin B, 10 per cent sodium iodide and prednisone, has continued. The patient is working and doing well eight months after the initiation of aerosol therapy. In the last two months three sputum cultures have been negative or Aspergillus, and hyphae have not been seen on direct smear. METHODS AND MATERIALS Skin

Testing.

Intracutaneous skin tests with serial dilutions of A. fumigatus (Abbott Laboratories, N. Chicago, Ill., Lot No. 812-3308) from 1: 10,000 to 1: lO,OOO,OOOwere performed. Cross reactivity to other species of Aspergillus was assessed by scratch tests with A. niger, A. hortei and A. flavus. (Obtained through the courtesy of Dr. Samuel M. Feinberg, Winnetka, Ill.) Passiue Transfer of Skin Sensitivity. An intracutaneous injection of 0.1 cc. of the patient’s serum was made into the foreann of a negative recipient. Forty-eight hours later the site was challenged with 0.05 cc. of A. fumigatus 1: 1,000. Production of Aspergi2lu.s Antib.ody in the Guinea Pig. An adult male guinea pig of the Swiss strain was immunized subcutaneously with Abbott Laboratory A. fumigatus extract in a 1:1

i\llergic

Bronchopullnonary

:~sI’eraillosis---~~‘lurlz’rr t’/ al.

FIG. I. Double gel diffusion comparing the patient’s prccipitin I~anda against aspergillus a positive acpergilloma serum. (Slide courtesr of Dr. Jordan N. Fink, Marquette School \. fumigatu? 20 mg. per ml. B, positive control serum from a patirnt with aspergilloma cipitins to ayrrgillus. C, patient’s serum. D, normal human erum.

111nlig;i I II\ wit II of %14icirx.) .\, and Lno~ II prp-

gel diffusion showing the patient’s two fjrecipitin bands against 2. Double aspcrgillus one of which crossreacts with the guinea pig band. A, $1. fumigatus 20 mg per ml. B, par&f’\ sermn from guinea pig immunized with A. fmnigatus. D. no1 ma1 human serum. E. rlolIna1 guinc;l

1°K.

ratio with Difco complete Freund’s adjuvant, according to the following weekly schedule: 10 mg., 10 mg., 50 mg. and 100 mg. Llernonstration of Precipitating Antib.ody to Aspergillus. Ordinary gel diffusion was performed according to the Ouchterlony double gel diffusion technic [13]. Immunoelectrophoresis was carried out by tfle microtechnic of Scheidegger [I#]. Reverse Passive Arthus Reaction. Ten milligrams of a purified A. fumigatus antigen (obtained from Dr. Joan L,ongbottom, through the courtesy of Dr. Jordan N. Fink, Marquette School of Medicine) was injected into the heart of an adult male guinea pig. Thirty minutes later an intracutaneous injection of 0.1 cc. of the patient’s serum concentrated by ammonimn sulfate precipitation was made in the flank. Intrabronchinl Challenge. After routine ventilatory function tests, including ti’med vital capacity, the patient inhaled a nebulized mist of buffered saline solution for sixty seconds. Fifteen minutes later ventilatory function was tested. Following this the patient inhaled a nebulized mist of A. fumigatus 1:250,000 for sixty seconds and ventilatory funrtion ~h’astested again after fifteen minutes. RESULTS

Skin Tests. Direct titration of the patient’s immediate skin reactivity to intracutaneous A. fumigatus showed a 4-I reaction to a 1:10,000 dilution, 4+ reaction to l:lOO,OOO, 3+ to 1:250,000, 2f to 1:500,000, I+ to 1: 1,000,000, 2 to 1:5,000,000 and negative to 1: 10,000,000. The marked immediate skin reaction to the

fu1nigatuy x’tum. (:, pig WI‘IIIII.

1: 10,000 dilution abated in two hours. At six hours a “late” reaction manifested by erythema and edema appeared, reaching a maximum at twenty-four hours and then resolving. A skin biopsy specimen obtained eight hours after the injection showed marked perivascular infiltration with polymorphonuclear Ileutrophils, including many eosinophils. A twenty-four hour biopsy specimen still showed many eosinophils with some increase in mononuclear cells. Skin testing to other Aspergillus species showed 3f scratch reaction to A. niger. 2+ to .\. hortei and 1 + to A. flavus. Passive Transfer of Skin Sensitivity. Skin sensitizing antibody to A. fumigatus was transferred to the nonsensitive recipient, as demonstrated by a 3+ immediate wheal and erythema reaction at the transfer site after challenge with Aspergillus. Precipitating Antibody. Figure 1 shows two distinct bands formed on the double gel diffusion plate between the patient’s serum and A. fumigatus extract. A similar reaction occurs with serum from a patient with an aspergilloma. Figure 2 indicates the relationship of the patient’s Aspergillus antibody to the antibody of the immunized guinea pig. The fusion of lines, or reaction of identity, indicates that one of the antigens had induced the production of a specific precipitating antibody in both man and the guinea pig. Immunoelectrophoresis of the patient’s serum and guinea pig serum in

Allergic Bronchopulmonary the two wells is shown in Figure 3. The antibody in both is seen in the gamma globulin region. Reverse Passive Arthus Reaction. Four hours after intracutaneous injection of the patient’s concentrated serum, the site became erythematous and indurated. The reaction became progressively more intense in the succeeding twenty hours. A biopsy of the skin at eighteen hours showed a marked vasculitis with perivascular accumulation of polymorphonuclear leukocytes and eosinophils. An injection into a control site using the patient’s concentrated serum was negative. Zntrabronchial Challenge. After the patient’s inhalation of buffered saline mist, there was no significant change in ventilator-y function, the one second forced expiratory volume (FEV,) increasing from 39 to 43 per cent. Fifteen minutes after the patient inhaled a mist of 1:250,000 A. fumigatus, FEVl was reduced to 23 per cent and wheezing was evident. COMMENTS

Airborne fungal spores are being increasingly recognized as allergens in man. In 1927 v. Leeuwen et al. [25] first reported A. fumigatus as an important fungal cause of respiratory tract allergy. Since then, A. fumigatus has been ranked high on the list of inhaled fungal spore allergens. We have already mentioned infection produced by A. fumigatus, and the condition of aspergillosis in man, animals and birds is well known. In 1952 Hinson et al. [IO], reporting on pulmonary infections in man, described several cases of transient lung shadows with eosinophilia in the peripheral blood which he attributed to hypersensitivity. More recently, Pepys and his workers [5], and Campbell and Clayton [II] have described many more patients with allergic bronchopulmonary aspergillosis, and have contributed greatly to a better definition of this syndrome. The essential features of allergic bronchopulmonary aspergillosis are as follows: (1) (2) (3) (4) (5) (6) (7)

Episodic wheezing Low grade fever Expectoration of brown plugs Transient pulmonary infiltrates Eosinophilia of blood and sputum Positive immediate skin tests Positive precipitating antibody

Low grade fever and wheezing are always seen. Several investigators [5,10,12] have VOL.

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FIG. 3. Immunoelectrophoresis showing both human and guinea pig antibody to A. fumigatus in the gamma globulin range. A, patient’s serum. B, A. fumigatus 20 mg. per ml. C, serum from guinea pig immunized with A. fumigatus.

stressed the history of long-standing asthma due to other causes in these patients before the development of aspergillosis. Bronchiectasis may also be a predisposing factor in allowing the establishment of the saprophytic infestation. The only exposure of our patient to a concentrated source of Aspergillus was a compost pile several months before the development of symptoms. In our patient, it is difficult to say whether bronchiectasis antedated aspergillosis or followed it. It would appear that aspergillosis caused the bronchiectasis for several reasons. First, there was no history of productive cough before the development of aspergillosis. Secondly, the type of bronchiectasis in our patient would favor the latter view. Citron and Pepys [16] have commented on the development of a specific type of bronchiectasis in allergic aspergillosis, a localized sacular dilatation occurring proximally in bronchial branches at the site of a previous segmental pulmonary infiltration. In this type, the more peripheral branches fill normally, which was the case in our patient. Citron and Pepys emphasized that the localized nature of the dilatation suggests a direct “toxic” action resulting from the presence of fungus rather than the usual sequence of events leading to bronchiectasis, i.e., bronchial obstruction, atelectasis and infection. The expectoration of brown plugs is characteristic of allergic aspergillosis. Several examinations of our patient’s sputum revealed numerous eosinophils and septate hyphae imbedded in mucous and fibrin debris. As already mentioned, the mere presence of Aspergillus in the sputum is not definite evidence of aspergillosis. The presence of septate hyphae in the

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1.1(.. -4. Transient pulmonary infiltrates were &dent in the patient. In 111~ upper left, on the first hospital atlmission. an inhltrate is seen on the loam pat! of the left lobe. In the upper right film, eleven months later, a second infiltrate is seen in the upper part of the left lobe. In the lower left film, four months later, both areas habe cleared and an area of atelectasis appears. In the lower right film, on the last hospital admission, a new infiltrate in the upper pa~-t of the right lobe appears together with reappearance of infiltrates in both the upper al;d lower paits of thcleft lobe

sputum, as well as repeated positive cultures, established without any question the active growth of Aspergillus in the lung or bronchi. A characteristic pattern of allergic aspergillosis on chest roentgenograms is described as transient pulmonary infiltrates. Over the one and a half year period that we have followed the patient roentgenoCqaphically, multiple sites and configurations of pulmonary infiltrates have been demonstrated (Fig. 4). Eosinophilia of peripheral blood and sputum is another characteristic of allergic asper-

gillosis. In our patient, peripheral blood eosinophilia was present as early as the summer of 1966. During his last hospital admission the per cent of eosinophils in the peripheral blood ranged from 18 to 31. With successful treatment. eosinophilia has been reduced to 3 per cent. On many occasions, eosinophils were much in evidence in the sputum. The presence of skin sensitizing antibody to Aspergillus, as manifested by the development of a positive immediate wheal and erythema reaction to an Aspergillus skin test, is a necesAMERICAN

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Allergic Bronchopulmonary sary finding in allergic aspergillosis. Elder and Smythe [17] state that a negative immediate skin test in a patient with precipitating antibody to Aspergillus tends to exclude the diagnosis of the hypersensitivity form. Our patient responded to as small an amount as a 1: l,OOO,000 dilution of A. fumigatus, and he further showed cross reactivity to a number of other Aspergillus species. Passive transfer of the nonprecipitating skin sensitizing antibody by the classic Prausnitz-Kiistner [18] technic was also demonstrated. The role of precipitating antibody to Aspergillus in allergic aspergillosis is controversial. Pepys et al. [5] first suggested that precipitins were participating in the hypersensitivity reaction in allergic subjects and were producing reactions in the lung of an Arthus type, as manifested by pulmonary infiltrates. Campbell and Clayton [II] took issue with this and thought that precipitins bear no direct relationship to the allergic manifestations of ASpergillus infection. They pointed out the high incidence of precipitating antibody in patients with aspergilloma with no evidence of allergic hypersensitivity to Aspergillus and concluded that the pulmonary infiltrates were on the basis of a gross allergic response mediated by nonprecipitating skin sensitizing antibody. More recently, Pepys [29] suggested that in patients with Aspergillus hypersensitivity the presence of skin sensitizing antibody and precipitins together may cause reactions not found in patients with skin sensitizing antibody alone or precipitins alone. The presence of skin sensitizing antibody and precipitins together is closely related to the development of pulmonary eosinophilia, i.e., transient pulmonary infiltrates and eosinophilia. Our patient demonstrated antibody to two distinct antigens of A. fumigatus and showed a reaction of identity to Aspergillus antibody produced in guinea pig. Further demonstration of our patierrt’s precipitating antibody was the successful reversed passive Arthus reaction in a normal guinea pig. The presence of a probable Arthus reaction in our patient was seen in the “late” response to the skin test of l:lO,OOO A. fumigatus. Several hours after complete abatement of the wheal and erythema immediate reaction, erythema and edema began and reached a peak at twenty-four hours. A skin biopsy obtained eighteen hours after the test revealed perivasculitis with accumulation of polymorphonuVCILC

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clear leukocytes and eosinophils. The absence of thrombosis and hemorrhage was probably due to the fact that we could not test with a concentration of A. fumigatus stronger than 1: 10,000 because of the violent immediate reaction. Pepys et al. [5] believe that the “late” skin reaction is a dermatologic manifestation of the pulmonary infiltrates and point out that cortical steroids cause a marked decrease in both the “late” skin reaction and the pulmonary infiltrates. A final demonstration of Aspergillus hypersensitivity was seen in our patient’s response to intrabronchial challenge. Inhalation of a dilute solution of A. fumigatus caused a significant decrease in FEV,. The presence of two distinct antibodies to Aspergillus in patients with allergic aspergillosis would seem to be related to the clinical manifestations. The nonprecipitating skin sensitizing antibody is responsible for the immediate skin reaction, the eosinophilia, the clinical features of asthma and the positive intrabronchial challenge. The precipitating antibody, on the other hand, could well account for the “late” skin reaction and the pulmonary infiltrates. The treatment of pulmonary aspergillosis is generally ineffective. Isolated instances of cures have followed the administration of nystatin orally [20] and by aerosol [21], stilbamidine intravenously [22], potassium iodide orally [23] and amphotericin B intravenously [24]. The intravenous administra.tion of amphotericin B has been reported to result in many complications, especially renal, but its use intrabronchially has been shown to be safe [25] and particularly effective when combined with sodium iodide [26’j. Tube dilution sensitivity tests to nystatin in our patient failed to show inhibition of growth. We therefore chose the amphotericin B-sodium iodide combination because of its ease of use and safety to the patient. In addition, we began hyposensistization injection therapy with mixed molds, including A. fumigatus. After an initial response with lessening of sputum production, wheezing and eosinophilia, the patient persisted with a productive cough and occasional wheezing. Prednisone, 5 mg. three times a day, was then added. Cortical steroids have been shown ,to decrease the pulmonary infiltrates [5], and other investigators [27] have used steroids in allergic aspergillosis with a view to lessening the allergic re-

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action and reducing airway obstruction resulting in a more rapid expectoration of remaining antigen. On tllis program the patient leas done well and has returned to work. Since the initial description of allergic aspergillosis in 3952 [IO], most reports have come from England. A recent paper from the Johns Hopkins University [28] states that “the syndrome of astltma with pulmonary infiltrates, eosinophilia of blood and sputum, and sometimes fever has ofteil been described in the L!nited States, but never to our knowledge related to Aspergillus infection, although secondary aspergillosis is well known.” Spotnitz and Overholt [27] have reported a case of allergic aspergillosis in an American, but the exposure to the fungus was in Korea. The question then arises, “Why is aspergillosis such a rarity in the United States?” It would seem that the climate and geography of England does not make a profound difference since A. fumigatus is commonly reported in air sampling surveys in this country [29]. In addition, as stated previaspergillosis is not uncomously, secondary mon. It appears then that a failure of recognition and errors of omission account for the rarity of allergic aspergillosis in the United States, Septate hyphae may be missed on the sputum smear. transient pulmonary infiltrates not appreciated and skin sensitizing and prec,ipitating antibodies not evaluated. With the proper appreciation of the characteristics of allergic aspergillosis, both laboratory and clinical, this disease may be more frequently recognized and take its place with such hypersensitivity pneumonitides as pigeon breeders’ disease [30], bagassosis [3I] and farmers’ lung [32]. ADDENDUM

Since this manuscript was accepted for publication, we have come across a second case of allergic aspergillosis in a nine year old girl whose clinical and laboratory findings fulfill all the criteria of this syndrome. Acknowledgment: We wish to thank Dr. ,James Martin for reviewing the chest roentgenograms, Drs. Frederick Germuth and George Gantner for their review of the biopsy material, Dr. Leonard Laskowski for morphologic studies and Dr. Herbert Sweet for his assistance in the insufflation studies. The techni-

.\spergillosis--,Rnz,in (,a1 assistance knowledged.

of

P/ N/. ,L[irs @heil;l

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I. ~IACARI.NEY, J. N. Puhnonary aspctgilloG\; a leview and a description of thwe nc\t cast’\. Tllortru, I!): 287, 1964. 2. GCKETAIN, G. Pulmonary asprrgillosiu. 1.0h. Iwest., 11: 1046, 1962. 3. RAPE& K. B. and FENNEL, I). 1.. ‘I’he GCIIUS Aspen. gillus. Baltimore, 1965. Williams & r\‘ilkins Co. 4. S%wH, I). T. Miscellaneous l’ungal tliseascs. 1. Cl,ron. Dis., 5: 528, 1957. 5. PEPYS, J., RIDDEL, R. IV., CITRON, K. M., CLAYTON, Y. N. and SHORT, E. I. Clinical and immunologic significance of aspergillus fumigatus in sputum. Atn. Rev. Resp. Dis., 80: 167, 1959. 6. SLUYTON, T. De vegetalibus organismi animalis parasitis. Diss. Inaug. Berolini, page 14, 1847. 7. REDON, I,. Etude sur l’aspergillose chez les animaux et chez l’ltomme. Paris, 1897. Masson et Cit. 8. FINEGOLD, S. M., WILL, D. and MURRAY, J. F. Aspergillosis, a review and report of twelve casts. Am. 1. d\Jed., 27: 463, 1959. 9. \\‘~HNER, H. W., HEPPER, N., ANDERSON, H. and \VEED, L. Pulmonary aspergillosis. .$nn. Znf. Med., 58: ,472, 1963. 10. HINSON, K. F. W., MOON, A. J. and PLL’,MMTK, N. S. Bronchopulmonary aspergillosis; a review and a report of eight new cases. Thorax, 73: 317, 1952. 1I. CAMPBELL, J. and CLAYTON, Y. M. Bronchopulmonarv aspergillosis; a correlation of the clinical and laboratory findings in 272 patients investigated for bronchopultnonary aspergillosis. Am. licv. Rap. Dis., 89: 186, 1964. 12. FRANKLAND, A. W. and HAMILTON, E. D. .4llcrgic pulmonary aspergillosis. .4 Ilergie ti ,4sthmn, 4: 202, 1959. 13. OUCHTERLONY, 0. Diffusion-in-gel methods for immunological analysis. Progr. .4llergy, 5: 1, 195X. 14. SCHEIDEGGER, J. J. Une micro-mcthode de l’inrnlunoelectrophorese. Znfernrrt. .4r&. .4l/qy, 7: 103, 1955. 15. v. LEEUWEN, W. S., BREN, Z., KREMEK, TV. anti VAREKAMP, H. On the significance of small spored types of aspergilli in the etiology of bronchial asthma. Ztschr. Immunitiitsforsch., 44: I, 1925. 16. CITRON, K. M. and PEPYS, J. Direct bronchial sensitivity testing in bronchopulmonar\ aspergillosis. In: Fungous Diseases and Their ‘l’reatment. p. 134. Edited hy Riddcll, R. W. and Stewart. G. T. London, 1958. Butterworth & Co. 17. ELDER, J. L. and SMYTHE, J. T. ,411rrgic hronchopulmonary aspergillosis. hf. 1. 4 flstmlio. 1: 2.71. 1967. 18. PR.~L!SNITZ, C. and K~;STNER, H. Studien iiher die llcherempfindlichkeit. Zmtmlhl. Bnkt., 86: 160. 1921. 19. PEPYS, J. Possible role of precipitins against aspergillus fumigatus. .lor. Rat. Rmp. Dis., 90: 465, 1964. 20. :\NTONFLI.I, J., MATHEY, I., HERW!, J,. COURNOT, L.. and CHOLLET, M. Aspergillonle hronchique: AMERICAN

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traitement prolong4 par la mycostatine: Gu&ison. Bull. et m&m. Sot. me’d. h8p. Paris, 75: 564, 1959. 21. START, J. E. Allergic pulmonary aspergillosis successfully

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chronic asthma. J. Allergy, 40: 319, 1967. 29. FEINBEKG, S. M. Allergy in Practice, p. 241. Chicago,

1946. Yearbook Publishers, Inc. 30. FINK, J. N., SOSMAN, A. J., BARBORIAK, J. J., SCHLUETER, D. P. and HOLMES, R. A. Pigeon breeders disease. Ann. Int. Med., 68: 1205, 1968. 31. BUECHNER, H. A., PREVATT, A. L., THOMPSON, J. and BUTZ, 0. Bagassosis. A review with further historical data, studies of pulmonary function and results of adrenal steroid therapy. Am. J. Med., 25: 234, 1958. 32. DICKIE, H. A. and RANKIN, J. Farmer’s lung-an acute granulomatous interstitial pneumonitis curring in agricultural workers J.A.M.A.,

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