- Email: [email protected]
Invasive aspergillosis diagnosed by immunohistochemistry with monoclonal and polyclonal reagents
Invasive Aspergillosis Diagnosed by Immunohistochemistry with Monoclonal and Polyclonal Reagents PETERPHILLIPS,MD, AND MARC H. WEINER, MD Therapies differ for invasive infection due to the various filamentous fungi. However, histopathologic identification of Asper-
gillus, Pseudallescheria, Fusarium, Trichosporon, dematiacious Hyphomycetes, Candida, and Zygomycetes may be confused, and microbiologic isolation is often delayed. To improve diagnosis, we examined the utility of polyclonal and monoclonal antibodies to identify Aspergillus organisms by peroxidase immunohistochemical techniques. Tissues obtained from 68 infected patients were examined. Fungal antigen was detected in all 26 cases of aspergillosis by the monoclonal immunohistochemical test. Hyphae of AspergiUus organisms exhibited strong intracellular and cell wall staining, and extracellular localization was also observed. In contrast, negative tests were observed in tissues from five cases of infection with Pseudallescheria boydii, three with Trichosporon, three with Zygomycetes, six with Candida species, and one with Curvularia, from as well as 20 cases of nonfungal pneumonia. However, cross-reactivity to Coccidioides immitis spherules was noted in three cases, Staining of Candida was observed with the polyclonal antibody but not the monoclonal reagent. This is the first immunohistochemical demonstration of aspergillus antigen with a monoclonal reagent. The study indicates that the monoclonal immunohistochemical technique can distinguish Aspergillus species from other filamentous fungi and may facilitate the clinical diagnosis of invasive aspergillosis. HUM PATHOL 18:1015--1024, 1987.
T h e diagnosis o f invasive fungal disease frequently relies on histologic examination and culture of a tissue biopsy specimen. Because microbiologic isolation and identification may take several days or weeks, a presumptive etiologic diagnosis o f mycotic infection depends on the morphologic appearance of the organism in tissue. 1 T h e histologic appearance of some fungi (e.g., spherules of Coccidioides immitis) is so characteristic as to be diagnostic. However, numerous filamentous fungi cannot be differentiated reliably with any of the commonly used fungal stains.
From the Division of Infectious Diseases, Department of Medicine, Audie L. Murphy Memorial Veterans Administration Hospital and The University of Texas Health Science Center, San Antonio, Texas. Accepted for publication 21 November 1986. A preliminary report of this work was presented at the 25th Annual Meeting of the Interscience Conference on Antimicrobial Agents and Chemotherapy. Grant support: from the National Institutes of Health (ROI AI13770-09) and the Research Service of the Veterans Administration. Dr. Phillips is supported by a British Columbia Health Care Research Foundation Postdoctoral Fellowship. His current address is the Division of Infectious Diseases, Vancouver General Hospital, 910 West 10th Avenue, Vancouver, British Columbia
VSZ IM9, Canada. Address correspondenceand reprint requests to Dr. VCeiner: Department of Medicine,The Universityof Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio,TX 78284-7881. 0046-8711187 $0.00 + .25
C o n s e q u e n t l y , i n a p p r o p r i a t e empiric antifungal therapy may be initiated on the basis of a mistaken histologic diagnosis. For example, amphotericin B remains the drug of choice for invasive aspergillosis, but infections caused by Pseudallescheria boydii or Tr/chosporon species may respond better to an imidazole derivative. 2,3 Kaplan et al.,4 Jackson et al., 5 and Hotchim et al. 6 have used polyclonal antibodies for direct immunofluorescence of tissue sections in the diagnosis of various systemic mycotic infections. To improve specificity of staining, the authors preadsorbed the polyclonal reagents with other cross-reacting organisms. Fungi that have been successfully identified with this technique include C. immitis, Histoplasma capsulatum, Blastomyces dermatitidis, Candida species, Aspergillus species, Zygomycetes, P. boydii, and Cryptococcus neofonnans. However, antibodies against fungi have not been widely available for clinical diagnostic purposes. As a result, fungal immunohistochemical testing has remained a service provided by few reference laboratories. In this study, we examine the diagnostic utility of polyclonal and monoclonal antibodies to identify Aspergillus species in human tissues using peroxidase immunohistochemical techniques.
METHODS Classification of Cases
Pathology records from three hospitals in San Antonio (Medical Center Hospital, Audie L. Murphy Veterans Administration Hospital, and the Wilford Hall USAF Medical Center) were reviewed to identify cases of invasive infections due to filamentous fungi and to identify control cases o f nonfungal pneumonia. Ten cases of fungal infection were provided by six medical centers outside Texas. Cases o f invasive aspergillosis were initially identified histologically and then subclassified by microbiologic criteria. If aspergilli were isolated from a single tissue that was also positive on histologic examination, then the case was classified as culture positive. I f aspergilli were isolated from a different site, then the case was classified as culture supported. If cultures did not yield a fungus or if no cultures were obtained, then the case was designated culture negative. Cases of nonfungaI pneumonia were defined as demonstrating an inflammatory pulmonary infiltrate but having negative results on methenamine silver stains.
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HUMAN PATHOLOGY
Volume 18, No. 10 [October 1987]
in 1 per cent swine serum in Tris saline. This was followed by incubation with the horseradish peroxidase/rabbit antiperoxidase immune complex (Dako) for 30 minutes. Tissue sections were washed with 1 per cent swine serum in Tris saline following the application o f each o f the immunoglobulin reagents. Final washings were performed with Tris saline and then Tris buffer prior to the application of 3,3'-diaminobenzidine and h y d r o g e n p e r o x i d e in Tris b u f f e r (10 minutes). This solution resulted in a brown color change at the sites where horseradish peroxidase had localized in the tissue sections. Perm a n e n t m o u n t s were made after cotmterstaining with hematoxylin for 1 minute.
Polyclonal Immunohistochemistry
Tissues (in the San Antonio hospitals) were placed in a 10 per cent buffered formalin for fixation. Paraffin-embedded tissue specimens from 11 cases were stained with a three-layer horseradish p e r o x i d a s e - a n t i p e r o x i d a s e (PAP) m e t h o d , according to the method of Sternberger. 7 The procedure for the preparation of the primary antibody, polyclonal anti-aspergillus rabbit serum, has been described previously, s Tissue sections 3 rtm thick were mounted on glass slides with 1 per cent aqueous polylysine hydrobromide (Polysciences, Inc., Warrington, Pennsylvania). Preliminary incubations immediately following deparaffinization included the application of a m e t h a n o l - 3 per cent hydrogen peroxide solution (5:1 by volume) for 30 minutes and then a methanol-hydrochloric acid solution (500:1 by volume) for 30 minutes. Tissues were then incubated with 3 per cent normal swine serum in Tris saline for 10 minutes, followed by the primary anti-aspergillus rabbit serum for 2 hours at room temperature. T h e optimal dilution of the primary antibody was determined to be 1:500 in a solution of 3 per cent normal swine serum in Tris saline. The tissues were overlaid with a commercial swine anti-rabbit immunoglobtdin (Dako Corp, Santa Barbara, California) diluted l:100
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Specimens from 68 cases were stained with a three-layer avidin-biotin-peroxidase complex (ABC) technique, 9 employing a murine monoclonal, antiaspergillus IgM antibody (M. Weiner, unpublished data). Both the mounting of tissue sections on glass slides with polylysine and the initial methanol-hydrogen peroxide incubation were carried out as for the PAP method. Then, tissues were washed in phosphate-buffered saline for 20 minutes. An incubation with 2 per cent normal goat serum in phosphate-
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FIGURE 2 (top). Invasive pulmonary aspergillosis stained with normal rabbit serum by'the peroxidase/antiperoxidase [PAP) method [panel /9); polyclonal anti-aspergllus rabbit serum by the PAP method [panel a); and monoclonal anti-aspergiIlus antibody by the avidin-biotin method [panels c and d]. (Original magnification: a and b, x 2; c, x 60; d, x 240.) FIGURE 3 [bottom). Invasive pulmonary aspergllosis, stained with monoclonal anti-aspergilius antibody by the avidin-biotin method. The left panel shows ceil wall and intracelular antigen localization, and the right panel shows hypha[ and extracelular staining. (Original magnification: left,, x 640; right, x 240.}
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HUMANPATHOLOGY
Volume 18, No. 10 (October 1987)
Interpretation of Peroxidase Immunohistochemlstry
TABLEt,
Cases and Controls Studied by Immunohlstochemlstry
Organism
Cases
Aspergillus spp.
25
Pseudallescheria boydii Trichosporon beigelii Zygomycetes Candida spp.
Curvularia lunata Coccidioides immitis Total mycotic infections Nonfungal pneumonias
5 3 3 6 1 3 46 20
Sites o f Infection Lung, 22; brain, 1; sinus, 1; subcutafieous tissue, 1 Brain, I; dura, 1; lung, 1; paranasal sinus, 1; kidney, 1 Kidney, 2; lung, 1 Sinus, 2; lung, 1 Lung, 6 Paranasal sinus, 1 Lung, 2; cervical node, 1
buffered saline for 20 minutes was followed by the murine monoclonal anti-aspergillus IgM antibody for 2 hours at 4~ T h e optimal concentration of the monoclonal antibody was found to be 1:1,000. Then, biotinylated goat anti-mouse IgM (Vector Laboratories, Burlingame, California) was applied for 30 minutes. Tissues were washed in phosphate-buffered saline after each step. Finally, the ABC complex (Vector Laboratories) was applied for 1 hour, after which the specimens were treated with 3,3'-diaminob e n z i d i n e - h y d r o g e n peroxide in Tris buffer, as in the PAP technique. Counter-staining was performed with hematoxylin, and permanent mounts were prepared.
Tissue sections were examined for the presence o f the characteristic brown staining. T h e intensity and distribution of staining for the positive cases were assessed as follows. Intensity was graded on a scale o f 0 to 3, where 0 indicated absence o f staining; 1, minimal staining only appreciated with a magnification of greater than 100x; 2, moderate staining detectable with the low-power objective (100 x); and 3, intense staining identified with a magnification of 40 • or less. T h e location of staining was indicated as hyphal, if fun.gal organisms were Stained. If staining was present m areas either adjacent to or remote from fungal organisms, then it was designated as extracellular. S p e c i m e n s that were positive by the ABC m e t h o d were f u r t h e r evaluated by repeating the technique with a control murine monoclonal IgM antibody. T h e control reagent was directed against the bacterium Gardnerella vaginalis. Two cases o f nonfungal pneumonia demonstrated nonspecific staining with control murine antibody and were not included in the subsequent analysis. RESULTS
Immunohistochemistry with the Polyclonal Reagent T h e polyclonal anti-aspergillus antibody was used in the PAP method in 11 cases: five o f aspergilTABLE 2. Clinical and Immunohtstochemtcal
Case
Age/Sex (yr)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
78/F 57/F 44/F 57/M 70/M 60/M 65IF 65IF 38IF 251M 65tM 45IF 26/M 59/M 40/M 231M 53/M 72/F 31/F 731M 69/M 65/M 52/F 3 IIF 57/F
Underlying disease
Broadspectrum Antibiotics
Imnmnosuppressive Therapy
Neutropenia*
Bronchitis Hemolysis Leukemia (ANLL) Asthma Hodgkin's Dysmyelopoiesis COPD COPD Hepatitis Leukemia (ANLL) Cirrhosis Leukemia (ANLL) Aplastic anemia Lung carcinoma Systemic lupus Leukemia (ALL) Cirrhosis Leukemia (ANLL) Renal transplant Nephritis Tongue carcinoma Leukemia (ANLL) Lymphoma Leukemia (ANLL) Leukemia (ANLL)
+ + + NA + + + + + + + + + + + + + + NA + NA + + + +
+ + + + + + + + + + + + + + + + + + + + + + +
+ + + ,+ + + + + + + NA + + +
ABBREVIATIONS" ANLL, acute nonlymphocytic leukemia; COPD, chronic obstructive pulmonary disease; ALL, acute lymphocytic leukemia; NA, not available. * Neutropenia refers to a leukocyte count
1018
INVASIVEASPERGILLOSIS[Phillips& Weiner] losis, two o f candidiasis, o n e o f coccidioidomycosis, a n d t h r e e o f n o n f l m g a l p n e u m o n i a . Aspergilhts antigen was detected, as indicated by a m o d e r a t e to intense staining o f f u n g a l h y p h a e , in all five s p e c i m e n s of invasive aspergillosis. However, cross-reactive s t a i n i n g was n o t e d f o r all cases o f candidiasis a n d coccidioidomycosis. F i g u r e s i a n d 2 show, at low magnification, c o m p a r a b l e tissue sections f r o m the s a m e s p e c i m e n o f invasive aspergillosis stained with m e t h e n a m i n e silver (fig. 1), n o r m a l rabbit s e r u m in the PAP m e t h o d (fig. 2b), a n d polyclonal rabbit antiaspergillus a n t i b o d y in the PAP m e t h o d (fig. 2a).
Immunohistochemistry with the M o n o c l o n a l Antibody Sixty-six cases w e r e e v a l u a t e d with the a n t i a s p e r g i l l u s m o n o c l o n a l a n t i b o d y u s i n g the avidinbiotin system. Forty-six o f these were invasive fungal infections; the o t h e r 20 cases were n o n f u n g a l p n e u m o n i a . T w e n t y - f i v e o f the fungal infections were d u e to Aspergillus species. T h e o t h e r 21 cases w e r e d u e to P. boydii (five cases), Trichosporon species (three cases), Z y g o m y c e t e s (three cases), Candida species (six cases), Curvularia lunata (one case), a n d C. immitis (three cases). T h e a n a t o m i c sites o f fungal i n v o l v e m e n t f o r the 46 cases a r e listed in table 1.
Aspergillus Infections T h e clinical a n d i m m u n o h i s t o c h e m i c a l data o f the 25 cases o f aspergillus infection are s u m m a r i z e d
TABLE 3.
Monoclonal lmmunohistochemical Findings in Aspergillosis
Microbiologic Status
Cases
Hyphal Staining
Extracellular Staining
Culture positive Culture supported Culture negative TOTALS
9 3 13 25
9 3 13 25
3 2 4 9
in table 2. T h e culture-positive cases included f o u r with A. fumigatus, f o u r with A. flavus, a n d o n e with A. terreus. T w e n t y patients (80 p e r cent) h a d b e e n receiving b o t h i m m u n o s u p p r e s s i v e a n d b r o a d - s p e c t r u m antibiotic t h e r a p y . Tissues studied by i m m u n o histochemical techniques included lung, brain, p a r a nasal sinus, a n d soft tissue. Aspergillus antigen was d e t e c t e d in all 25 cases o f invasive aspergillosis by the m o n o c l o n a l i m m u n o h i s t o c h e m i c a l technique (Fig. 2c a n d d), in b o t h fungal cell wall a n d intracellular locations (fig. 3 left). I n n i n e cases a n t i g e n was detected in e x t r a c e l l u l a r areas, usually adjacent to aspergillus h y p h a e (fig. 3 right). T h e s e results are s u m m a r i z e d in table 3. S p e c i m e n s f r o m five cases (cases 5, 11, 14, 20, a n d 21) w e r e e v a l u a t e d with b o t h t h e p o l y c l o n a l (PAP) a n d m o n o c l o n a l (ABC) techniques. Aspergillus a n t i g e n was readily detectable with either m e t h o d , b u t s t a i n i n g was s o m e w h a t less i n t e n s e with the m o n o c l o n a l r e a g e n t in cases 14 a n d 21. All 20 cases
Data for Cases of Invasive Aspergillosis I mmunohistochemistryw Tissue
Outcome
Fungal Infectiont
Cuhure~
Studied
PAb
MAb
Dead Dead Alive Alive Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Alive Dead Dead Dead
Dissem asp Dissem asp Inv asp sinusitis Soft tissue inv asp Inv pulm asp Inv pulm asp Inv pulm asp Inv puhn asp Inv pulm asp Inv pulm asp Dissem asp Dissem asp Dissem asp Inv pulm asp Dissem asp Inv pulm asp Inv pulm asp Inv pulm asp Dissem asp Inv pulm asp Inv pulm asp Inv pulm asp Inv'pulm asp Inv pulm asp Inv pulm asp
A. flavus (P) A. fumigatus (P) A. flavus (P) A. fumigatus (P) A. terreus (P) A.flavus (P) A. fumigatus (P) A. fumigatus (P) A. flavus (P) A.flavus (S-sputum) Aspergillus sp. (S-sputum) A. flavus (S-vitreous) Aspergillus sp. (S-skin biopsy) Aspergillus sp. (S-sputum) N N N N N N N N N N N
Lung Lung Sinus Neck mass Lung Lung Lung Lung Lung Lung Lung Brain Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung
----3 -----3 , --3 -----3 2 -----
3 3 3 3 3 3 2 3 3 3 3 3 3 2 3 3 3 3 3 3 1 3 3 3 3
Cultures were graded as positive (P), supportive (S), or negative (N). See Methods for details. wImmunohistochemical tests were done on the indicated tissue with either polyclonal (PAb) or monoclonal (MAb) antibodies. Staining was graded as 0 (none) to 3 (intense). See Methods for details. Tile dashes under PAb indicate not studied. 1019
HUMAN PATHOLOGY
Volume 18, No. 10 (October 1987] TABLE 4.
Case
Age/Sex (yr)
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
19/F 62/F 24lF 43/M NA/F 15/M 23/M 62/M 63/M 86/M NA/NA 78/F 0.6/F 55/F 32/F 17/M 65/M 23/M 25/M 21/M 57/M
Clinical and ImmunohistOchemical
Underlying Disease
Broadspectrum Antibiotics
Immunosuppressive T h e r a p y
Neutropenla*
Leukemia (ALL) Diabetes None Leukemia (ANLL) Seizures Leukemia (ALL) Aplastic anemia Glomerulonephritis Leukemia (ANLL) Diabetes NA COPD Prematurity Lymphoma Leukemia (ANLL) Leukemia (ANLL) Leukemia (ANLL) Allergic rhinitis None Renal transplant Hodgkin's
+ + NA + + + + NA NA + + + + + NA +
+ + NA + + + + NA + + + + + +
+ + NA + + + NA + + + + + +
ABBREVIATIONS: ANLL, acute nonlymphocytic leukemia; COPD, chronic obstructive p u l m o n a r y disease; ALL, acute lymphocytic leukemia; NA, not available. * N e u t r o p e n i a refers to a leukocyte count < l , 0 0 0 / m m s. t Dissem P. boydii, disseminated Pseudallescheria boydii; dissem coccid, disseminated coccidioidomycosis.
of aspergillosis that were tested with only the antiaspergillus monoclonal antibody showed intense (grade 3) hyphal staining.
Nonaspergillus Mycoses and Nonfungol Pneumonlas The clinical and immunohistochemical data for the cases of nonaspergillus mycoses and nonfungal pneumonias are summarized in tables 4 and 5. None of 20 control specimens of nonfungal pneumonia demonstrated aspergillus antigen (fig. 4). Cross-reactive staining was absent in the candidiasis cases, although this result was noted with the polyclonal reagent. All three cases of coccidioidomycosis exhibited cross-reactive staining with the monoclonal antibody. Aspergillus antigen could not be identified in 17 cases of fungal infection caused by filamentous organisms other than Aspergillus species. One additional case (case 33) was not easily classified. Two, morphologically distinct, filamentous fungi were distinguished on Gomori's methenamine silver stain of pulmonary tissue and suggested to the attending.pathologist the presence of a mixed fungal pneumonia. However, only Trichosporon beigelii was isolated from postmortem lung cultures. With the monoclonal immunohistochemistry, only fungal abscesses of one of the two morphotypes were stained with the ABG method (figs. 5-1A, 5-1B, and 6). To characterize this case better and to determine whether this represented a coinfection or cross-reactive staining with a shared antigen of T. beigelii, we did further immunohistochemical stains of this specimen. Rabbit polyclonal antibody against Cryptococcus
neoformans had been noted to cross-react with antigens of T. beigelii, lo We tested this case with a rabbit anti-cryptococcal reagent (kindly provided by Dr. Leo Kaufman, Centers for Disease Control) as the primary antibody in the PAP method. All areas (both fungal morphotypes) of fungal involvement distinguished by Gomori's methenamine silver method were stained with the unabsorbed cryptococcal antibody (figs. 5-2A and 5-2B). However, when the test was repeated using the cryptococcal reagent preadsorbed with a polyvalent aspergillus antigen mixture (figs. 5-3A and 5-3B), the only areas that stained positively were of the second morphotype which had not stained positively with the aspergillus monoclonal antibody. These immunochemical studies indicated a coinfection with two filamentous fungi rather than cross-reactivity of the monoclonal antibody to a T. beigelii epitope. DISCUSSION
Diagnostic problems regarding invasive fungal infections are common in immunocompromised patients. Culture results are often delayed, and the first opportunity to make a specific etiologic diagnosis may be provided by the histopathologic examination of a biopsy specimen. The combination of a positive mucicarmine stain and the detection of organisms morphologically consistent with Cryptococcus neoformans in tissue Is diagnostic for cryptococcosis, even though the stain is not absolutely specific for that organism. However, with
t020
INVASIVEASPERGILLOSIS[Phillips& Weiner] Data for 21 Cases of Nonasperglllus Mycoses Immunohistochemistryw Tissue
Outcome Dead Alive Alive Dead Dead Dead Dead Dead Dead Dead NA Dead Dead Dead Dead Dead Dead Alive Alive Alive Dead
Fungal Infectiont
Culture*
Studied
PAb
MAb
Dissem P. boydii P. bto'dii dural abscess P. bo)'dii sinusitis Dissem P. boydii Dissem P. boydii Dissem Trichosporon Dissem Trichosporon Dissem Trichosporon Zygomycosis Zygomycosis Zygomycosis Dissem candidiasis Dissem candidiasis Dissem candidiasis Dissem candidiasis Candida pneumonia Dissem candidiasis Curvularia sinusitis Dissem coccid Dissem coccid Dissem coccid
Pseudallescheria boydii (P) P. boydii (P) P. boydii (P) P. boydii (P) P. boydii (S) T. beigelii (P) 7". beigelii (P) T. beigelii (P) RUmpus sp. (P) N N Candida tropicalis (P) C. tropicalis (S-liver) C. albicans (S-blood) N N N Curvularia lunata (P) Coccidioides immitis (P) C. immitis (P) C. immitis (P)
Lung Dura Sinus Kidney Brain Kidney Kidney Lung Sinus Sinus Lung Lung Lung Lung Lung Lung Lung Sinus Neck node Lung Lung
m -m --m -~ m ---~ -3 -3 ~ -~ 3
0 0 0 0 0 0 0 0/382 0 0 0 0 0 0 0 0 0 0 2 3 3
* Cultures were graded as positive (P), supportive (S), or negative (N). See Methods for details. wImmunohistochemical tests were done on the indicated tissue with either polyclonal (PAb) or monoclonal (MAb) antibodies. Staining was graded as 0 (none) to 3 (intense). See Methods for details. 82See Results for discussion of this case. the widely used fungal stains, such as G o m o r i ' s m e t h e n a m i n e silver, periodic a c i d - S c h i f f , a n d Gridley fungus, it is the m o r p h o l o g i c a p p e a r a n c e o f the org a n i s m in tissue, r a t h e r t h a n its staining p r o p e r t i e s , that enables o n e to m a k e a p r e s u m p t i v e identification. U n f o r t u n a t e l y , m a n y o f the f i l a m e n t o u s fungi c a n n o t be reliably d i f f e r e n t i a t e d f r o m o n e a n o t h e r with a n y o f the a b o v e - m e n t i o n e d stains. Several techniques h a v e b e e n d e m o n s t r a t e d to aid in the h i s t o p a t h o l o g i c identification o f fungi. T h e s e include f u n g a l a u t o f l u o r e s c e n c e Ira2 a n d f l u o rescent stains such as acridine o r a n g e , t3 c a l c o f l u o r white, 14 2 - h y d r o x y s t i l b a m i d i n e isethionate, t5 Blankop h o r BA 267 p e r cent (Bayer, L e v e r k u s e n , West G e r many), 1~ a n d d i a e t h a n o l . 1 7 I n general, these m e t h o d s are m o r e r a p i d b u t s o m e w h a t less sensitive o r specific t h a n c o n v e n t i o n a l f u n g a t stains. E x c e p t f o r the Zygo-
TABLE 5.
m y c e t e s , w h i c h d o n o t a u t o f l u o r e s c e , t h e s e techn i q u e s d o n o t p r o v i d e d i f f e r e n t i a l staining o f the various f u n g a l genera. T h e f l u o r e s c e n t m e t h o d s m a y c o m p l e m e n t r a t h e r t h a n replace the c o m m o n l y used fungal stains. F u n g a l i m m u n o h i s t o c h e m i s t r y has facilitated the early diagnosis o f systemic mycoses in which the hist o p a t h o l o g i c findings are not p a t h o g n o m o n i c . 1 T o d a t e , p o l y c l o n a l a n t i b o d i e s , u s u a l l y p r e p a r e d in rabbits, h a v e b e e n used f o r such studies. U n f o r t u nately, p r o b l e m s relating to r e a g e n t availability a n d the necessity f o r p r e a d s o r p t i o n s with various crossr e a c t i n g f u n g i have limited the application o f these techniques. Monoclonal antibodies offer potential advantages over polyclonal reagents, including g r e a t e r specificity a n d u n i f o r m activity f r o m lot to lot. Utilization o f m o n o c l o n a l r e a g e n t s in fungal ira-
Monoclonal Immunohistochemlcal Findings In Control Cases of Filamentous Fungal Infection and Nonfungal Pneumonla Organism
PseudaUescheria boydii Trichosporon sp. Zygomycetes Candida sp. Curvularia sp. Total filamentous mycoses Nonfungal pneumonia
Microbiologic Status Culture pos. Culture pos. Culture pos. Culture neg. Culture pos. Culture neg. Culture pos.
Cases
Hyphal Staining
Extracellular Staining
5 3* 1 2 3 3 1 18 20
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
* One case with probable coinfection by Trichosporon and AspergiUus spp. identified by morphologic and microbiologic criteria. 1021
HUMAN PATHOLOGY
Volume 18, No. 10 (October 1987)
FIGURE 4 [top). Negative stainingof nonfungal pneumonia with monoclonal anti-aspergiilusantibody by the avidin-biotin method. [Original magnification: left;, x240; right, x4.]
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1022
FIGURE5 (bottom). Magnification of boxed areas A and B shown in figure 6. From contiguous sections of pulmonary tissue from case 33 stained with anti-aspergillus monoclonal antibody [panels 5-1A and 5-1B, x800], unadsorbed polyclonal anti-cryptococcal antibody by peroxidase-antlperoxidase method [panels 5-2A and 5-2B, x 800], and polyclonal anticryptococcal reagent preadsorbed with a polyvalent aspergiilus antigen mixture [panels 5-3A and 5-3B, x 320).
INVASIVEASPERGILLOStS[Phillips & Weiner]
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munohistochemical studies should obviate the preadsorption steps required to eliminate the cross-reactive staining seen with polyclonal antibodies. Our study o f fungal immunohistochemistry indicates that aspergillus antigen is readily detected in formalin-fixed tissues by a monoclonal antibody; cross-reactive staining was not observed for nonaspergillus filamentous fungi, with the possible exception o f one case o f Trichosporon beigelii pneumonia. This specificity allowed us to differentiate histologically Aspergillus species from other morphologically similar fungi. The intensity of staining of aspergillus antigen was similar with both polyclonal and monoclonal antibodies. Staining with the monoclonal technique was somewhat less intense in two of the five specimens in which both methods were employed (table 2). Nineteen of the 20 remaining aspergillosis cases all demonstrated intense (grade 3) staining of fungal hyphae with the monoclonal immunohistochemical technique. This result suggests that the monoclonal reagent is directed to one of the predominant epitopes expressed in vivo. E n h a n c e d recognition of one abundant epitope by a monoclonal antibody may compensate for its high specificity, i.e., its failure to recognize other antigenic sites. Aspergillus fumagatus, A. flav us, and A. terreus were isolated from culturepositive cases in this series. Staining of equal intensity was noted for all three species using the monoclonal reagent, indicating that the epitope identified is shared by at least those three members of the genus. The monoclonal antibody was prepared by immunizing Balb/c mice with a cell wall extract of A. fumigatus. However, abundant antigen was detected in both the hyphal cell wall and intracellular locations. This may be explained by the intracellular disposi-
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tion o f a cell wall component during synthesis or degradation. In nine of 25 cases of invasive aspergillosis, antigen was also detected in areas adjacent to, but clearly removed from, identifiable fungal hyphae. We have referred to this finding as "extracellular" fungal antigen. It was not found with control stains employing a monoclonal antibody to the bacterium Gardnerella vaginalis. Extracellular staining may represent the in vivo dissemination o f aspergillus antigen in tissue. However, the possibility that this represents antigen diffusion postmortem due to delayed or poor fixation technique cannot be excluded. 18 The relationship between aspergillus antigen disseminated in tissue and tlmt which has been detected in human serum by radioimmunoassay 1~ has not been examined. Cross-reactivity was observed in all three cases of coccidioidomycosis, with both the polyclonal and monoclonal antibodies. Tile dis'tinctive morphologic a p p e a r a n c e o f coccidioides spherules, however, readily ,distinguishes these spherules from the filamentous fungi and should not represent an immunodiagnostic problem. Cross-reactive staining also occurred with the polyclonal anti-aspergillus antibody in cases of invasive candidiasis. However, the monoclonal reagent was specific and did not produce cross-reactivity.' 9 'Negative, specific resuks were obtained with the monoclona! reagent in 17 control cases infected with filamentous organisms other than Aspergillus species. In one additional pulmonary case, histochemical differences suggested a coinfection with two filamentous fungi, although only T. beigelii was isolated from a postmortem culture. The monoclonal immunohistochemical technique also suggested coinfection. Fur-
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HUMAN PATHOLOGY
Volume 18, No. 10 (October 1987)
t h e r i m m u n o c h e m i c a l studies were d o n e to characterize the case better. W h e n an anti-cryptococcal rea g e n t cross-reactive to T. beigelii was p r e a d s o r b e d with aspergillus antigen, staining was only observed in the fungal m o r p h o t y p e that had not b e e n stained with the anti-aspergillus m o n o c l o n a l r e a g e n t (figs. 5-3A and 5-3B). This finding supports the explanation t h a t a positive r e s u l t in this case was d u e to staining o f coexistent Aspergillus species which h a d n o t been isolated by p o s t m o r t e m culture. Mixed invasive fungal infections are well d o c u m e n t e d in imm u n o c o m p r o m i s e d p a t i e n t s , 2~ a n d f a l s e - n e g a t i v e p o s t m o r t e m fungal cultures in histologically p r o v e n cases are not u n c o m m o n . An alternative premise that the anti-aspergillus m o n o c l o n a l antibody is directed against an epitope s h a r e d by both organisms appears unlikely in light o f the above studies. An increasing n u m b e r o f filamentous fungi are being recognized as opportunistic pathogens in the i m m u n o c o m p r o m i s e d patient, T h e s e organisms include Pseudallescheria boydii, Fusarium species, Trichosporon species, a n d several o f the dematiacious Hyp h o m y c e t e s such as Bipolaris species. T h e antifungal susceptibilities o f t h e s e o r g a n i s m s a n d Aspergillus species differ. F o r example, T. beigelii appears to be most susceptible to the imidazole derivatives miconazole and ketoconazole b u t relatively resistant to amphotericin and 5-flucytosine. a T h e d r u g o f choice f o r invasive P. boydii infection is miconazole. H o w e v e r , the c u r r e n t l y available imidazole d r u g s have b e e n disappointing in aspergillosis, for which a m p h o t e r icin B remains the d r u g o f choice. As the n u m b e r o f effective a n t i f u n g a l agents continues to increase, it will b e c o m e even m o r e imp o r t a n t to d e t e r m i n e the specific etiologic diagnosis o f invasive mycotic infection so that p r o m p t and app r o p r i a t e t h e r a p y can be initiated. T h e d e v e l o p m e n t o f a panel o f m o n o c l o n a l antibodies directed against the most c o m m o n l y e n c o u n t e r e d filamentous fungi m a y p r o v e useful in this respect, particularly f o r those institutions caring for significant n u m b e r s o f i m m u n o c o m p r o m i s e d patients.
Acknowledgments. T h e a u t h o r s t h a n k Drs. L. Kaufman, L. Ajello, and W. Chandler, Centers for Disease Control, Atlanta, Georgia, for providing the polyclonal anti-cryptococcal antibody; and the following persons for providing clinical case information and tissue specimens: Dr. James Orr and Dr. J. W. Rippon, University of Chicago, Chicago, Illinois; Dr. David A. Schwartz, Hahnemann Medical College, Philadelphia, Pennsylvania; Dr. J. Bagnail, Wilford Hall USAF Medical Center, San Antonio, Texas; Dr. Y. Tsukada and Dr. EI-Ani, Roswell Park Memorial Institute, Buffalo, New York; Dr. H. M. Haupt, Johns Hopkins Medical Institutions, Baltimore, Maryland; Dr. Bustamante, Baptist Memorial Hospital, San Antonio, Texas; Dr. C. Green, Michael Reese Hospital, Chicago, Illinois; Col. D.J. Wear, MC, Armed Forces Institute of Pathology, Washington, DC; and Dr. Stanton Gerson, Case Western Reserve Hospital, Cleveland, Ohio. The authors
also thank Dennis A. Havel for the photography of the immunohistochemistry, and Catherine Gonzalez, Judy Fox, and Sarah Lock-Lim for technical assistance.
REFERENCES 1. Chandler FW, Kaplan W, Ajello L: Histopathological diagnosis. In Color Atlas and Text of the Histopathology of Mycotic Diseases. Chicago, Year Book Medical Publishers, 1980 2. Rippon JW: Pseudallescheriasis. In Medical Mycology: The Pathogenic Fungi and The Pathogenic Actinomycetes, ed 2. Philadelphia, WB Saunders Co, 1982 3. Lew MA, Potter K, Levin M: Susceptibilityof Trichosporonbeigelii to systemic antifungal agents [Abstract 1037]. In Proceedings of the Interscience Conference on Antimicrobial Agents and Chemotherapy, 1984 4. Kaplan W, Kraft DE: Demonstration of pathogenic fungi in formalin-fixed tissues by immunofluorescence. Am J Clin Pathol 52:420, 1969 5. Jackson JA, Kaplan W, Kaufman L, et al: Development of fluoroescent-antibody reagents for demonstration of PseudaUescheriaboydii in tissues.J Clin Microbiol 18:668, 1983 6. Hotchim M, SchwarzJ, Kaplan W: Limitations of fluorescent antibody staining ofHistoplasma capsulatumin tissue sections. Sabouraudia 10:157, 1972 7. Sternberger LA: Immunochemistry, ed 2. New York, John Wiley & Sons, 1979, p 104 8. Weiner MH, Coats-Stephen M: Immunodiagnosis of systemic aspergillosis: antigenemia detected by radioimmunoassay in experimental infection. J Lab Clin Med 93:111, 1979 9. Hsu S, Raine L, Fanger H: Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577, 1981 10. McManus EJ, Bozdech MJ,JonesJM: Role of the latex agglutination test for cryptococcal antigen in diagnosing disseminated infections with Trichosporon beigelii. J Infect Dis 151:1167, 1985 11. Graham AR: Fungal autofluorescence with ultraviolet illumination. AmJ Clin Pathol 79:231, 1983 12. Mann JL: Autofluorescence of fungi: an aid to detection in tissue sections. AmJ Clin Pathol 79:587, 1983 13. Pickett JP, Bishop GM, et al: A simple fluorescent stain for fungi. Selective staining of fungi by a fluorescent stain for mucin. AmJ Clin Pathol 34:197, 1960 14. Monheit JE, Cowan DF, Moore DG: Rapid detection of fungi in tissues using calcofluor white and fluorescence microscopy. Arch Pathol Lab Med 108:616, 1984 15. Murgatroyd LB: 2-Hydroxystilbamidine isethionate: a new fluorochrome for use in general pathology: II. The selective demonstration of fungi. Diag Histopathol 5:219, 1982 16. Hollander H, Keilig W, et al: A reliable fluorescent stain for fungi in tissue sections and clinical specimens. Mycopathologia 88:131, 1984 17. Koch HA, Pimsler M, Walsh GP: Nonspecific fluorescent stains for detecting and identifying fungal elements in tissues [abstract F-62]. In Program of the Annual Meeting of the American Society for Microbiology, VCashington, DC, 1986 18. DeLellis RA, Sternberger LA, eiali-Immunoperoxidase techniques in diagnostic pathology: report of a workshop sponsored by the National Cancer Institute. Am J Clin Pathol 71:483, 1979 19. Weiner MH, Talbot GH, et al: Antigen detection in the diagnosis of invasive aspergillosis: utility in controlled blinded trials. Ann Intern Med 99:777, 1983 20. Reyes CV, Rippon Jw: Localized oxalosis associated with simulataneous Aspergillus and Mucor infection in diabetic foot gangrene. HUM PATHOL15:89, 1984
1024
gillus, Pseudallescheria, Fusarium, Trichosporon, dematiacious Hyphomycetes, Candida, and Zygomycetes may be confused, and microbiologic isolation is often delayed. To improve diagnosis, we examined the utility of polyclonal and monoclonal antibodies to identify Aspergillus organisms by peroxidase immunohistochemical techniques. Tissues obtained from 68 infected patients were examined. Fungal antigen was detected in all 26 cases of aspergillosis by the monoclonal immunohistochemical test. Hyphae of AspergiUus organisms exhibited strong intracellular and cell wall staining, and extracellular localization was also observed. In contrast, negative tests were observed in tissues from five cases of infection with Pseudallescheria boydii, three with Trichosporon, three with Zygomycetes, six with Candida species, and one with Curvularia, from as well as 20 cases of nonfungal pneumonia. However, cross-reactivity to Coccidioides immitis spherules was noted in three cases, Staining of Candida was observed with the polyclonal antibody but not the monoclonal reagent. This is the first immunohistochemical demonstration of aspergillus antigen with a monoclonal reagent. The study indicates that the monoclonal immunohistochemical technique can distinguish Aspergillus species from other filamentous fungi and may facilitate the clinical diagnosis of invasive aspergillosis. HUM PATHOL 18:1015--1024, 1987.
T h e diagnosis o f invasive fungal disease frequently relies on histologic examination and culture of a tissue biopsy specimen. Because microbiologic isolation and identification may take several days or weeks, a presumptive etiologic diagnosis o f mycotic infection depends on the morphologic appearance of the organism in tissue. 1 T h e histologic appearance of some fungi (e.g., spherules of Coccidioides immitis) is so characteristic as to be diagnostic. However, numerous filamentous fungi cannot be differentiated reliably with any of the commonly used fungal stains.
From the Division of Infectious Diseases, Department of Medicine, Audie L. Murphy Memorial Veterans Administration Hospital and The University of Texas Health Science Center, San Antonio, Texas. Accepted for publication 21 November 1986. A preliminary report of this work was presented at the 25th Annual Meeting of the Interscience Conference on Antimicrobial Agents and Chemotherapy. Grant support: from the National Institutes of Health (ROI AI13770-09) and the Research Service of the Veterans Administration. Dr. Phillips is supported by a British Columbia Health Care Research Foundation Postdoctoral Fellowship. His current address is the Division of Infectious Diseases, Vancouver General Hospital, 910 West 10th Avenue, Vancouver, British Columbia
VSZ IM9, Canada. Address correspondenceand reprint requests to Dr. VCeiner: Department of Medicine,The Universityof Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio,TX 78284-7881. 0046-8711187 $0.00 + .25
C o n s e q u e n t l y , i n a p p r o p r i a t e empiric antifungal therapy may be initiated on the basis of a mistaken histologic diagnosis. For example, amphotericin B remains the drug of choice for invasive aspergillosis, but infections caused by Pseudallescheria boydii or Tr/chosporon species may respond better to an imidazole derivative. 2,3 Kaplan et al.,4 Jackson et al., 5 and Hotchim et al. 6 have used polyclonal antibodies for direct immunofluorescence of tissue sections in the diagnosis of various systemic mycotic infections. To improve specificity of staining, the authors preadsorbed the polyclonal reagents with other cross-reacting organisms. Fungi that have been successfully identified with this technique include C. immitis, Histoplasma capsulatum, Blastomyces dermatitidis, Candida species, Aspergillus species, Zygomycetes, P. boydii, and Cryptococcus neofonnans. However, antibodies against fungi have not been widely available for clinical diagnostic purposes. As a result, fungal immunohistochemical testing has remained a service provided by few reference laboratories. In this study, we examine the diagnostic utility of polyclonal and monoclonal antibodies to identify Aspergillus species in human tissues using peroxidase immunohistochemical techniques.
METHODS Classification of Cases
Pathology records from three hospitals in San Antonio (Medical Center Hospital, Audie L. Murphy Veterans Administration Hospital, and the Wilford Hall USAF Medical Center) were reviewed to identify cases of invasive infections due to filamentous fungi and to identify control cases o f nonfungal pneumonia. Ten cases of fungal infection were provided by six medical centers outside Texas. Cases o f invasive aspergillosis were initially identified histologically and then subclassified by microbiologic criteria. If aspergilli were isolated from a single tissue that was also positive on histologic examination, then the case was classified as culture positive. I f aspergilli were isolated from a different site, then the case was classified as culture supported. If cultures did not yield a fungus or if no cultures were obtained, then the case was designated culture negative. Cases of nonfungaI pneumonia were defined as demonstrating an inflammatory pulmonary infiltrate but having negative results on methenamine silver stains.
1015
HUMAN PATHOLOGY
Volume 18, No. 10 [October 1987]
in 1 per cent swine serum in Tris saline. This was followed by incubation with the horseradish peroxidase/rabbit antiperoxidase immune complex (Dako) for 30 minutes. Tissue sections were washed with 1 per cent swine serum in Tris saline following the application o f each o f the immunoglobulin reagents. Final washings were performed with Tris saline and then Tris buffer prior to the application of 3,3'-diaminobenzidine and h y d r o g e n p e r o x i d e in Tris b u f f e r (10 minutes). This solution resulted in a brown color change at the sites where horseradish peroxidase had localized in the tissue sections. Perm a n e n t m o u n t s were made after cotmterstaining with hematoxylin for 1 minute.
Polyclonal Immunohistochemistry
Tissues (in the San Antonio hospitals) were placed in a 10 per cent buffered formalin for fixation. Paraffin-embedded tissue specimens from 11 cases were stained with a three-layer horseradish p e r o x i d a s e - a n t i p e r o x i d a s e (PAP) m e t h o d , according to the method of Sternberger. 7 The procedure for the preparation of the primary antibody, polyclonal anti-aspergillus rabbit serum, has been described previously, s Tissue sections 3 rtm thick were mounted on glass slides with 1 per cent aqueous polylysine hydrobromide (Polysciences, Inc., Warrington, Pennsylvania). Preliminary incubations immediately following deparaffinization included the application of a m e t h a n o l - 3 per cent hydrogen peroxide solution (5:1 by volume) for 30 minutes and then a methanol-hydrochloric acid solution (500:1 by volume) for 30 minutes. Tissues were then incubated with 3 per cent normal swine serum in Tris saline for 10 minutes, followed by the primary anti-aspergillus rabbit serum for 2 hours at room temperature. T h e optimal dilution of the primary antibody was determined to be 1:500 in a solution of 3 per cent normal swine serum in Tris saline. The tissues were overlaid with a commercial swine anti-rabbit immunoglobtdin (Dako Corp, Santa Barbara, California) diluted l:100
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Specimens from 68 cases were stained with a three-layer avidin-biotin-peroxidase complex (ABC) technique, 9 employing a murine monoclonal, antiaspergillus IgM antibody (M. Weiner, unpublished data). Both the mounting of tissue sections on glass slides with polylysine and the initial methanol-hydrogen peroxide incubation were carried out as for the PAP method. Then, tissues were washed in phosphate-buffered saline for 20 minutes. An incubation with 2 per cent normal goat serum in phosphate-
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FIGURE 2 (top). Invasive pulmonary aspergillosis stained with normal rabbit serum by'the peroxidase/antiperoxidase [PAP) method [panel /9); polyclonal anti-aspergllus rabbit serum by the PAP method [panel a); and monoclonal anti-aspergiIlus antibody by the avidin-biotin method [panels c and d]. (Original magnification: a and b, x 2; c, x 60; d, x 240.) FIGURE 3 [bottom). Invasive pulmonary aspergllosis, stained with monoclonal anti-aspergilius antibody by the avidin-biotin method. The left panel shows ceil wall and intracelular antigen localization, and the right panel shows hypha[ and extracelular staining. (Original magnification: left,, x 640; right, x 240.}
1016
INVASIVEASPERGILLOSIS[Phillips & Weiner]
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1017
HUMANPATHOLOGY
Volume 18, No. 10 (October 1987)
Interpretation of Peroxidase Immunohistochemlstry
TABLEt,
Cases and Controls Studied by Immunohlstochemlstry
Organism
Cases
Aspergillus spp.
25
Pseudallescheria boydii Trichosporon beigelii Zygomycetes Candida spp.
Curvularia lunata Coccidioides immitis Total mycotic infections Nonfungal pneumonias
5 3 3 6 1 3 46 20
Sites o f Infection Lung, 22; brain, 1; sinus, 1; subcutafieous tissue, 1 Brain, I; dura, 1; lung, 1; paranasal sinus, 1; kidney, 1 Kidney, 2; lung, 1 Sinus, 2; lung, 1 Lung, 6 Paranasal sinus, 1 Lung, 2; cervical node, 1
buffered saline for 20 minutes was followed by the murine monoclonal anti-aspergillus IgM antibody for 2 hours at 4~ T h e optimal concentration of the monoclonal antibody was found to be 1:1,000. Then, biotinylated goat anti-mouse IgM (Vector Laboratories, Burlingame, California) was applied for 30 minutes. Tissues were washed in phosphate-buffered saline after each step. Finally, the ABC complex (Vector Laboratories) was applied for 1 hour, after which the specimens were treated with 3,3'-diaminob e n z i d i n e - h y d r o g e n peroxide in Tris buffer, as in the PAP technique. Counter-staining was performed with hematoxylin, and permanent mounts were prepared.
Tissue sections were examined for the presence o f the characteristic brown staining. T h e intensity and distribution of staining for the positive cases were assessed as follows. Intensity was graded on a scale o f 0 to 3, where 0 indicated absence o f staining; 1, minimal staining only appreciated with a magnification of greater than 100x; 2, moderate staining detectable with the low-power objective (100 x); and 3, intense staining identified with a magnification of 40 • or less. T h e location of staining was indicated as hyphal, if fun.gal organisms were Stained. If staining was present m areas either adjacent to or remote from fungal organisms, then it was designated as extracellular. S p e c i m e n s that were positive by the ABC m e t h o d were f u r t h e r evaluated by repeating the technique with a control murine monoclonal IgM antibody. T h e control reagent was directed against the bacterium Gardnerella vaginalis. Two cases o f nonfungal pneumonia demonstrated nonspecific staining with control murine antibody and were not included in the subsequent analysis. RESULTS
Immunohistochemistry with the Polyclonal Reagent T h e polyclonal anti-aspergillus antibody was used in the PAP method in 11 cases: five o f aspergilTABLE 2. Clinical and Immunohtstochemtcal
Case
Age/Sex (yr)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
78/F 57/F 44/F 57/M 70/M 60/M 65IF 65IF 38IF 251M 65tM 45IF 26/M 59/M 40/M 231M 53/M 72/F 31/F 731M 69/M 65/M 52/F 3 IIF 57/F
Underlying disease
Broadspectrum Antibiotics
Imnmnosuppressive Therapy
Neutropenia*
Bronchitis Hemolysis Leukemia (ANLL) Asthma Hodgkin's Dysmyelopoiesis COPD COPD Hepatitis Leukemia (ANLL) Cirrhosis Leukemia (ANLL) Aplastic anemia Lung carcinoma Systemic lupus Leukemia (ALL) Cirrhosis Leukemia (ANLL) Renal transplant Nephritis Tongue carcinoma Leukemia (ANLL) Lymphoma Leukemia (ANLL) Leukemia (ANLL)
+ + + NA + + + + + + + + + + + + + + NA + NA + + + +
+ + + + + + + + + + + + + + + + + + + + + + +
+ + + ,+ + + + + + + NA + + +
ABBREVIATIONS" ANLL, acute nonlymphocytic leukemia; COPD, chronic obstructive pulmonary disease; ALL, acute lymphocytic leukemia; NA, not available. * Neutropenia refers to a leukocyte count
1018
INVASIVEASPERGILLOSIS[Phillips& Weiner] losis, two o f candidiasis, o n e o f coccidioidomycosis, a n d t h r e e o f n o n f l m g a l p n e u m o n i a . Aspergilhts antigen was detected, as indicated by a m o d e r a t e to intense staining o f f u n g a l h y p h a e , in all five s p e c i m e n s of invasive aspergillosis. However, cross-reactive s t a i n i n g was n o t e d f o r all cases o f candidiasis a n d coccidioidomycosis. F i g u r e s i a n d 2 show, at low magnification, c o m p a r a b l e tissue sections f r o m the s a m e s p e c i m e n o f invasive aspergillosis stained with m e t h e n a m i n e silver (fig. 1), n o r m a l rabbit s e r u m in the PAP m e t h o d (fig. 2b), a n d polyclonal rabbit antiaspergillus a n t i b o d y in the PAP m e t h o d (fig. 2a).
Immunohistochemistry with the M o n o c l o n a l Antibody Sixty-six cases w e r e e v a l u a t e d with the a n t i a s p e r g i l l u s m o n o c l o n a l a n t i b o d y u s i n g the avidinbiotin system. Forty-six o f these were invasive fungal infections; the o t h e r 20 cases were n o n f u n g a l p n e u m o n i a . T w e n t y - f i v e o f the fungal infections were d u e to Aspergillus species. T h e o t h e r 21 cases w e r e d u e to P. boydii (five cases), Trichosporon species (three cases), Z y g o m y c e t e s (three cases), Candida species (six cases), Curvularia lunata (one case), a n d C. immitis (three cases). T h e a n a t o m i c sites o f fungal i n v o l v e m e n t f o r the 46 cases a r e listed in table 1.
Aspergillus Infections T h e clinical a n d i m m u n o h i s t o c h e m i c a l data o f the 25 cases o f aspergillus infection are s u m m a r i z e d
TABLE 3.
Monoclonal lmmunohistochemical Findings in Aspergillosis
Microbiologic Status
Cases
Hyphal Staining
Extracellular Staining
Culture positive Culture supported Culture negative TOTALS
9 3 13 25
9 3 13 25
3 2 4 9
in table 2. T h e culture-positive cases included f o u r with A. fumigatus, f o u r with A. flavus, a n d o n e with A. terreus. T w e n t y patients (80 p e r cent) h a d b e e n receiving b o t h i m m u n o s u p p r e s s i v e a n d b r o a d - s p e c t r u m antibiotic t h e r a p y . Tissues studied by i m m u n o histochemical techniques included lung, brain, p a r a nasal sinus, a n d soft tissue. Aspergillus antigen was d e t e c t e d in all 25 cases o f invasive aspergillosis by the m o n o c l o n a l i m m u n o h i s t o c h e m i c a l technique (Fig. 2c a n d d), in b o t h fungal cell wall a n d intracellular locations (fig. 3 left). I n n i n e cases a n t i g e n was detected in e x t r a c e l l u l a r areas, usually adjacent to aspergillus h y p h a e (fig. 3 right). T h e s e results are s u m m a r i z e d in table 3. S p e c i m e n s f r o m five cases (cases 5, 11, 14, 20, a n d 21) w e r e e v a l u a t e d with b o t h t h e p o l y c l o n a l (PAP) a n d m o n o c l o n a l (ABC) techniques. Aspergillus a n t i g e n was readily detectable with either m e t h o d , b u t s t a i n i n g was s o m e w h a t less i n t e n s e with the m o n o c l o n a l r e a g e n t in cases 14 a n d 21. All 20 cases
Data for Cases of Invasive Aspergillosis I mmunohistochemistryw Tissue
Outcome
Fungal Infectiont
Cuhure~
Studied
PAb
MAb
Dead Dead Alive Alive Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Alive Dead Dead Dead
Dissem asp Dissem asp Inv asp sinusitis Soft tissue inv asp Inv pulm asp Inv pulm asp Inv pulm asp Inv puhn asp Inv pulm asp Inv pulm asp Dissem asp Dissem asp Dissem asp Inv pulm asp Dissem asp Inv pulm asp Inv pulm asp Inv pulm asp Dissem asp Inv pulm asp Inv pulm asp Inv pulm asp Inv'pulm asp Inv pulm asp Inv pulm asp
A. flavus (P) A. fumigatus (P) A. flavus (P) A. fumigatus (P) A. terreus (P) A.flavus (P) A. fumigatus (P) A. fumigatus (P) A. flavus (P) A.flavus (S-sputum) Aspergillus sp. (S-sputum) A. flavus (S-vitreous) Aspergillus sp. (S-skin biopsy) Aspergillus sp. (S-sputum) N N N N N N N N N N N
Lung Lung Sinus Neck mass Lung Lung Lung Lung Lung Lung Lung Brain Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung
----3 -----3 , --3 -----3 2 -----
3 3 3 3 3 3 2 3 3 3 3 3 3 2 3 3 3 3 3 3 1 3 3 3 3
Cultures were graded as positive (P), supportive (S), or negative (N). See Methods for details. wImmunohistochemical tests were done on the indicated tissue with either polyclonal (PAb) or monoclonal (MAb) antibodies. Staining was graded as 0 (none) to 3 (intense). See Methods for details. Tile dashes under PAb indicate not studied. 1019
HUMAN PATHOLOGY
Volume 18, No. 10 (October 1987] TABLE 4.
Case
Age/Sex (yr)
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
19/F 62/F 24lF 43/M NA/F 15/M 23/M 62/M 63/M 86/M NA/NA 78/F 0.6/F 55/F 32/F 17/M 65/M 23/M 25/M 21/M 57/M
Clinical and ImmunohistOchemical
Underlying Disease
Broadspectrum Antibiotics
Immunosuppressive T h e r a p y
Neutropenla*
Leukemia (ALL) Diabetes None Leukemia (ANLL) Seizures Leukemia (ALL) Aplastic anemia Glomerulonephritis Leukemia (ANLL) Diabetes NA COPD Prematurity Lymphoma Leukemia (ANLL) Leukemia (ANLL) Leukemia (ANLL) Allergic rhinitis None Renal transplant Hodgkin's
+ + NA + + + + NA NA + + + + + NA +
+ + NA + + + + NA + + + + + +
+ + NA + + + NA + + + + + +
ABBREVIATIONS: ANLL, acute nonlymphocytic leukemia; COPD, chronic obstructive p u l m o n a r y disease; ALL, acute lymphocytic leukemia; NA, not available. * N e u t r o p e n i a refers to a leukocyte count < l , 0 0 0 / m m s. t Dissem P. boydii, disseminated Pseudallescheria boydii; dissem coccid, disseminated coccidioidomycosis.
of aspergillosis that were tested with only the antiaspergillus monoclonal antibody showed intense (grade 3) hyphal staining.
Nonaspergillus Mycoses and Nonfungol Pneumonlas The clinical and immunohistochemical data for the cases of nonaspergillus mycoses and nonfungal pneumonias are summarized in tables 4 and 5. None of 20 control specimens of nonfungal pneumonia demonstrated aspergillus antigen (fig. 4). Cross-reactive staining was absent in the candidiasis cases, although this result was noted with the polyclonal reagent. All three cases of coccidioidomycosis exhibited cross-reactive staining with the monoclonal antibody. Aspergillus antigen could not be identified in 17 cases of fungal infection caused by filamentous organisms other than Aspergillus species. One additional case (case 33) was not easily classified. Two, morphologically distinct, filamentous fungi were distinguished on Gomori's methenamine silver stain of pulmonary tissue and suggested to the attending.pathologist the presence of a mixed fungal pneumonia. However, only Trichosporon beigelii was isolated from postmortem lung cultures. With the monoclonal immunohistochemistry, only fungal abscesses of one of the two morphotypes were stained with the ABG method (figs. 5-1A, 5-1B, and 6). To characterize this case better and to determine whether this represented a coinfection or cross-reactive staining with a shared antigen of T. beigelii, we did further immunohistochemical stains of this specimen. Rabbit polyclonal antibody against Cryptococcus
neoformans had been noted to cross-react with antigens of T. beigelii, lo We tested this case with a rabbit anti-cryptococcal reagent (kindly provided by Dr. Leo Kaufman, Centers for Disease Control) as the primary antibody in the PAP method. All areas (both fungal morphotypes) of fungal involvement distinguished by Gomori's methenamine silver method were stained with the unabsorbed cryptococcal antibody (figs. 5-2A and 5-2B). However, when the test was repeated using the cryptococcal reagent preadsorbed with a polyvalent aspergillus antigen mixture (figs. 5-3A and 5-3B), the only areas that stained positively were of the second morphotype which had not stained positively with the aspergillus monoclonal antibody. These immunochemical studies indicated a coinfection with two filamentous fungi rather than cross-reactivity of the monoclonal antibody to a T. beigelii epitope. DISCUSSION
Diagnostic problems regarding invasive fungal infections are common in immunocompromised patients. Culture results are often delayed, and the first opportunity to make a specific etiologic diagnosis may be provided by the histopathologic examination of a biopsy specimen. The combination of a positive mucicarmine stain and the detection of organisms morphologically consistent with Cryptococcus neoformans in tissue Is diagnostic for cryptococcosis, even though the stain is not absolutely specific for that organism. However, with
t020
INVASIVEASPERGILLOSIS[Phillips& Weiner] Data for 21 Cases of Nonasperglllus Mycoses Immunohistochemistryw Tissue
Outcome Dead Alive Alive Dead Dead Dead Dead Dead Dead Dead NA Dead Dead Dead Dead Dead Dead Alive Alive Alive Dead
Fungal Infectiont
Culture*
Studied
PAb
MAb
Dissem P. boydii P. bto'dii dural abscess P. bo)'dii sinusitis Dissem P. boydii Dissem P. boydii Dissem Trichosporon Dissem Trichosporon Dissem Trichosporon Zygomycosis Zygomycosis Zygomycosis Dissem candidiasis Dissem candidiasis Dissem candidiasis Dissem candidiasis Candida pneumonia Dissem candidiasis Curvularia sinusitis Dissem coccid Dissem coccid Dissem coccid
Pseudallescheria boydii (P) P. boydii (P) P. boydii (P) P. boydii (P) P. boydii (S) T. beigelii (P) 7". beigelii (P) T. beigelii (P) RUmpus sp. (P) N N Candida tropicalis (P) C. tropicalis (S-liver) C. albicans (S-blood) N N N Curvularia lunata (P) Coccidioides immitis (P) C. immitis (P) C. immitis (P)
Lung Dura Sinus Kidney Brain Kidney Kidney Lung Sinus Sinus Lung Lung Lung Lung Lung Lung Lung Sinus Neck node Lung Lung
m -m --m -~ m ---~ -3 -3 ~ -~ 3
0 0 0 0 0 0 0 0/382 0 0 0 0 0 0 0 0 0 0 2 3 3
* Cultures were graded as positive (P), supportive (S), or negative (N). See Methods for details. wImmunohistochemical tests were done on the indicated tissue with either polyclonal (PAb) or monoclonal (MAb) antibodies. Staining was graded as 0 (none) to 3 (intense). See Methods for details. 82See Results for discussion of this case. the widely used fungal stains, such as G o m o r i ' s m e t h e n a m i n e silver, periodic a c i d - S c h i f f , a n d Gridley fungus, it is the m o r p h o l o g i c a p p e a r a n c e o f the org a n i s m in tissue, r a t h e r t h a n its staining p r o p e r t i e s , that enables o n e to m a k e a p r e s u m p t i v e identification. U n f o r t u n a t e l y , m a n y o f the f i l a m e n t o u s fungi c a n n o t be reliably d i f f e r e n t i a t e d f r o m o n e a n o t h e r with a n y o f the a b o v e - m e n t i o n e d stains. Several techniques h a v e b e e n d e m o n s t r a t e d to aid in the h i s t o p a t h o l o g i c identification o f fungi. T h e s e include f u n g a l a u t o f l u o r e s c e n c e Ira2 a n d f l u o rescent stains such as acridine o r a n g e , t3 c a l c o f l u o r white, 14 2 - h y d r o x y s t i l b a m i d i n e isethionate, t5 Blankop h o r BA 267 p e r cent (Bayer, L e v e r k u s e n , West G e r many), 1~ a n d d i a e t h a n o l . 1 7 I n general, these m e t h o d s are m o r e r a p i d b u t s o m e w h a t less sensitive o r specific t h a n c o n v e n t i o n a l f u n g a t stains. E x c e p t f o r the Zygo-
TABLE 5.
m y c e t e s , w h i c h d o n o t a u t o f l u o r e s c e , t h e s e techn i q u e s d o n o t p r o v i d e d i f f e r e n t i a l staining o f the various f u n g a l genera. T h e f l u o r e s c e n t m e t h o d s m a y c o m p l e m e n t r a t h e r t h a n replace the c o m m o n l y used fungal stains. F u n g a l i m m u n o h i s t o c h e m i s t r y has facilitated the early diagnosis o f systemic mycoses in which the hist o p a t h o l o g i c findings are not p a t h o g n o m o n i c . 1 T o d a t e , p o l y c l o n a l a n t i b o d i e s , u s u a l l y p r e p a r e d in rabbits, h a v e b e e n used f o r such studies. U n f o r t u nately, p r o b l e m s relating to r e a g e n t availability a n d the necessity f o r p r e a d s o r p t i o n s with various crossr e a c t i n g f u n g i have limited the application o f these techniques. Monoclonal antibodies offer potential advantages over polyclonal reagents, including g r e a t e r specificity a n d u n i f o r m activity f r o m lot to lot. Utilization o f m o n o c l o n a l r e a g e n t s in fungal ira-
Monoclonal Immunohistochemlcal Findings In Control Cases of Filamentous Fungal Infection and Nonfungal Pneumonla Organism
PseudaUescheria boydii Trichosporon sp. Zygomycetes Candida sp. Curvularia sp. Total filamentous mycoses Nonfungal pneumonia
Microbiologic Status Culture pos. Culture pos. Culture pos. Culture neg. Culture pos. Culture neg. Culture pos.
Cases
Hyphal Staining
Extracellular Staining
5 3* 1 2 3 3 1 18 20
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
* One case with probable coinfection by Trichosporon and AspergiUus spp. identified by morphologic and microbiologic criteria. 1021
HUMAN PATHOLOGY
Volume 18, No. 10 (October 1987)
FIGURE 4 [top). Negative stainingof nonfungal pneumonia with monoclonal anti-aspergiilusantibody by the avidin-biotin method. [Original magnification: left;, x240; right, x4.]
v
1022
FIGURE5 (bottom). Magnification of boxed areas A and B shown in figure 6. From contiguous sections of pulmonary tissue from case 33 stained with anti-aspergillus monoclonal antibody [panels 5-1A and 5-1B, x800], unadsorbed polyclonal anti-cryptococcal antibody by peroxidase-antlperoxidase method [panels 5-2A and 5-2B, x 800], and polyclonal anticryptococcal reagent preadsorbed with a polyvalent aspergiilus antigen mixture [panels 5-3A and 5-3B, x 320).
INVASIVEASPERGILLOStS[Phillips & Weiner]
r
/,.
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FIGURE 6. Low magnification of pulmonary tissue in case 33, stained with anti-aspergillus monoclonal antibody. The boxed areas A and B represent focal areas of infection by two different fungal morphotypes. [x 2.}
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munohistochemical studies should obviate the preadsorption steps required to eliminate the cross-reactive staining seen with polyclonal antibodies. Our study o f fungal immunohistochemistry indicates that aspergillus antigen is readily detected in formalin-fixed tissues by a monoclonal antibody; cross-reactive staining was not observed for nonaspergillus filamentous fungi, with the possible exception o f one case o f Trichosporon beigelii pneumonia. This specificity allowed us to differentiate histologically Aspergillus species from other morphologically similar fungi. The intensity of staining of aspergillus antigen was similar with both polyclonal and monoclonal antibodies. Staining with the monoclonal technique was somewhat less intense in two of the five specimens in which both methods were employed (table 2). Nineteen of the 20 remaining aspergillosis cases all demonstrated intense (grade 3) staining of fungal hyphae with the monoclonal immunohistochemical technique. This result suggests that the monoclonal reagent is directed to one of the predominant epitopes expressed in vivo. E n h a n c e d recognition of one abundant epitope by a monoclonal antibody may compensate for its high specificity, i.e., its failure to recognize other antigenic sites. Aspergillus fumagatus, A. flav us, and A. terreus were isolated from culturepositive cases in this series. Staining of equal intensity was noted for all three species using the monoclonal reagent, indicating that the epitope identified is shared by at least those three members of the genus. The monoclonal antibody was prepared by immunizing Balb/c mice with a cell wall extract of A. fumigatus. However, abundant antigen was detected in both the hyphal cell wall and intracellular locations. This may be explained by the intracellular disposi-
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tion o f a cell wall component during synthesis or degradation. In nine of 25 cases of invasive aspergillosis, antigen was also detected in areas adjacent to, but clearly removed from, identifiable fungal hyphae. We have referred to this finding as "extracellular" fungal antigen. It was not found with control stains employing a monoclonal antibody to the bacterium Gardnerella vaginalis. Extracellular staining may represent the in vivo dissemination o f aspergillus antigen in tissue. However, the possibility that this represents antigen diffusion postmortem due to delayed or poor fixation technique cannot be excluded. 18 The relationship between aspergillus antigen disseminated in tissue and tlmt which has been detected in human serum by radioimmunoassay 1~ has not been examined. Cross-reactivity was observed in all three cases of coccidioidomycosis, with both the polyclonal and monoclonal antibodies. Tile dis'tinctive morphologic a p p e a r a n c e o f coccidioides spherules, however, readily ,distinguishes these spherules from the filamentous fungi and should not represent an immunodiagnostic problem. Cross-reactive staining also occurred with the polyclonal anti-aspergillus antibody in cases of invasive candidiasis. However, the monoclonal reagent was specific and did not produce cross-reactivity.' 9 'Negative, specific resuks were obtained with the monoclona! reagent in 17 control cases infected with filamentous organisms other than Aspergillus species. In one additional pulmonary case, histochemical differences suggested a coinfection with two filamentous fungi, although only T. beigelii was isolated from a postmortem culture. The monoclonal immunohistochemical technique also suggested coinfection. Fur-
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HUMAN PATHOLOGY
Volume 18, No. 10 (October 1987)
t h e r i m m u n o c h e m i c a l studies were d o n e to characterize the case better. W h e n an anti-cryptococcal rea g e n t cross-reactive to T. beigelii was p r e a d s o r b e d with aspergillus antigen, staining was only observed in the fungal m o r p h o t y p e that had not b e e n stained with the anti-aspergillus m o n o c l o n a l r e a g e n t (figs. 5-3A and 5-3B). This finding supports the explanation t h a t a positive r e s u l t in this case was d u e to staining o f coexistent Aspergillus species which h a d n o t been isolated by p o s t m o r t e m culture. Mixed invasive fungal infections are well d o c u m e n t e d in imm u n o c o m p r o m i s e d p a t i e n t s , 2~ a n d f a l s e - n e g a t i v e p o s t m o r t e m fungal cultures in histologically p r o v e n cases are not u n c o m m o n . An alternative premise that the anti-aspergillus m o n o c l o n a l antibody is directed against an epitope s h a r e d by both organisms appears unlikely in light o f the above studies. An increasing n u m b e r o f filamentous fungi are being recognized as opportunistic pathogens in the i m m u n o c o m p r o m i s e d patient, T h e s e organisms include Pseudallescheria boydii, Fusarium species, Trichosporon species, a n d several o f the dematiacious Hyp h o m y c e t e s such as Bipolaris species. T h e antifungal susceptibilities o f t h e s e o r g a n i s m s a n d Aspergillus species differ. F o r example, T. beigelii appears to be most susceptible to the imidazole derivatives miconazole and ketoconazole b u t relatively resistant to amphotericin and 5-flucytosine. a T h e d r u g o f choice f o r invasive P. boydii infection is miconazole. H o w e v e r , the c u r r e n t l y available imidazole d r u g s have b e e n disappointing in aspergillosis, for which a m p h o t e r icin B remains the d r u g o f choice. As the n u m b e r o f effective a n t i f u n g a l agents continues to increase, it will b e c o m e even m o r e imp o r t a n t to d e t e r m i n e the specific etiologic diagnosis o f invasive mycotic infection so that p r o m p t and app r o p r i a t e t h e r a p y can be initiated. T h e d e v e l o p m e n t o f a panel o f m o n o c l o n a l antibodies directed against the most c o m m o n l y e n c o u n t e r e d filamentous fungi m a y p r o v e useful in this respect, particularly f o r those institutions caring for significant n u m b e r s o f i m m u n o c o m p r o m i s e d patients.
Acknowledgments. T h e a u t h o r s t h a n k Drs. L. Kaufman, L. Ajello, and W. Chandler, Centers for Disease Control, Atlanta, Georgia, for providing the polyclonal anti-cryptococcal antibody; and the following persons for providing clinical case information and tissue specimens: Dr. James Orr and Dr. J. W. Rippon, University of Chicago, Chicago, Illinois; Dr. David A. Schwartz, Hahnemann Medical College, Philadelphia, Pennsylvania; Dr. J. Bagnail, Wilford Hall USAF Medical Center, San Antonio, Texas; Dr. Y. Tsukada and Dr. EI-Ani, Roswell Park Memorial Institute, Buffalo, New York; Dr. H. M. Haupt, Johns Hopkins Medical Institutions, Baltimore, Maryland; Dr. Bustamante, Baptist Memorial Hospital, San Antonio, Texas; Dr. C. Green, Michael Reese Hospital, Chicago, Illinois; Col. D.J. Wear, MC, Armed Forces Institute of Pathology, Washington, DC; and Dr. Stanton Gerson, Case Western Reserve Hospital, Cleveland, Ohio. The authors
also thank Dennis A. Havel for the photography of the immunohistochemistry, and Catherine Gonzalez, Judy Fox, and Sarah Lock-Lim for technical assistance.
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