USE OF PERIODIC ACID-SCHIFF STAIN IN IDENTIFICATION OF PATHOGENIC FUNGI IN TISSUES

Transcription

1 USE OF PERIODIC ACID-SCHIFF STAIN IN IDENTIFICATION OF PATHOGENIC FUNGI IN TISSUES GRIER F. STARR, M.D., CLYDE J. DAWE, M.D., AND LYLE A. WEED, M.D. Departments of Pathology, Surgical Pathology and Bacteriology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota Need still exists for more productive and less erring methods of identification of causative agents in granulomatous inflammations. This fact is illustrated by a study as recently as 1951/ in which it was found possible to identify a causative agent in the tissues in only 21 of 65 cases of pulmonary granuloma using standard bacteriologic and histologic methods. The apparent ineffectiveness of laboratory methods as presently used is particularly regrettable in view of the fact that many of the agents concerned are susceptible to antibiotic or chemotherapeutic attack, the proper treatment varying according to the organism present. The problem has been particularly intensified by advances in thoracic surgery, since pulmonary granulomas now frequently come to the hands of the pathologist as a by-product of surgical explorations for possible pulmonary malignant lesions. The introduction of the periodic acid-schiff (PAS) method of staining by McManus 7 8 and by Hotchkiss 2 led very shortly to the application of this method to the staining of fungi in tissues. It is probably accurate to say that at the present time the PAS procedure has been turned to this purpose by the majority of histologic laboratories. Indeed, the impression is gained from reports during the past few years that the method has revolutionized the approach to the study of mycotic infections. For example, it has been stated: "It also seems that the diagnosis of fungous disease will no longer require specialized knowledge and that this procedure [PAS staining] will become a diagnostic method in the routine laboratory." There can be no doubt among those who have used the PAS method that it is an effective means of tinting those microorganisms, such as some fungi, which possess prominent carbohydrate components. An entirely different question, however, is the extent to which the pathologist, on the basis of the PAS method, may alter his approach to the individual case-at-hand in which a granulomatous or chronic inflammatory lesion is recognized or suspected. Previous reports have demonstrated that histologically indistinguishable lesions may contain any of a number of organisms or chemical agents including Mycobacterium tubercidosis, Brucella, Pasteurella hdarensis, Coccidioides immitis, Blastomyces dermatitidis, silica, beryllium and oils. Only the fungous agents of this group can be demonstrated by the PAS method, a fact which would automatically categorize the method as one with more or less reliable exclusion-potentials. Granting this to be the case, the PAS method would still be of considerable value should it be proved (1) that the method is capable of demonstrating or- Received for publication September 13, 1954; accepted October 7,

2 Jan PAS STAIN FOR FUNGI IN TISSUE 77 ganisms when they would be missed by other means or (2) that failure to demonstrate an organism by this means is equivalent to excluding mycotic infection. It is evident that evaluation of the effectiveness of the method in the foregoing sense would have to be expressed in relative terms, using as a standard some other method or methods of recognized worth, such as culture on laboratory mediums, animal inoculation, staining by other stains, chemical analysis or combinations of these. Some information relevant to the problem has been reported by Puckett 10 in a study of pulmonary granulomas. Employing the PAS stain, he reported finding Hisloplasma capsalatum in 22 pulmonary lesions from which the same organism could be cultured on blood-heart infusion medium in only 1 instance. In companion sections of the same material stained with hematoxylin and eosin, Puckett was unable to identify organisms "with any degree of certainty." Such findings are of utmost interest, since they would indicate, assuming the cultural methods to have been adequate and the morphologic interpretations correct, that (1) the PAS staining method is more sensitive than cultural methods, or (2) that the organisms in Puckett's cases were dead or otherwise incapable of growth on the laboratory mediums used. It lias been the experience in our laboratories that cultural methods have been relatively more successful in the demonstration of mycotic agents than morphologic methods, using hematoxylin and eosin, Brown-Gram, acid-fast, Stpughton, and Best-Bodian staining methods, the last 4 stains being used somewhat sporadically at the histologic examiner's discretion. Experience with the PAS method over the past few years showed considerable promise, so that the present study was undertaken in order to assess its value. MATERIALS AND METHODS This study was designed primarily to show what difference in results might have been obtained in the histologic examination of tissues for fungi, had the simple addition of the PAS method been applied. It was not designed for the purpose of determining whether or not cultural methods of identification of pathogenic fungi could be discarded in favor of using staining methods alone. Comparison of results using cultural methods as against staining methods has been made because a standard is necessary in any evaluation of method. The investigation was carried out in such a way as to minimize the advantages that would accrue to the PAS or any other method merely as a result of more extensive sampling of the tissue. It is recognized that in the problem of identifying microorganisms in tissues, the fruits of the search bear a direct relationship to the time expended. The present studies do not, therefore, involve serial block sampling of the tissues, a method that is practicable in relatively few laboratories, but only the sampling generally thought feasible in routine histopathologic work. The study should not, therefore, be regarded as measuring the value of the PAS method as exhaustively applied, but as practically applied. Neither does the study attempt to prove or disprove the occurrence of dead fungous organisms in granulomatous lesions.

3 78 STARR ET AL. Vol. 25 Two series of studies were carried out. In Series I an attempt was made to find fungi by the PAS method in lesions that had previously.yielded no organisms by culture or animal inoculation or by histologic examination with other stains. Cultural methods have been published in detail elsewhere. 6 Fifty-four pulmonary lesions were examined in Series I, including 2 of the 44 culturally negative cases previously reported by Good and his co-workers. 1 Four of the 54 cases were in the nature of positive controls, in that 2 were examples of culturally and microscopically confirmed pulmonary histoplasmosis. Two others were cases in which cultures had been negative, but organisms thought to be H. capsulatum and C. immitis (Fig. 1), respectively, had been found in the tissues with hematoxylin and eosin stains. The remaining cases were arbitrarily selected to include the 3 main morphologic types of granulomas. Twenty-two of the lesions were circumscribed, solitary, noncavitating, caseous lesions, exemplary of the so-called tuberculoma. In 21 cases the specimens contained multiple foci with caseous and noncaseous, noncavitating granulomatous reaction. The remaining 7 specimens contained cavitating caseous granulomas. In all cases in this series the histologic material was limited to sections cut from the original paraffin-embedded blocks taken at the initial pathologic examination of the specimen. One to 3 blocks were available in each case (101 blocks from 54 cases). Sections were stained by the PAS method, using Lillie's modification in preparation of the Schiff reagent. 4 The total area of each section was examined under the oil-immersion lens (magnification 970), an average time of 45 minutes being spent on each case. Sections were numerically coded to avoid recognition of the known positive cases. Series II was composed of 31 cases of granuloma culturally positive for pathogenic fungi, but in which no fungi had previously been found by examination of tissue sections stained with hematoxylin and eosin, occasionally, by Brown- Gram, Stoughton, or Best-Bodian methods. These cases actually represented all of those available over the past 7 years in which a fungous agent had been cultured but in which no organism had been demonstrated morphologically by staining methods other than the PAS method. Since fungi had been demonstrated in 61 cases during this period, these 31 cases give an indication of the relative defectiveness of histologic methods without the benefit of PAS staining. In Series II sections were cut only from the original paraffin-embedded blocks, with the exception of 2 surgical cases and 2 necropsy cases in which the original blocks had been lost or completely used in previous sectioning, or in which the blocks available were obviously not representative. In these 4 cases new blocks of tissues were cut from formalin-preserved specimens. In no case was serial block sectioning employed. A total of 128 blocks was taken from the 31 cases. The lesions were not all pulmonary and 2 cases involved necropsy material, 9 blocks being taken from several organs (spleen, lungs, liver, adrenal glands and lymph nodes) in each of these. Organisms previously cultured from these 31 cases were as follows: //. capsulatum in 7; B. dermatilidis in 6; Sporotrichum schenckii in 3; C. immitis in 14, and Cryptococcus neoformans in 1.

4 Jan PAS STAIN FOR FUNGI IN TISSUE 79 r ig. 1 (upper left). Organisms believed to be C. immilis from a culturally negative pulmonary lesion. Endosporulating spherule upper left, developing spore lower right. P A S. X 400. F I G. 2 (upper right). Organisms culturally proved to be H. capsukuum, but originally misinterpreted as leishmaniae. PAS. X F I G. 3 (lower left). Organisms believed to be H. capsidatum histologically demonstrated in a pulmonary lesion t h a t was culturally negative. Histoplasmin skin test positive. PAS- X F I G. 4 (lower right). Organisms culturally proved to be B. brasiliensis. These organisms, demonstrated histologically in a gingival lesion, were presumed to be of the South American type on the basis of the onest of disease while the patient was in Venezuela. PAS. X 1500.

5 80 STARR ET AL. Vol. 25 In the entire first group and in 10 cases of the second group, 3 adjacent sections were cut from each block; 2 were stained by the PAS technic, one being counterstained with hematoxylin and the second with fast green, while the third was stained with hematoxylin and eosin. In the 21 remaining cases of the second group no counterstain was used. OBSERVATIONS Series I. In the 54 cases examined, it was believed that H. capsidatum was present in 4 and C. immitis in 1. Three of the 4 cases showing H. capsulatum proved, on decoding, to be the positive controls, as was the case showing C. immitis (Fig. 1). Thus, in only 1 case (Fig. 3) was an organism (believed to be H. capsulatum) found that had not previously been recognized by routine histologic or bacteriologic methods or both. Series II. Of the 7 cases from which H. capsulatum had been cultured, the organisms were demonstrated in 5 with the PAS stain. Two of these were necropsy cases. In one of the latter, organisms were found only in the lungs, in the other in both the lungs and spleen. Of the 6 cases in which B. dermatitidis had been proved by culture, 5 were shown to contain the organism in the PAS preparations. In the 3 cases yielding S. schenckii by culture, organisms were found in only 1 case utilizing the specially stained sections. In the 14 cases from which C. immitis had been cultured, the organism was identified correctly in the PAS-stained tissues in 9. C. neoformans was recognized morphologically as such in the single case from which it had been cultured. Thus, of the 31 cases in which pathogenic fungi were shown by culture but not hy histologic methods other than the PAS technic, the corresponding organism was demonstrated in 21 with the PAS technic (Table 1). It is of interest that after the fungi stained by the Schiff reagent had been detected, it was possible to find the organisms in the companion slides stained with hematoxylin and TABLE 1 DEMONSTRATION OP FUNGI IN STAINED SECTIONS OF TISSUES AMONG CASES HAVING POSITIVE CULTURES FOR FUNGI* AT THE MAYO CLINIC FROM 1946 THROUGH 1953 Number Positive by Histologic Examination Organisms Total Routine examination without PAS Added bv PAS ' Total Bliistonwcesf H. capsulatum C. immitis C. neoformans S. schenckit Total * The term "fungi" is used applying only to the organisms mentioned, f North and South American types.

6 Jan PAS STAIN FOR FUNGI IN TISSUE SI eosin in all 5 cases of blastomycosis, 6 cases of coccidioidomycosis, 3 of the cases of histoplasmosis and the single case of cryptococcosis by careful re-examination of the corresponding areas in the sections. This fact illustrates the ease with which organisms may be overlooked, even though readily demonstrable, when they are not brought into sharp color contrast with their surroundings. AVith regard to the counterstain used, it was found that no difference was apparent whether fast green or hematoxylin was employed. Actually, sections in which no counterstain was used seemed to be the most satisfactory. COMMENT It seems fair to interpret the findings reported in the preceding section as indicating a definite but limited sphere of usefulness for the PAS stain in the routine histologic examination of granulomatous lesions. One is, in fact, impressed by the finding that the cultural successes exceeded the morphologic ones onl}' 10 times in a total of 61. fungous infections examined by both methods. Indeed, when one takes into consideration the fact that in Series I the PAS staining procedures permitted identification (presumably correct) of organisms in 1 case in which cultures were negative and examination by hematoxylin and eosin staining in 2 additional cases among the controls had been sufficient to identify organisms, advantages do not all appear to be in favor of bacteriologic methods. Three important points must not be lost from view, however. First, the PAS procedure is capable at best of demonstrating only mycotic pathogens. When one is confronted with a granulomatous lesion, the practical problem is not that of demonstrating a fungus or of not demonstrating a fungus, bid of demonstrating one of a wide variety of agents that may stimulate granidomatous reaction, of which the fungous group forms only a small segment. Obviously, large numbers of infections by the tubercle bacillus or Brucella would go unproved if resort to cultural methods were abandoned. Secondly, a pitfall inherent in morphologic methods is the presence of artifacts that may closely simulate certain pathogenic fungi, especially Histoplasma and Sporotrichum. Puckett has described and illustrated these in some detail. Artifacts were a constant problem in the search for organisms in the present study. The number of artifacts one encounters is largely reflective of the lack of specificity of the PAS stain. It should be recognized that the PAS method as used in the search for fungi is a contrast method and not a specific histochemical procedure. We do believe, however, that the presence of completely typical organisms in large numbers does permit a probable diagnosis in certain instances, depending on the organism concerned. Thirdly, advantages inherent to cultural methods cannot be supplanted by morphologic methods. Even when a fungous organism can be unquestionably demonstrated it is often difficult and sometimes impossible to identify the species with certainty. The current debate as to the existence of small forms of Blastomyces 3, 6i 12 and large forms of Histoplasma 11 is illustrative of this fact, and cannot be settled without the accumulation of information on the cultural characteristics of the organisms in question. Indeed, in the face of such problems

7 82 STARR ET AL. Vol. 25 one might question the validity of any purely morphologic identification of Histoplasma or Blastomyces in tissues. This observation takes more meaning when one notes that organisms seen in tissues removed at operation have been misinterpreted on occasion by the surgical pathologist. In the present group of cases, for example, fl. capsidatum was cultured from a tissue said by the surgical pathologist to contain leishmaniae (Fig. 2), C. immitis was cultured from tissue of a patient following a microscopic diagnosis of blastomycosis, and conversely B. dermatitidis was cultured from tissue said to contain Coccidioides. In another instance B. dermatitidis was cultured from tissue histologically examined and diagnosed as containing cryptococci. Finally, one diagnosis was made on the basis of geography rather than morphology in a case of South American blastomycosis, which was ultimately proved on the basis of the cultural characteristics of the organism (Fig. 4). The foregoing points are cited because current literature indicates some tendency toward the view that improved staining methods could replace cultural ones. It is our attitude that the methods are, on the contrary, largely supplementary, and in our laboratories the handling of granulomatous lesions routinely includes, in addition to PAS and acid-fast staining of histologic material, rather extensive bacteriologic investigation. As a minimum, this includes cultures for the commonly encountered pyogens, culture and guinea-pig inoculation for tubercle bacilli, cultures for fungi with the plates incubated at 37 C. and room temperature, and cultures for Brucella. If the history or other laboratory data indicate, special studies may be made for viruses, rickettsiae, spirochetes or Leptospira, Toxoplasma or leishmaniae. SUMMARY Enthusiasm in recent years for the periodic acid-schiff (PAS) staining procedure as a method of identifying fungi in tissues has not been paralleled by critical attempts to evaluate the limitations as well as the potentialities of the method. In the present study an attempt has been made to determine what the PAS method alone, without advantage of increased sampling, can add to the finding and identification of fungi in tissues in routine histopathologic work. In a group of 50 pulmonary granulomas in which no organisms had previously been identifiable either by histologic, cultural, or animal inoculation methods, organisms believed to be Hisloplasnia capsidatum were found in 1 instance, utilizing the same tissue blocks that had originally been cut. Four positive control cases, 3 of //. capsidatum and 1 of Coccidioides immitis, were correctly identified among this group (total of 54 cases). Two of the control cases (C. immitis and 1 of H. capsidatum) were culturally negative, histologic diagnosis having been made during initial routine examination. Pathogenic fungi were identified, using the PAS staining method, in 21 of 31 granulomas in which the organisms had not previously been demonstrated in routine histologic examination, but from which fungi had been isolated in cultures. In 15 of the 21, companion sections stained with hematoxylin and eosin were also shown to contain the organisms, which had been overlooked, pre-

8 Jan PAS STAIN FOR FUNGI IN TISSUE S3 sumably because of poor contrast. In this group, organisms were found with the PAS stain in 5 of 6 cases of blastomycosis, in 5 of 7 cases of histoplasmosis, in 1 of 3 cases of sporotrichosis, in 9 of 14 cases of coccidioidomycosis and in a single case of cryptococcosis. By the application of the PAS method to routine sampling of the tissues (not serial blocking), the discrepancy between positive results in culture and in histologic methods so far as fungi are concerned has thus been reduced to 10 out of a total of Gl cases over 7 years. Organisms have been found histologically in 3 instances where culture and animal inoculation were negative (2 cases apparently of //. capsulatum and 1 of C. immitis). The observations are believed to indicate definite value, with certain limitations, in the routine use of the PAS method in histologic examination of granulomatous lesions. The method must remain supplementary to cultural procedures, since (1) negative results do not exclude a fungous etiology, (2) even when fungi are found, it is not always possible to identify the species correctly by morphologic methods in tissues alone and (3) perhaps even more important is the fact that over-enthusiasm for the PAS method, carried to the extent of neglect of bacteriologic technics, can lead to failure of identification of nonmycotic organisms that are also commonly the cause of granulomatous inflammation. REFERENCES 1. GOOD, C. A., CIJAGETT, 0. T., AND WEED, L. A.: Granuloma of the lung: A problem of differential diagnosis. Tr. Nat. Tu.berc._A., 47: , FIOTCIIKISS, R. D.: A microchemical reaction resulting in the staining of polysaccharide structures in fixed tissue preparations. Arch. Biochem., 16: , 194S. 3. LAYTQN,.). M\, MCKBB, A. P., AND STAMLER, F. W.: Dual infection with Blastomyces dermatitidis and Histoplasma capsulatum: Report of a fatal case in man. Am. J. Clin. Path., 23: , Li LI.I E, R. D.: Simplification of the manufacture of Schiff reagent for use in histochemicnl procedures. Stain Technol., 26: , MANWAKING,,J. H.: Unusual forms of Blastomyces dermatitidis in human tissues. Arch. Path., 48: , MCDONALD, J. R., AND WEED, L. A.: Problems concerned with the histologic diagnosis of tuberculosis of lymph nodes. Am. J. Clin. Path., 21: , MCMANUS, J. F. A.: Histological demonstration of mucin after periodic acid. Nature, 158: 202, S. MCMANUS, J. F. A.: Histological and histochemical uses of periodic acid. Stain Technol., 23: 99-10S, 194S. 9. Pn.i.snunv, D. M., AND KIJGMAN, A. M.: A new histochemical tool for the definitive diagnosis of fungus infections. Tr. New York Acad. Sc, 13: S, PUCKUTT, T. F.: Pulmonary histoplasmosis: A study of twenty-two cases with identification of //. capsulatum in resected lesions. Am. Rev. Tuberc, 67: , SCMWAHZ,./.: Giant forms of Histoplasma capsulatum in tissue explants. Am. J. Clin. Path., 23: S9S-903, TUTTI.E, J. G., LICHTWAKDT, H. 13., AND ALTSIIULBR, C. H.: Systemic North American blastomycosis: Report of a case with small forms of blastomvoetes. Am. J. Clin. Path., 23: S90-897, 1953.