840
A.J.C.P. • November 1978
UDOJI AND FRIGY
References
1. Atkinson K, McElwainTJ, MacKay AM: Myeloma of the heart,
Brit Heart J 36:309-312, 1974
2. Churg J, Gordon AJ: Multiple myeloma. Lesions of extraosseous hematopoietic system. Am J Clin Pathol 20:934945, 1950
3. Cohen AS, Calkins E: Electron microscopic observations on
a fibrous component in amyloid of diverse origins. Nature
(London) 183:1202-1203, 1959
4. DePetris S, Karlsbad G, Pernis B: Filamentous structures in
the cytoplasm of normal mononuclear phagocytes. J Ultras
Research 7:39-55, 1962
5. Freeman JA, Samuels MS: The ultrastructure of a fibrillar
formation of leukemic human blood. Blood 13:725-731, 1958
6. Goldberg E, Mori K: Multiple myeloma with isolated visceral
(epicardial) involvement and cardiac tamponade. Chest 57:
584-587, 1970
7. Maldonado JE: Cytoplasmic and intranuclear electron-dense
bodies in myeloma cells. Arch Pathol 81:484-500, 1966
8. Osserman EF: Plasma cell myeloma. N Engl J Med 261:952960, 1965
9. Puchtler H, Sweat F: Congo red as a stain for fluorescent microscopy of amyloid. J Histochem Cytochem 13:693-694, 1965
10. Smetana K, Hermansky F, Koblizkova H, et al: A further note
on the ultrastructure of myeloma plasmacytes. Neoplasma
18:3-13, 1971
11. Tanaka Y: Fibrillar structures in the cells of blood forming
organs. J Natl Cancer Inst 33:467-485, 1964
12. Welsh RA: Electron microscopic localization of Russell bodies
in the human plasma cell. Blood 16:1307-1312, 1960
13. Zucker-Franklin D, Franklin EC: Intracellular localization
of human amyloid by fluorescence and electron microscopy.
Am J Pathol 59:23-42, 1970
Allergic Bronchopulmonary Aspergillosis Caused by
Aspergillus ochraceus
HAROLD S. NOVEY, M.D., AND IAN D. WELLS, B.Sc. (TECH.)
Novey, Harold S., and Wells, Ian D.: Allergic bronchopulmonary Aspergillosis caused by Aspergillus ochraceus. Am J
Clin Pathol 70: 840-843, 1978. A case with the characteristics
of allergic bronchopulmonary aspergillosis is described. The
species of Aspergillus involved, A. ochraceus, has not previously
been found in this disorder. The organism had antigenic properties distinct from five other species of Aspergillus most commonly associated with allergic bronchopulmonary aspergillosis.
The patient had immediate skin test, immunodiffusion, and
radioallergosorbent reactivity to the species. Four per cent
of 112 serum samples from others suspected of having allergic
bronchopulmonary aspergillosis had precipitins to A. ochraceus
only. It may be necessary in some cases to prepare and test
extracts of the patient's Aspergillus isolate in order to confirm
the diagnosis. (Key words: Allergic bronchopulmonary aspergillosis; Aspergillus; Aspergillus ochraceus.)
ASPERGILLUS ORGANISMS have been associated
with human disease as tissue invaders, cavity colonizers,
and antigenic stimulators of the immune system leading
to hypersensitivity syndromes. Of the some 150 species,8 relatively few have been incriminated in disease.
Three citations were found in the medical literature for
A. ochraceus. Two cases were associated with otomyReceived August 3, 1977; received revised manuscript September
13, 1977; accepted for publication September 13, 1977.
Address reprint requests to Dr. Novey: Department of Medicine,
Division of Allergy and Immunology, University of California
Irvine Medical Center, 101 City Drive South, Orange, California
92668.
Department of Medicine, Division of Allergy and
Immunology, University of California Irvine
Medical Center, Orange, California
cosis,8 and the other was associated with pellagra
in 1905.5
The purpose of this report is to present the case of
a patient who had allergic bronchopulmonary aspergillosis associated with sensitization to A. ochraceus,
together with the results of immunoserologic tests
supportive of his diagnosis and progress, and studies
of the prevalence of precipitins to this species in selected populations.
Report of a Case
A 29-year-old black man was referred to the medical center in
December 1972, because of asthma of increasing severity and cough
productive of greenish sputum of three weeks' duration. There was
associated anorexia and diffuse arthralgia, without joint swelling.
Despite self-medication with tetracycline for six days, the illness
did not abate.
Past history revealed that the patient's health had been good,
except that he had had asthma since the age of 6 years. The asthma
was relatively mild and easily controlled by occasional use of an
orally administered bronchodilator until the patient's employment
at the age of 23 years at a glass and bottle factory. During his four
years there, the asthma progressed to a chronic disease incompletely
controlled by high-dose bronchodilators and prednisone at an average
0002-9173/78/1100/0840 $00.70 © American Society of Clinical Pathologists
Vol. 70 • No. 5
dose of 10 mg daily. His work entailed heavy exposures to dusts
thought to contain silica, wood, and arsenicals.
The patient had been hospitalized by his physician in March 1972
for asthma, at which time a report of roentgenographic evidence of
apical infiltrates in the chest suggestive of tuberculosis had been
made. Such evidence had not been present on a roentgenogram
of the chest in 1967. During hospitalization a complete blood count
had disclosed 10,250 leukocytes with 9% eosinophils. Sputum culture for bacteria grew no pathogens. Sputum cytology was not
reported. A tuberculin skin test, type unspecified, had produced
2.5 cm induration in 48 hours. The patient had been treated with
antibiotics and injectable and oral steroids, and after 11 days the
abnormalities on the roentgenogram of the chest had cleared.
The family history was negative for asthma, tuberculosis, and
allergic disorders. On examination, the patient was well-developed
and well-nourished, with the facial rounding, rubor, and acne of
Cushing's syndrome. He had clinical signs of asthma and a temperature of 37.8 C and appeared to be in mild distress.
Pulmonary function test results were consistent with airway obstruction, which was alleviated by bronchodilator inhalation. Diffusing capacity was slightly greater than predicted, also compatible
with asthma. A roentgenogram of the chest was interpreted as
showing stranding in both apices. Skin tests produced immediate
reactions to several grass, weed, dust, and mold antigens. The following tests showed no abnormality: sputum cultures x 3 for mycobacteriaceae, complete blood count, urinalysis, SMA 12, T 3 and
T< by column, a, antitrypsin, quantitative immunoglobulins, and
determination of sweat chlorides.
Although the patient became afebrile, he continued to experience
asthma, and noticed the appearance of tiny yellow plugs in his sputum,
which were not made available for examination by physicians. On
January 10, 1973, the patient was examined by a visiting professor,
Dr. Charles Reed, of the University of Wisconsin, who concurred
with the suggested diagnosis of allergic bronchopulmonary aspergillosis. Sputum cultured in Sabouraud's medium produced a large
growth of an Aspergillus species, later identified by the California
State Health Department as A. ochraceus, based on the morphology
described by Raper and Fennell. 5
A skin test with Aspergillus mixture 1-10,000*, 0.02 ml, injected
intracutaneously, produced a 4+ reaction with pseudopodia. There
was no Arthus reaction. The patient was taking prednisone, 60 mg,
four times a day, at the time. Serum precipitins to Aspergillus fumigatus and four other species were all negative.
An extract prepared from the patient's A. ochraceus culture,
however, yielded four distinct precipitin bands to his serum (Table 1).
The patient has been followed by the same service to the present
time. He has continued to experience steroid-dependent asthma.
In March 1975, a bronchogram of the right side only was consistent
with diffuse cystic bronchiectasis. Skin tests with extracts of Aspergillus species produced positive wheal-and-flare reactions to A.
ochraceus and A. niger, 1-1,000, and to A. fischeri, A. fumigatus,
and the commercial Aspergillus mixture at 1-10,000 dilution. The
most recent culture of A. ochraceus occurred despite the fact that
the patient had been receiving amphotericin B, 1 mg, four times
a day by intermittent positive-pressure breathing for three months,
and was relatively asymptomatic. His dose of steroids had been
reduced from 100 mg every other day four months before to 25 mg
every other day.
A bronchial challenge test was performed with increasing concentrations of Penicillium extract shortly after the organism appeared
in the sputum culture. There was no bronchial reactivity as measured by examination and forced expiratory flow rate in 1 second
(9 1. Collins timed Vitalometer). Challenges with Aspergillus extracts were not performed.
* Hollister-Stier, Spokane, Washington.
841
CASE REPORTS
Materials and Methods
Mycology
Sputum specimens were collected in sterile containers
and cultured for fungal species on Sabouraud's dextrose
agar and mycobiotic agar at 30 C for two weeks, and
on a 5% sheep blood-brain-heart infusion agar at 35 C
for four weeks. Subcultures were transferred to Czapek
Dox agar plates for identification. For bacterial flora,
sputum samples were cultured on sheep blood, MacConkey, and chocolate agars, each at 35 C in a 5%
C02-enriched atmosphere.
lmmunoserologic Studies
Antigens. Pure culture isolates of A. fumigatus, A.
niger, A. flavus, A. terreus, A. fischeri, and A. ochraceus were grown on histoplasmin-asparagine synthetic medium for twelve weeks at room temperature.
An equal volume of Coca's fluid (x2 concentration)
was then added, and extraction was allowed to proceed
for seven days at room temperature. The supernatants
were Seitz-filtered, dialysed 24 hours against running
tap water, and lyophilized.
Precipitin Tests. The lyophisates were reconstituted
at 30 mg/ml in physiologic saline solution with 0.05%
sodium azide added as preservative. The tests were
performed on agar gel by the double-diffusion Ouchterlony method.3
Tests for specific IgE antibody: Aspergillus immunosorbent conjugates were prepared by mixing 20-mg
amounts of the lyophisates with 100 mg of CNBr-activated cellulose particles, and IgE antibody was determined by the radioallergosorbent technic.7 A scoring
system of 0 for counts per minute equal to the range
of controls, +/-for cpm over upper limits to 2.5 x controls, 1+ for 2.5-5X, 2 + = 5 - 1 0 x , 3+ = 10-20x,
and 4+ = >20x mean of control cpm was used.
Test Sera. Test sera included (1) sequential samples
from the subject over the study period, (2) samples
from 260 patients attending the adult allergy clinic at
UCIMC, (3) samples from 112 subjects throughout
California suspected by their physicians of having
allergic bronchopulmonary aspergillosis, (4) samples
from 22 healthy control subjects, and (5), for the radioallergosorbent technic only, 22 samples from cystic
fibrosis patients with (10) and without (12) IgE antibodies to Aspergillus species.
Results
Mycology
The two initial sputum isolates, as well as two others
43 and 50 months later, grew Aspergillus identified as
A.J.C.P. • November 1978
NOVEY AND WELLS
842
Table I. Precipitin and IgE Antibody to Aspergillus ochraceus in Serum from Patient
with Sputum Cultures of A. ochraceus and Other Fungi
Date
No.
Precipitin
Bands
2/73
9/73
3/74
8/74
1/75
3/75
10/75
6/76
9/76
4/77
4 (0,0)t
3 (0,0)
2 (0,0)
2(1,1)
1 (1,0)
1(1,1)
0(1,0)
0 (0,0)
0 (0,0)
0 (0,0)
IgE Antibody*
Sputum Cultures
2+
4+
ND
1+
ND
1+
A. ochraceus
Trichosporum spp.
Fungus isolated, not identified
Streptococcus
pneumoniae
Cephalosporin and Penicillium spp.
Negative (transtracheal)
Dermatiacae non sporulants
Not done
A. ochraceus, Fusarium, and Penicillium spp.
A. ochraceus, A. niger, and Penicillium spp.
+/1+
2+
2+
* Radioallergasorbenttechnic. 1+ = cpm2V4-5x control, 2+ = 5 - 1 0 X . 3 + = 10-20X,
and 4+ = > 2 0 x control.
t First number in parentheses refers to precipitin bands for A. fumigatus; second refers
to A. niger. The following antigen sources were also examined and were always negative: A. terreus, A.fischeri, A.flavus, Penicillium spp., and Candida albicans.
A. ochraceus by mycologists with the California State
Health Department, Berkeley.
The subject had examinations of 23 other sputum
samples over a four-year period, none of which grew
this species. Many samples grew either some bacteria
other than normal throat flora or other fungal organisms,
as shown in Table 1.
serum activity occurred early in the patient's course,
but has continued positive throughout most of his follow-up examinations. The 2+ or 4+ scores by the
radioallergosorbent technic were present with and
without precipitin activity, but appeared to be coincident with sputum isolates of A. ochraceus. None of
the controls, some of whom had IgE antibodies to
various Aspergillus species, had a positive radioallergosorbent reaction to A. ochraceus.
Precipitins to A. ochraceus and five other species
of Aspergillus in three population groups are tabulated
in Table 2. Of the 19 subjects whose specimens produced precipitins to A. ochraceus, six did not react
to any of the other Aspergillus species.
Immunoserologic Studies
The results of the double-diffusion tests for precipitating antibody and the radioallergosorbent technic
for IgE antibody in most of the sequential serum samples
are listed in Table 1. The first three samples obtained
(months 1,7, and 13) reacted specifically and to A.
ochraceus only. By the eighteenth month, serum precipitins to A. fumigatus and A. niger had appeared
also. At 23 months there was a single line to A. ochraceus and A. fumigatus, and three months later, to all
three species again. Six specimens obtained after this
(only four shown) were precipitin-negative to A. ochraceus.
The immediate skin test reactivity to an extract of
A. ochraceus (see the case report) was confirmed as
probably representing specific IgE antibody by the
results of the radioallergosorbent technic. The highest
Discussion
Aspergillus ochraceus has rarely been associated
with disease in man. To our knowledge, it has not been
reported as being cultured from sputum, or been causally
related to any of the forms of pulmonary aspergillosis,
i.e., allergic, invasive, or aspergilloma. The patient had
many of the criteria used to establish the diagnosis of
allergic bronchopulmonary aspergillosis. He was in his
twenties, with a long history of asthma, expectorated
small yellow plugs, had eosinophilia, abnormal chest
Table 2. Incidence of Serum Precipitins to Aspergillus ochraceus Compared with Five Other Aspergillus Species*
+ to A.
ochraceus Alone
Population Studied
Suspected ABPAt
Adult allergy clinic
(consecutive patients)
Normal adults
* Same as in Table 1.
t Allergic bronchopulmonary aspergillosis.
No. +/
No. Tested
%
No.
13/112
12
4
4
46
6/260
0/22
2
0
2
0
0.8
0
15
0
%
% + to Other
Aspergillus Spp.
Vol. 70 • No. 5
CASE REPORTS
films, repeated cultural evidence of viable Aspergillus
organisms, and an immunologic response in the form
of positive immediate skin tests and specific precipitating and IgE antibodies in the serum. The disease
did not have features of an aspergillus toxicosis. The
sources of the patient's exposure to the fungi are unknown. His clinical state deteriorated during his workrelated exposure to dust, which may have been contaminated with fungal spores.
Testing for Aspergillus precipitins is helpful in the
diagnosis of allergic bronchopulmonary Aspergillosis
and perhaps in the establishing the prognosis of the
disease. M In our patient's case, the suspected diagnosis could not be confirmed by using extracts of A.
fumigatus or other Aspergillus species reported to be
involved in allergic bronchopulmonary aspergillosis.
The immunodiffusion and radioallergosorbent technic
results suggest that the subject initially reacted chiefly
to antigenic determinants unique to the ochraceus
species. That his response itself is not unique is shown
by six others who also reacted solely to A. ochraceus,
albeit with only one sample each studied.
Thus, in those patients suspected of having allergic
bronchopulmonary aspergillosis but negative to a battery of Aspergillus antigens, isolation and extraction
of the patient's own strain or species may be necessary
in order to elicit precipitin activity. Even in carefully
studied series of allergic bronchopulmonary aspergillosis, precipitins were not found in at least 4% of the
patients.2 Although it is conceivable that high steroid
dosage might interfere with immunoglobulin synthesis
and be responsible for absent precipitins, this patient
had precipitins during prednisone dosage as high as
843
100 mg every other day for four weeks. Interestingly,
oxidases extracted from/4, ochraceus have been used
to convert various portions of the sterol molecule,
and thus, the organisms conceivably could inhibit the
pharmacologic effects of the corticosteroids directed
against them.5
It should also be noted that after four years of close
medical supervision and excellent cooperation, the
patient has finally had a remission, sustained for more
than four months, during which the asthma has abated,
well-being, and pulmonary function tests have improved, and steroid dosage has been decreased to its
lowest level. Improvement was coincident with a loss
of precipitin activity, and despite the presence of specific IgE antibodies and sputum isolates of Aspergillus.
Acknowledgments. Louis E. Marchioli, M.D., and Paul Cloninger,
M.D., referred the patient to us.
References
1. Hanson G, Flod N, Wells I, et al: Bronchocentric granulomatosis: a complication of allergic bronchopulmonary aspergillosis. J Allergy Clin Immunol 59:83-90, 1977
2. McCarthy DS, Pepys J: Allergic bronchopulmonary aspergillosis. Clin Allergy 1:261-286, 1971
3. Ouchterlony O: Precipitin tests for the detection of circulating
antibody to specific antigen. Acta Pathol Microbiol Scand
32:231-240, 1953
4. Pepys J: Hypersensitivity Diseases of the Lungs Due to Fungi
and Organic Dusts. Basel, Karger, 1969
5. Raper KB, Fennell DI: The Genus Aspergillus. Baltimore,
Williams and Wilkins, 1965
6. Sinshi JT: The Epidemiology of Human Mycotic Diseases.
Springfield, 111., Charles C Thomas, 1975
7. Wide L, Bennich H, Johannson SGO: Diagnosis of allergy in
vitro test for allergen antibodies. Lancet 2:1105-1106, 1967
8. Wolf FT: Relation of various fungi to otomycosis. Arch Otolaryngol 46:361-374, 1947