Are postmortem studies of glossal candidal infection useful? An experimental study Edmund Peters,” Ronald Whitehouse,b and Colleen Murdoch,” Edmonton, Alberta, Canada

UNIVERSITY OF ALBERTA

Autopsy studies have suggested that candidal pseudohyphae may be found in dorsal glossal epithelium in

33% to 42% of cases. Candidal yeast forms, and occasionally free pseudohyphae, are found as oral

commensals in about 44% of the population. This study examined the possible distorting influence in

autopsy studies that could be caused by postmortem hyphal transformation of candidal yeast forms followed by saprophytic infestation. Candida albicans yeast forms were topically applied to the middorsal

glossal mucosa of five healthy pigs, immediately after killing. Biopsy specimens from this mucosa were

subsequently maintained, in vitro, for periods of 12 and 24 hours in humid conditions at different

temperature regression rates chosen to approximate those of the oral cavity after death. Biopsy specimens

subjected to a temperature regression of 35” C (oral temperature) to 23” C (room temperature) over 11

hours showed infestation of epithelium by pseudohyphae in all cases. Biopsy specimens subjected to a

similar temperature regression over 5 hours showed infestation in two of five cases. Control biopsy

specimens showed that there was no candidal infection at the time of killing. The results indicate that in

vitro saprophytic candidal infestation is possible in the time intervals and the declining oral temperatures preceding autopsy. It suggests that postmortem saprophytic candidal infestation may distort results from

autopsy studies that do not anticipate this problem. (ORAL SURG ORAL MED ORAL PATHOL 1989;67:177-80)

A utopsy studies have consistently demonstrated candidal pseudohyphae within glossal epithelium in 38% to 42% of cases.‘-3 Attempts to correlate ante- mortem factors, such as the concomitant prevalence of malignant*, 3 or generalized3 disease, ‘with these findings have been negative in two of the most recent studies. This has suggested that such postmortem data in the absence of correlated systemic disease could be clinically applicable. Accordingly, although these results have been interpreted with great cau- tion, they have been used to implicitly support2 or directly dispute3 the controversial question of wheth- er median rhomboid glossitis represents a localized middorsal candidal infection rather than a develop- mental anomaly.4 It has also been suggested that the presence of Candida albicans in glossal epithelium can be regarded as a common findings3 However, the additional possibility that postmortem saprophytic infestation by commensal candidal yeast forms could

“Department of Oral Biology, Faculty of Dentistry. bDepartment of Medical Microbiology and Infectious Diseases, Faculty of Medicine.

have distorted these findings has apparently not been considered.

Candida organisms are present in the oral cavity, as commensals in the nonpathogenic yeast form and occasionally as free hyphae, in approximately 44% of the population; the tongue is the primary oral reser- voir.5 Furthermore, there is a period before autopsy, usually not greater than a day, during which yeast could transform into the pathogenic hyphal form and infest the oral mucosa. These theoretical considera- tions suggest that an unduly high prevalence of candidal pseudohyphae within epithelium could be found at autopsy and that extrapolations from these data to the clinical situation could be unreliable. The purpose of this study, therefore, was to examine this possibility by assessing the potential of candidal yeast to change to the hyphal form and invade nonvital glossal mucosa under the conditions preced- ing autopsy.

MATERIALS AND METHODS

An aqueous suspension of C. albicans yeast in tryptic soy broth (1 X 10’ cells/ml) was prepared

177

178 Peters, Whitehouse, and Murdoch Oral Surg February 1989

Table I. Candidal hyphae infestation in group 1 (35” to 23” regression in 11 hours) and group 2 (35” to 23” regression in 5 hours); biopsy specimens maintained at room temperature (group 3) were uniformly negative ~-...__ --.---

Exp. group I (n = IO) T---- -~~ ----Exp.groupj -L-:7---- .-- n Control group 3 (n = IOJ

Case 12 h

No. (n = 5)

24 h

(n = 5) I I2 h

I I

24 h 12 h 24 h

in = 5) (n = SJ In = 5) (n = S)

I + ++

2 + +

3 + +

4 + + +

5 + ++ +

-, Absence of pseudohyphae or pseudohyphae not penetrating epithelium. f, Pseudohyphae penetrating epithelium. ++, Pseudohyphae in tangled masses or extending to lamina propria.

with an isolate cultured from a patient with candidi- asis; identification of C. albicans was established by microscopic appearance, chlamydospore formation, and germ tube formation in human sera within 2 hours at 37” C. The suspension was smeared and examined microscopically to ensure that only yeast forms of Candida organisms were present. Subse- quently, a sterile cotton swab was dipped in the suspension and applied topically to the glossal muco- sae of five healthy 30 kg female Yorkshire pigs that had been killed with an intravenous pentobarbital overdose.

After the topical application, four biopsy speci- mens, each about 2 cm long, were obtained from the middorsal aspect of each tongue. The biopsy speci- mens were separated into two pairs, and each pair was placed in a separate covered partitioned Petri dish. Thus, two groups of five dishes were formed, with each dish containing two biopsy specimens. Each of the two groups was then subjected to different conditions of declining temperatures, as follows:

1. The first group of dishes was placed into a beaker, which in turn was suspended in a semi- insulated 6 liter water-filled container. The water in the 6 liter container had been previously warmed to 37’ C. This arrangement corresponded to 35” C (the oral temperature) within the immersed beaker con- taining the dishes and permitted a gradual decline to room temperature (23’ C) over 11 hours.

2. The second group was similarly placed into a warmed water-filled 1 L container that permitted a decline from 35” C to room temperature over 5 hours.

Humidity in each dish was maintained by placing a wet gauze sponge into one of the empty compart- ments contiguous to the biopsy specimens. After 12

- .- - . - - - + - + -

hours, one of the two biopsy specimens was removed from each dish, and after 24 hours, the second was removed. After removal, the biopsy specimens were immediately fixed in neutral buffered formalin and routinely processed. A single 5 I.trn section was then cut, stained with periodic acid-Schilf (PAS) reagent, and examined for infesting candidal pseudohyphae.

As controls, in each of the five animals, two further biopsy specimens were obtained at the same time and from the same inoculated area as the experimental biopsy specimens. Each biopsy speci- men was maintained at room temperature for 12 and 24 hours, respectively, in humid conditions identical to those of the experimental groups. These biopsy specimens were subsequently processed and exam- ined in a manner similar to that of the experimental biopsy specimens.

Controls to examine for preexisting infection con- sisted of single biopsy specimens from each animal taken before inoculation with the candidal suspen- sion; these were immediately fixed and then routine- ly processed and examined for candidal pseudohy- phae. In addition, the glossal mucosae were swabbed before the inoculations and cultured for Candida organisms on Phytone yeast extract agar.

A probability value was determined with the use of Fisher’s exact test (one-tailed) for proportionate comparison between positive experimental biopsy specimens and the control .biopsy specimens.

RESULTS

Microscopic examination of the smeared yeast suspensions used for the topical application revealed only yeast forms in concentrations of about 600 per square millimeter.

The cultures taken from the tongues, before topi- cal application of the yeast suspension, demonstrated

Volume 67

Number 2 Postmortem candidal infection 179

Fig. 1. Biopsy specimen maintained for 24 hours with temperature regression occurring over 5 hours (group II). Candidal pseudohyphae are evident penetrating deep into epithelium. (Periodic acid-Schiff stain. Original magnification, x250.)

Fig. 2. Resection edge of biopsy specimen maintained for 24 hours with temperature regression occurring over 11 hours (group 1). A massive proliferation of yeast forms and pseudohyphae is evident. (Periodic acid-Schiff stain. Original magnification, x250.)

a single endogenous yeast colony in a single case. mens that were maintained in vitro for 12 to 24 hours None of the control biopsy specimens, taken to under varying temperature conditions. No infesta- determine preexisting infection, showed pseudohy- tion was found in control biopsy specimens main- phae either free or penetrating the epithelium.

Table I shows the results from the biopsy speci- tained at room temperature from the onset (group 3). All biopsy specimens that were subjected to the

180 Peters, Whitehouse, and Murdoch Oral Surg February 1989

1 l-hour temperature decrease (group 1) showed infestation after 12 and 24 hours (p = 0.01). Four biopsy specimens from two cases, which were sub- jected to the 5hour temperature decrease (group 2), showed infestation after 12 and 24 hours (p = 0.22). Fig. 1 shows an infested biopsy specimen after 24 hours. Infestation was generally most florid close to the resected edges of the biopsy specimen, as shown in Fig. 2.

DISCUSSION

The model used in this study attempted to approx- imate the oral environment after death. A humid atmosphere was maintained with variable conditions of declining temperature. Although somewhat capri- cious, estimation of oral temperature regression was extrapolated from data indicating a postmortem decline of about 1 o C per hour in the axilla and the rectum.6 Accordingly, a decline to room temperature might be anticipated in about 12 hours. Application of candidal yeast forms to the experimental mucosa was necessary to ensure initial standardized condi- tions of infectivity since preliminary work with cultures had shown that Candida organisms were rarely present in our animal model. In comparison, as previously noted, oral candidal yeast forms are present in approximately 44% of the healthy adult population, and it is possible that this prevalence is higher in a debilitated hospitalized population.’

This study indicates that in vitro candidal infesta- tion of nonvital glossal mucosa is easily accomplished during the warmer transitional periods of tempera- ture regression comparable to postmortem intervals. In contrast, none of the mucosal samples that were immediately brought to room temperature, and only two cases from the group in which the temperature decline occurred quickly (group 2), showed infesta- tion. The ease with which infestation could be de- monstrated (in positive cases) and the florid nature of some infestations, which occasionally extended into lamina propria, were probably influenced by the numbers of topically applied yeast forms. The unex- pected predilection for infestation of epithelium proximal to the resected edges of the experimental biopsy specimens may have been because the dis- rupted epithelium permitted easier penetration of

hyphae or as a result of enhanced growth due to nutrient elements from contiguous connective tissue. This raises the clinical possibility that mucosa, ulcer- ated or atrophied by disease or the inanition fre- quently preceding death, may be similarly predis- posed.

These theoretical considerations and the experi- mental results reinforce the cautions previously put forward with respect to the usefulness of postmortem data. The microbial characteristics of glossal mucosa can be expected to change after death. When studies are undertaken to examine the issue of glossal candidal infection it would seem prudent to use biopsy specimens of vital tissue, or the biopsy speci- mens should be taken immediately after death. In either situation, previous work would dictate that patients with prolonged illnesses involving any form of immune compromise or who have received treat- ment with agents known to promote candidiasis, such as wide spectrum antibiotics or steroids, should be excluded.’

The technical assistance of Mr. Ted Germaine and Mr. Dennis Carmel is gratefully acknowledged.

REFERENCES

1. Soames JV. A review of the histology of the tongue in the region of the foramen cecum. ORAL SURG ORAL MED ORAL PATHOL 1973;36:220-4.

2. Wright BA, Fenwick F. Candidiasis and atrophic tongue lesions. ORAL SURC ORAL MED ORAL PATHOL 1981;51:55- 61.

3. Van der Wal N, Van der Waal I. Cundida albicans in median rhomboid glossitis. Int J Oral Maxillofac Surg 1986;15: 322-5.

4. Cooke BED. Median rhomboid glossitis-candidiasis and not a developmental anomaly. Br J Dermatol 1975;93:399-405.

5. Arendorf TM, Walker DM. The prevalence and intraoral distribution of Candida albicans in man. Arch Oral Biol 1980;25:1-10.

6. Ludwig J. Current methods of autopsy practice. Philadelphia: WB Saunders Co, 1972;15.

7. Lehner T. Oral thrush, or acute pseudomembranous candidi- asis. ORAL SURG ORAL MED ORAL PATHOL 1964;18:27-37.

Reprint requests to.

Dr. E. Peters Department of Oral Biology, Dental/Pharmacy Bld. University of Alberta Edmonton, Alberta, T6G 2N8 Canada