Diagnosis of Experimental Fungal Corneal Ulcers by Fluorescent Antibody Techniques

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Only for #o=0 (meridional line) does the measured distance ds on the Haag-Streit chart correspond to the visual angle dcr. Also, for 0o<3C9O°and for \d — 0o|
NOVEMBER, 1966

ional direction can be used without transformation. For nonmeridional (transversal) measurements, the intervals must be corrected according to the above formula. Further explanations will be given in a subsequent paper.

DIAGNOSIS OF EXPERIMENTAL FUNGAL CORNEAL ULCERS BY FLUORESCENT ANTIBODY TECHNIQUES M E L V I N S. R H E I N S , P H . D . , P A T R I C I A A. P I X L E Y , M . S C , T E D S U I E , P H . D . , AND R I C H A R D H . K E A T E S ,

M.D.

Columbus, Ohio Ocular mycoses have become more preva­ lent with the advent of the widespread use of steroids and antibiotics in the treatment of ocular diseases. Corticosteroids have been shown to potentiate fungal growth in vivo 1 and Ley 2 concluded that antibiotics en­ hanced the virulence of Candida albicans in experimental ocular infections. Although not all workers agree as to the role of anti­ biotics in potentiation of ocular mycoses, 1 evidence is accumulating which indicates this is the case. 3 The ophthalmologists are now more aware of the capacity of fungi to invade various parts of the ocular apparatus and, with more efficient methods of isolation and identification at their disposal, these my­ coses are more often correctly diagnosed. Early identification of the etiologic agents may not be forthcoming due to the slow growth of many fungi in vitro and the difficulty of recognizing fungal structures in stained clinical material. Therefore, it would be advantageous if a rapid method were de­ veloped which would allow detection of fun­ gal agents during the early stages of the ocular lesion. T h e adaptability of a fluorescent antibody technique for the rapid diagnosis of experi­ mental fungal corneal ulcers is the subject of this paper. From the Departments Ophthalmology, The Ohio study was supported by a port grant of the United Service.

of Microbiology and State University. This general research sup­ States Public Health

MATERIALS AND METHODS ORGANISMS

A strain of Candida albicans isolated from a human eye infection seen at T h e Ohio State University Hospital was used. T h e other or­ ganisms (Sporotrichum schenckii, Scopulariopsis brevicaulis, Aspergillus fumigatus, and Allescheria boydii) were obtained from the Ohio State Health Department Labora­ tory. These fungi were maintained on deep Sabouraud-dextrose agar slants, transferred periodically, incubated at room temperature and stored at 4°C. ANIMALS

Female albino rabbits, weighing 3.5-5.0 kg, were employed for immunization, while adult albino rabbits of either sex and weigh­ ing 2.0-3.0 kg were used for the experimen­ tal mycotic ocular infections. VACCINE

PREPARATION

T h e organisms were treated according to the following procedures: Sporotrichum schenckii was grown on moist Francis-cystine agar at 37°C for one week to obtain the yeast-phase of this orga­ nism. T h e growth was harvested in 0.4% formol saline. Allescheria boydii, Aspergillus fumigatus, and Scopulariopsis brevicaulis were cultivat­ ed in Sabouraud-dextrose broth at room temperature for two weeks on a rotary plat­ form shaker. T h e resultant mycelial mass of

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each was separated from the broth by means of nitration through a Buchner funnel, washed three times with sterile physiologic saline and stored in 0.4% formol saline. All four organisms were permitted to re­ main in the formol saline for two weeks at room temperature, after which time sterility tests were conducted. The formolized orga­ nisms were washed free of the formol saline and inoculated onto Sabouraud-dextrose agar, incubated one week at 37°C and then one week at room temperature. If no growth resulted at the end of this period, the stock suspensions were considered to be free of viable organisms. Sporotrichum schenckii was washed three times with sterile saline, the turbidity ad­ justed to a No. 7 nephelometer, and aqueous merthiolate added to a 1:104 concentration. This preparation constituted the final vaccine. The remaining three suspensions of orga­ nisms were washed free of formol saline, according to the same procedure and soni­ cated with a Bronson model SI 10 sonic vi­ brator until the preparations would pass through the lumen of a 25-gauge needle (1.0 hour for Allescheria boydii and Aspergillus fumigatus, 1.5 hours for Scopulariopsis brevicaulis). The system was cooled in an icewater bath. Each of these preparations was standardized to a No. 7 nephelometer and merthiolate added as already described. Candida albicans was cultivated on Sab­ ouraud-dextrose agar for 48 hours at room temperature and harvested into 0.4% for­ mol saline. This preparation was left at room temperature and sampled periodically for viability. When no growth occurred, the cells were washed three times in sterile sa­ line, adjusted to a No. 6 nephelometer and merthiolate added. All vaccines were stored at 4°C. IMMUNIZATION SCHEDULES AND METHODS

All vaccines were injected intravenously into the posterior auricular vein of the des­ ignated rabbits. Prior to immunization, 10 ml of blood were withdrawn from each animal by car­

893

diac puncture. After clotting, the sera were separated and stored in the frozen state until needed as controls for subsequent serologic tests. The vaccine preparations of Aspergillus fumigatus, Scopulariopsis brevicaulis, Al­ lescheria boydii and Sporotrichum schenckii were administered daily for three days dur­ ing each of three weeks in the amounts of 0.5, 0.5, 0.75; 0.75, 1.0, 1.0; 1.5, 1.5 and 2.0 ml. The Candida albicans vaccine was admin­ istered on alternate days in a series of seven 1-ml injections. One week after the final injection 20-35 ml of blood were withdrawn from each rab­ bit by cardiac puncture and the sera ob­ tained were titrated by yeast cell agglutina­ tion tests or by fungal complement-fixation techniques. The Candida albicans antiserum demon­ strated a low yeast cell agglutination titer. Therefore, a vaccine prepared in the man­ ner of the first and standardized to a No. 7 nephelometer was given in 0.5, 1.0 and 1.0 ml at weekly intervals to the rabbits which had previously received Candida albicans. This new Candida vaccine greatly stimu­ lated the production of antibody as shown by an increase in the yeast cell agglutination titer. SERUM COLLECTION

One week following the final injection in each series, 20-35 ml of blood were drawn from all rabbits by cardiac puncture and the sera were recovered by centrifugation from the clotted and refrigerated blood samples. GAMMA GLOBULIN PREPARATION

Gamma globulin to be used for conjuga­ tion with isothiocyanate containing antibody was fractionally recovered from serum aliquots by the method of Thurston, Rheins and Buehler.4 PROTEIN DETERMINATION

A standard Biuret method for determina­ tion of protein in the globulin fraction was used.

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The method of conjugating gamma globu­ lin with fluorescein isothiocyanate was es­ sentially that described by Goldstein and associates.5

ume of cells of each of the five organisms employed in this study. Formolized antigens were washed three times in 0.85% NaCl and one time with phosphate-buffered saline before the addi­ tion of the conjugate.

REMOVAL OF UNCONJUGATED FLUOROCHROME

OPTICAL EQUIPMENT

CONJUGATION OF GAMMA GLOBULIN

After conjugation, the various globulinisothiocyanate mixtures were passed through a Sephadex-25 column to remove unconju­ gated fluorochrome. Also, the multivalent antiserum prepara­ tion to be described was adsorbed with mouse liver powder to minimize nonspecific fluorescence. PREPARATION AND STAINING OF SLIDES

Slides to be used were cleaned by soaking in dichromate solution overnight, washed thoroughly in distilled water and air dried. Formolized antigens, viable organisms grown in vitro, and scrapings from the eyes of experimentally infected rabbits, served as the source of test materials which were affixed to the slides in various manners. The specimens were stained with the des­ ignated conjugates for 30 minutes in a 37° C water bath. During this process the speci­ mens were enclosed in a humidified cham­ ber. The slides were rinsed for 10 minutes in two changes of cold phosphate-buffered sa­ line (pH 7.4) and finally in cold distilled water. A drop of buffered glycerol was placed on the specimen, a cover slip (22 by 22 mm, No. 1 thickness) was applied, sealed with clear nail polish and the slide stored at 4°C until examined for fluorescence. CONTROLS

Normal serum conjugates and inhibition staining were employed as control measures. CROSS ADSORPTION

Each conjugate was adsorbed for 18 hours at 4°C against a one-half packed vol­

A Bausch and Lomb monocular mi­ croscope fitted with a cardioid darkfield con­ denser and an aluminized first-surface mir­ ror was used. An H B O 200 mercury arc lamp (Osram) enclosed in an American Op­ tical housing equipped with a heat adsorbing filter served as the light source. A No. 5840 Corning exciter filter and a No. 2A Wratten (Eastman Kodak) barrier filter were use in combination. PHOTOGRAPHIC EQUIPMENT

Kodachrome-2 film was used for photo­ graphing the corneal ulcers produced in rab­ bits. A Reichert microscope, equipped with a built-in ultraviolet light source, the filter combinations previously mentioned and a camera attachment, was used to photograph fluorescent microscopic fields of specimens prepared from material taken from the ex­ perimentally infected rabbit eyes. Anscochrome ASA-200 film was employed, with exposure times ranging from 10 seconds to 2.5 minutes. EXPERIMENTAL INFECTIONS

Eyes of each rabbit were cortisonized by injecting hydrocortisone subconjunctivally (2.5 mg contained in 0.1 ml/eye). Twentyfour hours later, under local anesthesia (Ophthaine), the epithelium of the cornea of each eye was scarified by a 4-6 cross-hatch pattern. Approximately 0.1 ml of a heavy saline suspension of the designated orga­ nism was placed into each eye and the eye­ lids held closed momentarily to facilitate penetration of the inoculum into the abraded area. One drop of Maxidex (a steroid prep-

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TABLE 1 CROSS REACTIVITY OF FLUORESCENT ANTISERA (VIABLE ANTIGENS)

Fluorescent Antibody Preparation Antigens C. albicans S. schenckii S. brevicaulis A. fumigatus A. boydii

S. schenckii

C. albicans +4

S. brevicaulis

A. fumigatus

A. boydii

+2

+4 +2

+4

+4

+

+ +

+2

Reactivity graded from ( —) to (+4).

aration) was introduced into each eye three times a day for three days postinoculation. RESULTS

Table 1 shows the results of reacting the various "tagged" antisera with both the ho­ mologous and heterologous antigens. It was evident that the fluorescent anti-Scopulariopsis brevicaulis serum was defective since it would not stain its homologous antigen and was, therefore, eliminated from further study as a single test agent. The other four antiserum preparations cross-reacted either not at all, minimally or to an extent permit­ ting easy distinction from the homologous test systems. Equal volumes of each conjugate were combined and tested as a multivalent prep­ aration. Table 2 illustrates that all five anti­ gens were stained with this preparation. Experimental infections were produced by the methods described previously. With­ in two days both rabbits which had received Aspergillus fumigatus had developed exten­ sive suppurative lesions in both eyes. Those receiving Allescheria boydii developed similar lesions in approximately one and one-half weeks, while punctiform ulcers ap­ peared in Candida albicans-infected rabbits

in IS days and developed into extensive opaque caseous lesions by the 20th day. By the 27th day, pinpoint white growths ap­ peared in the eyes of the Sporotrichum schenckii-infected animals. Those rabbits receiving Scopulariopsis brevicaulis revealed no evidence of ulceration by the 27th day so they were reinoculated at this time. Scrapings and cultures on Sabourauddextrose agar were made from the eyes of Allescheria and Aspergillus rabbits on the 12th day. This procedure was followed on the 20th day with the Candida inoculated rabbits, while the eyes of those rabbits re­ ceiving Sporotrichum were scraped and cul­ tured on Sabouraud-dextrose agar and BHI-dextrose-blood agar. All Sabouraud slants were incubated at room temperature while the BHI-dextrose-blood blood agar slants were incubated at 37°C. Scrapings and cultures were made at 3, 6, 9 and 14 days following reinoculation of rabbits with Scopulariopsis brevicaulis. However, ulcera­ tion did not occur in the eyes of these rab­ bits. It was found that both the multivalent and the specific adsorbed fluorescent anti­ sera stained the organisms in the scrapings from the eyes of rabbits infected with the

TABLE 2 STAINING SPECTRUM OF THE FLUORESCENT MULTIVALENT SERUM

Candida albicans

Sporotrichum schenckii

Scopulariopsis brevicaulis

A. fumigatus

A. boydii

+4

+4

+2

+4

+4

Reactivity graded from ( —) to (+4).

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TABLE 3 IMMUNOFLUORESCENT DETECTION OF FUNGAL ANTIGENS IN SCRAPINGS OF EYES OF EXPERIMENTALLY INFECTED RABBITS

Fluorescent Antibody Preparation Antigen C. albicans 20 days 30 days S schenckii 30 days 35 days 40 days

"Candida

*Sporotrichum

*Aspergillus

*Allescheria

+ (few)

Multivalent

+ + + +

+ +

A. fumigatus 12 days 20 days 30 days A. boydii 12 days 20 days 30 days

+

+ + + (few)

+ +

S. brevicaulis 3 through 14 days * Indicates specific adsorbed antiserum. Graded from ( —) to ( + ) .

first four organisms (table 3). It should be noted that nonspecific staining of debris and cells was extensive. However, since the fun­ gal elements sought were large and fluoresced intensely when stained, this fac­ tor did not materially detract from the de­ sired result. It was thought that partial elimi­

nation of background staining would short­ en the length of time required to read a pre­ pared slide. Accordingly, the multivalent fluorescent antiserum preparation was ad­ sorbed with powdered mouse liver. This procedure permitted the effective staining of the fungal elements, but the nonspecific

Fig. 1 (Rheins, Pixley, Suie and! Keates). Aspergillus fumigatus. Scrapings from experimental lesion (12 days postinoculation), stained". with multivalent conjugate, X40Q„ 25 seconds exposure.

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Fig. 2 (Rheins, Pixley, Suie and Keates). Allescheria boydii. Scrapings from experimental lesion (12 days postinoculation), stained with multivalent conjugate, X250, IS seconds exposure.

staining was reduced so that greater con­ trast was achieved. At no time was Scopulariopsis brevicaulis demonstrated by I F staining or culturing and no evidence of lesions was observed in the rabbits inoculated with this agent. Figures 1 through 4 photographically de­ pict the appearance of these fungi as found in immunofiuorescently stained scrapings. The Allescheria and Aspergillus occurred in short filaments while Sporotrichum occurred as a cigar-shaped yeast and Candida as an ovoid yeast.

Fig. 3 (Rheins, Pixley, Suie, and Keates), Sporotrichum schenckii. Scrapings from experimental le­ sion (30 days postinoculation), stained with liver absorbed multi­ valent conjugate, X2S0, 60 second exposure.

DISCUSSION

The principal purpose of this investiga­ tion was to devise a more rapid and accurate method for diagnosis of corneal ulcers of fungal etiology. It was thought that a direct fluorescent antibody procedure, that of Coons and Kaplan,6 might be applicable to meet these requirements. Therefore, five genera of fungi were cul­ tivated in a manner which resulted in phases of growth similar to those observed when these organisms are grown in vivo. Candida

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Fig. 4 (Rheins, Pixley, Suie and Keates). Candida albicans (specially prepared). Stained with multivalent conjugate, X400, 25 seconds exposure.

albicans may be a yeast or demonstrate mycelial elements according to the method em­ ployed for propagation, while Sporotrichum schenckii is a yeast at body temperature or on brain heart infusion-dextrose-blood agar at 37°C, although a sporulating fila­ mentous fungus at room temperature. Be­ cause of this, Sporotrichum was cultivated under the former conditions. Aspergillus fumigatus, Allescheria boydii and Scopular­ iopsis brevicaulis are not biphasic, but exist only as filamentous, sporulating fungi at all temperatures. However, in vivo, filaments do occur but sporulation is rare. It was nec­ essary, then, to cultivate these three fungi in a broth medium with constant agitation so that if sporulation occurred, germination would immediately follow. Formolized vaccines prepared from these cultures were administered intravenously to rabbits in order to obtain antibody sera. When these sera were titrated for the presence of antibody to the formolized anti­ gens by agglutination techniques, it was noted that the anti-Candida and anti-Sporo­ trichum sera reacted strongly with the homol­

NOVEMBER, 1966

ogous systems but exhibited some evidence of cross reactivity. However, of the three sera titrated by means of fungal complement fixation, only one, anti-Allescheria, pos­ sessed specific antibody demonstrable by this technique. This antiserum did not cross react. Anti-Aspergillus fixed complement in the presence of Scopulariopsis brevicaulis but no other antigen. Anti-Scopulariopsis serum possessed no detectable complement fixing antibody. Although these two antisera did not pos­ sess complement-fixing antibodies to their homologous antigens, they were processed and the gamma globulin fractions conjugat­ ed as were the other three. It was proposed that the specific complement fixing antibody had been formed earlier in the course of the immunization series and had disappeared by the time the serum samples were obtained although the agglutinating antibody re­ mained. The tagged gamma globulin preparations were used to stain both formolized and via­ ble organisms to determine the degree of cross reactivity. It was found that each preparation stained its specific formolized antigen, but exhibited very little cross reactivity. How­ ever, when these preparations were used to stain viable antigens, cultured in vitro, tagged anti-Scopulariopsis would not stain its homologous antigen, but did stain Asper­ gillus fumigatus. The other four prepara­ tions stained their specific antigens strongly. A greater range of cross reactivity was ob­ tained with viable antigens than with for­ molized antigens. These results indicated that perhaps the antigens had been altered by formolization. Results obtained when methods of fixation of specimens to slides were studied supported this view. It should be noted, however, that Asper­ gillus fumigatus, Allescheria boydii, and Scopulariopsis brevicaulis were sonicated for lengthy periods during which heat was generated within the system. Although an ice-water bath was employed for cooling,

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some denaturation of protein may have oc­ curred. This would more easily account for the total inability of the tagged anti-Scopulariopsis serum to stain its specific antigen, when viable. The other two organisms were not so drastically treated, so that the origi­ nal antigenic structure may not have been altered, or only slightly. This is indicated by the intense staining of specific viable orga­ nisms. Tagged gamma globulin obtained from animals which had not been immunized failed to stain both formolized and viable antigenic material. This suggested that the staining which occurred upon addition of fluorescein conjugates or similar prepara­ tions from immunized animals was not due to the presence of the fluorochrome itself but some other factor. Cross adsorption of each serum with its homologous formolized antigen resulted in preparations which were incapable of stain­ ing specific viable antigens. Adsorption with heterblogous antigens did not have this effect. Thus, it would seem that antibody was present in the preparations. A conjugated multivalent serum was pre­ pared which stained all five fungal antigens used in this study, in both the nonviable and viable state. It was postulated that this prep­ aration would react in a similar manner with viable antigens from scrapings of ex­ perimentally induced ocular infections. Specifically adsorbed antisera were also prepared and studied. Fungal antigens were demonstrable in scrapings using both the multivalent and the specific fluorescent antisera. However, non­ specific staining posed an area of confu­ sion. The multivalent conjugate was ad­ sorbed with mouse liver powder to minimize background staining. This greatly facilitated the screening of slides since there was less fluorescent material to be examined. Those rabbits which received Scopulariopsis brevicaulis did not develop corneal lesions. Animals receiving Aspergillus, Allescheria, and Candida developed extensive

899

and extremely destructive lesions, while those resulting from inoculation with Sporo­ trichum remained small and the surrounding tissues did not appear to be involved. Upon staining with the conjugated multi­ valent antiserum, fungal elements were found in all initial scrapings. However, those infections which appeared to be most de­ structive also presented significant amounts of cells in which the fungal elements were enmeshed. Candida albicans was demonstrable but the fluorescing yeast cells were few. As these infections progressed, the fungi dis­ cernible in stained preparations became more scarce and finally were inapparent. In the case of Sporotrichum, the organisms were readily observed for several weeks. It seems then that the time of sampling is critical with such a technique. It is suggest­ ed that if scrapings are taken early in the course of the lesion there is a better chance of demonstrating the etiologic agent. As the lesions progress, there is an increase in tis­ sue destruction and a concomitant reduction in the visualization of organisms. It has been assumed that progression of these fungal infections of the rabbit eye was analogous to the clinical situation encoun­ tered in patients. In a natural situation, the infective dosage of fungal agent introduced into the eye probably would not be so great as in these experimental infections. There­ fore, the progress of the ulcers possibly would be different and perhaps other resul­ tant factors would differ. As physicians become aware of the prob­ lem, and versed in the advantage of this method of diagnosis, and commercial tagged antifungal sera become available, this proce­ dure could serve a useful purpose in diag­ nosis. SUMMARY

1. It was shown that a direct fluorescent staining procedure is applicable for the iden­ tification of fungal elements from scrapings of corneal ulcers experimentally produced in rabbits.

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2. A conjugated multivalent antiserum was prepared which stained any one of the five fungi examined in this study, whether the agents were formolized or viable (culti­ vated in v i t r o ) . 3. This same multivalent antiserum stained four of the five fungi present on slides prepared from the scrapings from ex­ perimental lesions. All four of these fungi were also cultivated on media. T h e fifth or­ ganism failed to produce infection when in­ troduced into rabbit eyes. 4. It has been postulated that the time of sampling of these lesions for culturing and staining is important. 410 West

Tenth

Avenue

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NOVEMBER, 1966

REFERENCES

1. McLean, J. M.: Oculomycosis. Tr. Am. Acad. Ophth. Otolaryn. 67:149, 1963. 2. Ley, A. P.: Experimental fungus infections of the cornea: a preliminary report. Am. J. Ophth. 42 :S9, 19S6. 3. Rheins, M. S., Suie, T., Van Winkle, M. G., and Havener, W. H.: Potentiation of mycotic ocu­ lar infections by drugs. Brit. J. Ophth. In press. 4. Thurston, J. R., Rheins, M. S., and Buehler, E. V.: A rapid method for recovering serologically active globulins by sodium sulfate precipita­ tion. J. Lab. &€lin. Med. 49:647, 1957. 5. Goldstein, G., Sizys, I. S., and Chase, M. W.: Studies on fluorescent antibody staining: I. Non­ specific fluorescein coupled sheep anti-rabbit glob­ ulins. J. Exper. Med. 114:89, 1961. 6. Coons, A. H., and Kaplan, M. H.: Localiza­ tion of antigen in tissue cells: Improvements in a method of the detection of antigen by means of fluorescent antibody. J. Exper. Med. 91:1, 1950.

CEREBRAL PHYCOMYCOSIS (MUCORMYCOSIS) WITH OCULAR INVOLVEMENT J O S E P H GINSBERG, M.D.,

A B B O T G. S P A U L D I N G , M.D.,

AND V E R N O. L A I N G ,

M.D.

Cincinnati, Ohio Though the incidence of acute phycomycotic infections is rising 1 ' 3 and the various clinical patterns are usually characteristic, a significant number of cases are still not diagnosed during life or at gross autopsy. T h e cerebral form, the most common of the three major types (cerebral, pulmonary and intestinal), is of particular importance to ophthalmologists since the ocular manifesta­ tions are often among the earliest or most conspicuous clinical signs, appearing in ap­ proximately 8 0 % of cases with cerebral phycomycosis and including, chiefly, unilat­ eral orbital cellulitis with proptosis, ptosis, internal and external ophthalmoplegia, optic neuritis, posterior uveitis and obstruction of the central retinal artery. Marked visual imFrom the Departments of Pathology, Ophthal­ mology, Neurology and Medicine, University of Cincinnati, College of Medicine, the Cincinnati General Hospital and Cincinnati Veterans Admin­ istration Hospital.

pairment is usual and the ocular signs tend to be permanent. Excellent general reviews 1 " 11 and approx­ imately 55 case reports 1 - 6 1 of cerebral phy­ comycosis have appeared, mostly in the last 15 years. T h e term "cerebral" has been widely used to designate the form of acute phycomycosis which tends to involve some or all of the following a r e a s : the orbit (with complete ophthalmoplegia and blindness) ; the nose (with dark, blood-tinged discharge and reddish-black necrotic appearance of the turbinates and s e p t u m ) ; the paranasal sinuses (with a tendency to pansinusitis) ; the oral cavity (especially the hard palate with fistulas and sloughs) ; the soft tissues of the cheek (with gangrene and slough­ ing) ; and the central nervous system (with meningoencephalitis and motor and sensory deficits). Though clinical or laboratory evi­ dence of cerebral infection may be lacking, it may be anticipated that the b r a i n eventu-