Dr. O. Cameron. Gruner
Dr. Rife's First Microscope

 

Dr. Rife And Dr Gruners Cryptomyces Pleomorpha Fungus

On the Marsh Rife Audio CD's Dr. Rife talks at great length about his work with Dr. Guner on the Cryptomyces Pleomorpha Fungus. Below is Dr. Gurners written paper on this organism. 

CRYPTOMYCES PLEOMORPHA: A NEW ORGANISM ISOLATED FROM THE BLOOD OF A
CASE OF METASTASIZED CARCINOMA OF THE BREAST*


By: O. C. GRUNER, M.D.,
Montreal The Canadian Medical Association Journal, Jan, 1935

THIS report is presented for the following reasons:

(1) The organism was detected in the circulating blood by direct examination (Fig. 1).

(2) It was detected among the tumour cells in the original neoplasm, in sections which had been made four years previously (Fig. 2).

(3) An organism, evidently of the same type, has been found in seven previous cases, but its cultural characteristics and pathology had not presented the same degree of completeness.

(4) Though in part resembling other fungoid organisms which have been described before in human pathology, this one seems to present additional distinctive and interesting features of its own.

(5) Careful observations of the living cultures have shown that in some phases this organism exactly mimics the cell-elements of human blood, and also the so-called Plimmer bodies and Russell fuchsinophile bodies of some malignant tissues, and, in addition, has forms which are just like the free nuclei and various sized granules found in many sections of neoplasms. These mimicries explain how the organism may effectually escape detection in the routine observation of tissues and blood.

Isolation.-From the blood of a case of carcinoma of the breast (excised five years ago) with intrathoracic metastasis; previously, in other cases, from the tumour tissue.

Cultural characteristics.-The initial growth appeared only after seven days' incubation at room temperature. After developing a raised formation like that of sporotrichum, but extremely hard in consistence, it remained stationary for two weeks, despite repeated attempts at subculture on a great variety of media of all gradations of pH. Finally, the use of the patient's serum, and asparagus extract to enrich the glycerine-glucose-peptone water, proved to induce cultural success, good growths now appearing in all subsequent cultures in 48 hours. The organism was strictly aerobic. Room temperature was best. The optimum pH was 6.8 to 7.0. Bacterial contamination effectually antagonized the cultures.

On a.sparagus agar.-Within 48 hours a small round orange-tinted colony appears with elevated glistening centre. As this gradually enlarges it becomes coral pink. A somewhat hardened pellicle forms in the centre and delicate threads form at the periphery, at first whitish, and, later, faintly coral-pink (Fig. 1).

Cryptomyces Pleomorpha Fungus

Fig 1.-Petri dish culture, 12 days old. The central raised growth consists of the torula-like spore forms, and is of coral colour in the original. The delicate hairy outgrowth is the mycelial phase.

Only spore forms occur in the initial colony. Hyphae develop in from 6 to 10 days, showing various forms as the culture ages (Figs. 17-31). After 14 days the pink centre wrinkles up, much like sporotrichum, Mycobact. tuberculosis, etc., and the whole growth constitutes quite a firm membrane rather firmly adherent to the surface of the medium. Finally, a very fine white powdery deposit appears all over the growth, and no further change occurs.

On asparagus glucose glycerine peptone water, a pellicle forms in 48 hours; this is in the form of a ring at the surface, adhering to the glass, and made up almost entirely of mycelia, the spores falling to the bottom of the tube in large numbers. A pink colour appears in 7 to 10 days, and the ring can be detached from the glass, when it will remain intact and hanging in the medium.

Gelatine.-Not liquefied; very slow growth.

Meat-extract media.-Very slow growth; colonies very small; whitish; no colour.

Plain glucose glycerine agar.-Extremelyslow growth; small glistening colonies; no colour; no mycelium.

Sabouraud.-Very slow colourless growth; very small and few hyphre; spores much smaller than those on asparagus media.

Dextrose-tartaric acid.-No growth (in distinction from usual fungi).

Potato.-Very scanty dull yellow growth, slowly turning pinkish.

Tomato.-Very luxuriant growth in 48 hours; pink colour slightly paler than the flesh of the fruit. The condensation water rapidly fills with torula forms and turns pink.

Milk.-Budding torula forms develop in 72 hours; no coagulation; no acid.

Peptone water.-Noindol.

Nitrate media.-No nitrite formation.

Sugar-reactions.-No fermentation of glucose; gas, but no acid in lactose; saccharose, nil.

Stainability.-Dilutecarbol-fuchsin gives the best results. Methylene blue stains relatively feebly. It is Gram-positive. It is not acid-fast. Rossophilia is almost absent.

Morphology.-This organism is very pleomorphic. Thus at different times, in the same culture tube, one may see transient micrococci like Doyen's (1) M. neoformans of 1904 (Fig. 8), spherical forms like Sanfelice's blastomyces (1896) (Fig. 7), tubular forms apparently answering v. Brehmer's (2) description of his Siphonospora (Fig. 8) ; oval resting spores like those found in a specimen of Schmidt’s (3) "Blastolysin" (Fig. 9); combined with mycelial formations (Fig. 11).

Cryptomyces Pleomorpha Fungi

The following forms of reproduction are observable, as met with in other fungi: (a) fission forms or oidia, or diplospores (Fig. 10) suggestive of schiwsaccharomyces; (b) bud-formation as usual in torula or saccharomyces (Fig. 11); (c) ascospores, or "megaspores" (Fig. 12) in keeping with zygosaccharomyces; (d) very minute microspores (Fig. 14), which are highly refractile and arise by detachment from (b); these are metachromatic with Leishman and with methylene blue; (e) chlamydospore formation (Fig. 15); (f) lateral buds (Fig. 28), as in typical sporotrichum; (g) sessile sporangia in the aerial mat (Fig. 16), suggestive of some mucors; (h) still smaller particles occur, possibly filter-passing. There is a fairly regular sequence. Thus from b to d, from b to mycelium (first week), from mycelium to f, and to a, and to e (second week); from a to c and to g. The whole series ends in about three weeks, when a new subculture becomes essential.
The organism is therefore an ascomycete, having affinities with sporotrichum, cryptococcus and blastomyces. It is given the name of Cryptomyces because it combines mycelial formation with an obscure biology and incidentally an effective concealment when situated in invaded tissues.

Pathogenicity. - It is pathogenic for white rats ,when given intrapleurally, and for white mice when given into the submammary tissue. It was recovered true to its original cultural characteristics in pure form from the blood and all organs of these animals but developed very little mycelium. A high monocytosis was striking and early. The progeny of a female rat inoculated with the organism while pregnant developed generalized infection with the same organism, dying 21 days after birth. Subcutaneous inoculation produced indurated areas in which the organisms were plentiful. After 12 days nodules formed in which spindle-shaped connective-tissue cells were predominant, and megaspores were abundant among them (Fig. 26). Mycelium was not detected in the tissues.

Cuti-reaction.-Skinreactions have been obtained by using an emulsion of this organism on the patient whence it was derived, as well as on several other malignant cases, including sarcomas, but not on persons in ordinary health. Further studies on this point are in progress.

Relation to malignant disease.-Naturally, such an organism may be primary or secondary. If the former it would presumably act in virtue of some carcinogenic substance being formed among the products of its metabolism. In any case, the frequency of its association with new-growths remains to be established. The usual arguments against a microbic origin of the disease naturally arise.

DISCUSSION

An accidental admixture of two or more organisms would provide a simple explanation of the pleomorphism. The two main forms have already been separately described from time to time as being causal for cancer, to be subsequently rejected as "contaminants", namely, the yeast-like forms and the mucor-like forms. Moreover, in the present case, the cultures after passage through the infected rats and mice showed a dominantly blastomycetic form, with mycelial formation almost negligible. Further, the patient had been for a few weeks in a region where sporotrichosis occurs, though never having the slightest evidence of lesions of that kind.

The reasons for believing the organism to be single arc: (a) the constancy of the occurrence of the dual forms in succession, at the same time intervals in all subcultures; (b) the ability actually to observe the one form changing into the other; ( c) the inability to separate them permanently by plating; (d) the cultural characters; (e) the possession of pathogenicity; (f) its unique character; (g) the unlikelihood of a chance admixture of distinct organisms resulting in such a close symbiosis as actually to manifest conjugation.

Of some interest is the experience of the animal lesions produced, in that the organism is found closely mingled with the reactive infiltration of predominantly monocytic type, and shows in the sections appearances which strongly recall those seen in sections of carcinomas and sarcomas, though such particles are usually regarded as unessential bacterial or degenerative components. Applied to human tumour histology, the presence of such microscopic objects would 'seem to require an explanation.

Of further interest is the evidence of sexuality in this organism (in common with other fungi), though the question of haploid and diploid phases has not been taken up. The delay in obtaining good subcultures would be explained by the difficulty of finding a medium favourable for the conjugation of the + and - elements. This provides a useful suggestion that such occupants of tumour tissue (whatever their presence means) present a sexuality factor which determines ease or difficulty of culture outside the body. The repeated observation of the megaspore forms inside the phagocytic cells in the infected animals with their mimicry of the Plimmer-body and other inclusions in human cancers supports this suggestion quite definitely.

CONCLUSIONS

A report is given of a new organism, here called Cryptomyces pleomorpha, which is placed among the ascomycetes. The question of etiological relationship with malignant disease is necessarily left open.

Very grateful thanks are due to Dr. Archibald and to Dr. Mark Kaufmann for enabling this case to be worked out, and also to Dr. Pauline Beregoff, for recently undertaking further tests on animals.

REFERENCES

1. DOYEN: Ewing's Neoplastic Diseases, W. B. Saunders, Phila., 3rd ed., 1928, p. 122.

2. V. BREHMER, W.: Krebs eine Erregerkrankheit, Fortschrit. der Medizin, 1932, 50: 697; edit. in Brit. M. J., 1934, 2: 520; annotation in The Lancet, 1934, 2: 609.

3. SCHMIDT, O.: Ewing's Neoplastic Diseases, W. B. Saunders, Phila., 3rd ed., 1928, p. 125.

* From the Department of Surgery, McGill University, Montreal. Under a grant for Cancer Research contributed to Dr. E. 'V. Archibald by an anonymous donor.

 



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