GIANT FORMS OF H1STOPLASMA
CAPSULATUM
TISSUE EXPLANTS
IN
JAN SCHWARZ, M.D.
Clinical Laboratories, Jewish Hospital, and the Laboratory of Mycology (Departments oj
Dermatology and Pathology), Cincinnati General Hospital, Cincinnati, Ohio
The present report is the outgrowth of the finding of giant forms of Histoplasma
capsulalum in a tissue explant. A portion of a lymph node removed from a child
with histoplasmosis was placed on a blood agar plate and incubated at 37 C.
Ten clays after the culture was initiated, a fine warty growth was observed on the
surface of the tissue but not on the surface of the medium. The tissue was removed from the medium and histologic sections were prepared. Enormous fungus
forms (Fig. 1) were found in the superficial portions of the tissue, and this observation led us to set up several experiments in order (a) to determine if large cells
of //. capsulation can be produced at will in tissue explants, and (b) to demonstrate that the large forms observed were not contaminants or derivatives of a
second fungus present in the tissue.
In 1931, Crumrine and Kessel1 described ". . . larger extracellular phases of
the organism . . ." in the spleen of a patient with histoplasmosis, a diagnosis
verified by Meleney and other observers. >This report was made prior to De
Monbreun's studies2 of the first cultures of H. capsulatum, and, judging from the
description, one wonders if a Cryptococcus, also, may have been present: "The
halo is also much thicker than the capsule of the intracellular phase, attaining
a diameter of 10 or 12 microns in some instances." Similarly, the budding, which
was observed quite frequently in the extracellular forms, is indicative rather of
Cryptococcus. However, no culture was obtained, and the exact generic identity
of the large forms observed by Crumrine and Kessel is not certain.
DeMonbreun 2 observed similar large forms in cultures of H. capsulatum in
serum, as indicated by his description: "Instead, after 6 to 8 days, the few organisms that survive appear as spherical bodies 4 to 5 times as large as the young
forms. They are surrounded by a thick membrane within which may be seen
spherical globules. In fresh preparations the similarity of these structures to
asci is very striking, but appropriate staining methods prove the contained
spherules to be fat globules, and not spores. These large spherical cells behave
functionally like chlamydospores. When cultured at room temperature on
dextrose agar, or blood agar, they germinate, giving rise to the mycelial form of
the fungus. The same form, without the occurrence of yeastlike forms, has also
been obtained in cultures of these spores on blood agar maintained at 37 C."
The arthrospore-like forms described by Humphrey 4 may represent some fungus other than H. capsulatum. He describes the pulmonary cavity as being filled
Received for publication March 21, 1953.
Dr. Schwarz is Associate Director of Clinical Laboratories, Jewish Hospital, and Director of Laboratory of Mycology, Cincinnati General Hospital.
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GIANT FORMS OF H1STOPLASMA CAPSULATUM
899
with mycelial strands with certain forms which possibly represent spores; but
these are not sufficiently convincing.
Finally, Haley3 recently described tuberculated chlamydospores developing
from mycelia found in the necrotic liver of an infected mouse. Such forms differ
from those described in the present study. We have not identified tuberculate
spores in tissue, and we believe that the large forms described in this report
develop from the yeastlike cells without intermediate hyphal forms.
METHODS
Twelve mice were inoculated intravenously with mycelial phase of //. capsulalum. After 3 to 4 weeks, the liver, and sometimes the spleen, was removed from
the body by aseptic technic and cut into small pieces with scissors. Each piece
was then deposited on the surface of an agar medium (Difco blood-agar-base)
in a petri dish which was sealed subsequently with cellophane tape. Every 24
hours one of the small pieces of tissue was removed in order to obtain information
on the growth of organisms, including the development of large forms. The
tissues, together with the colonies developing on top, were fixed in formalin, and
sections were stained with hematoxylin and eosin and with periodic acid stain.
Subcultures from each piece were made in order to rule out the possibility of
contaminating fungi. In every instance, H. capsulatum was recovered as a pure
culture. The measurements of the organisms were made with a Spencer ocular
micrometer.
OBSERVATIONS
Twenty-four hours. The structure of the liver was identifiable although autolysis was present in the center of the tissue. There was marked, diffuse and focal
hepatitis with a fair number of organisms measuring 1 to 2 microns lying within
macrophages. No large forms were identified in sections stained by either hematoxylin or periodic acid. With the periodic acid stain, little clusters of yeastlike
cells in the macrophages could be made out clearly. At this early stage, there was
an obvious localization of organisms in the periphery of the tissue and no organisms were observed in the poorly stained, autolytic center. One could not determine if this absence of organisms was associated with poor fixation and autolysis, or if the organisms in the center were actually destroyed in 24 hours. No
hyphae were observed in the tissue or in the fungus growth.
Forty-eighl hours. The center of the liver was more autolyzed, but the periphery was still fairly well preserved and the histologic structure could be identified easily. The organisms had multiplied because in many areas one could find
clusters of yeastlike cells intracellularly. Some of these cells were considerably
larger than those observed after 24 hours, and the larger cells measured up to 5
and 6 microns. However, the overwhelming majority of organisms were considerably smaller (1 to 3 microns). Occasional budding of the small yeastlike
cells was observed and some of the phagocytic cells had ruptured and extracellular
forms were found. However, the great majority of organisms were still intracellular. No hyphae were seen.
900
SCHWARZ
Seventy-two hours. The structure of the liver was only slightly different from
that seen after 48 hours, but many ruptured cells and yeastlike structures could
be recognized in the sinusoids. Many organisms (Fig. 2) were considerably larger
than those observed after 24 hours. The larger and more numerous elements
were 5 to 6 microns in diameter, and occurred in clusters. Small yeastlike cells
were scarce, and no hyphae were seen.
One hundred twenty hours. The structure of the liver was not appreciably
different from that described above, but the number and size of large yeastlike
cells had increased considerably. The majority of the large cells (Fig. 3) were
now about 6 microns in diameter, with occasional single cells measuring 8 to
10 microns. A few septate hyphae were seen. These were short and obviously
took origin in some of the large cells described. The large forms developed
nearly exclusively on the surface of the tissue, in the region where relatively
more oxygen was available. The center of the tissue was completely autolyzed
and did not show any growth of fungus.
Seven days. At this time the autolysis of the liver was fairly complete but the
outline of the hepatic cells could still be recognized. Yeastlike cells were observed in locations regarded as remnants of sinusoids. In some areas one could
find considerable numbers of small yeastlike cells that apparently had multiplied
locally. Generally, the outstanding feature was the presence of large yeastlike
cells that could be recognized easily in sections stained with hematoxylin or
periodic acid. In examination of tissue prior to 7 days, the yeastlike cells in
hematoxylin-eosin sections stained poorly with indefinite outlines. In sections
made after 7 days the cells appeared with a distinct basophilic outline of the
cell wall. Many elements measured from 12 to 18 microns but the majority of the
large cells still measured 5 to 6 microns. Hyphae had increased in number in
some areas, although large portions of the surface of the liver showed only the
yeastlike cells.
Eleven days. Several yeastlike cells measured as much as 20 microns in diameter.
The majority of the larger yeastlike cells were still about 5 to 6 microns, but
there was a considerable number of larger forms. Hyphae were numerous and
mycelia were forming.
Nineteen days. Portions of the surface of the liver fragment were completely
covered by mycelial growth. Below the mycelium the larger and smaller yeastlike cells were seen. In other regions numerous yeastlike cells without appreciable
number of hyphal elements were seen. All the yeastlike cells were fairly large,
All photographs were taken from sections stained with periodic acid. Magnification X 600
(oil immersion). The time given refers to the period of incubation on sealed blood agar at
37 C.
FIG. 1 (left upper). Explant kept for 10 days on blood agar at 37 C , from culture of
lymph node from child with generalized histoplasmosis. Numerous small yeastlike cells,
predominantly in upper half of the figure. The large cell in the center is giving rise to a
germinal tube. The large adjacent cell on the right has a central dark spot.
FIG. 2 (right upper). Seventy-two hours. Clusters of cells of 5 to 6 n, present in the sinusoids of mouse liver (experimentally injected intravenously 4 weeks prior to death). One
cell is already considerably larger than the others.
FIG. 3 (left lower). From culture of mouse liver at 5 days. The average size of the fungus
cells has increased and large forms are developing. Possible budding of the largest cell.
FIG. 4 (right lower). From culture at 19 days. Entangled hyphae, small cells (in upper
half of picture) and large (budding?) forms. Liver was completely autolyzed.
GIANT FORMS OF HISTOPLASMA CAPSULATUM
FIGS. 1-4
901
902
SCHWARZ
the majority being at least 10 microns in diameter (Fig. 4). Some of the largest
forms had a considerably thickened wall, which gave the impression of a double
wall. Budding was observed only infrequently in either the small or larger elements. Frequently small and large yeastlike cells without hyphae were seen but,
wherever hyphae were noted, yeastlike cells were present. None of the large
forms showed any tuberculation, and their outlines were smooth and distinct.
Similar studies were made on naturally infected human tissue and, although
variations were noted in development of hyphae, the characteristic large cells
were not distinguishable from those observed in the tissues from experimental
animals. In other tissues from mice, as well as in the human material, some
variations were observed in the development of hyphae but the size and shape
of the large cells were identical in all instances.
DISCUSSION
The first thought upon finding the large cells unexpectedly in material from a
patient with histoplasmosis was that a contaminant was present. It is felt that
this possibility has been eliminated by the fact that morphologically identical
forms could be obtained in explants of tissues from experimentally infected animals, and by the fact that pure cultures of H. capsulatum were obtained by
subcultures of the tissue explants.
It is interesting to speculate regarding the mechanism by which growth of the
individual large cells occurs, and what these cells represent. It is apparent that
there is simply a growth in size of the original small yeastlike cells. We are not
dealing with chlamydospores borne on hyphae because the increase in size of the
cells is observed before hyphae appear. Furthermore, the frequent combination
of small yeastlike cells with the large elements indicates that they are probably
developmental phases of one single form. The fact that this has not been observed
except by DeMonbreun 2 in culture but can be so easily reproduced in tissue is
remarkable. It may be that certain enzymatic processes liberate substances in the
tissues which stimulate the growth of .the yeastlike cells or possibly antagonize
inhibitory effects of viable tissues. An additional factor may well be that the
large forms are aerophilic, reasoning from their localization a few millimeters
below the surface, where oxygen should be readily available. It is also remarkable
that the blood agar medium surrounding the tissue particle could contain great
numbers of hyphae and occasional larger cells, but the giant forms were seen
exclusively in the tissues.
These findings seem to be significant in morphologic diagnosis. In the past,
upon the assumption that II. capsulatum produces cells no larger than 1 to 5
microns, cases may have been mislabeled when larger yeastlike cells were observed. Such cases should be considered with great care before deciding the
nature of the organisms on morphologic grounds alone. This is especially important when only individual organisms are seen. As an example, we have recently observed a case of histoplasmosis with complete caseation of the adrenal,
glands, the sections of which were characterized by the presence of the classical'
intracellular organisms, measuring 1 to 3 microns. However, a large number of
GIANT FORMS OF HISTOPLASMA CAPSULATUM
903
yeastlike cells were present that were regarded by competent observers as
Blastomyces or Candida. Considering the observations presented herein, such
forms may represent / / . capsulahmi, similar to those described in the tissue
explants.
The findings suggest also that explants may provide data on growth more
quickly than can be obtained by the usual cultural methods. Tissue specimens
may be processed as whole tissue explants and sections made after 3 days. In our.
experience, organisms that can be detected only with difficulty in sections made
from promptly fixed tissue, may be found with ease after 72 hours in the explants
because of their multiplication and increase in their size. In this way, one can
reduce the interval necessary for conclusive diagnosis to about half the average
time required when ordinary cultures are used. Indeed, the culture need not be
destroyed when the explant is removed from the agar surface, and it may be
used to confirm the morphologic findings.
In view of the relative frequency of microforms of B. dermalilidis (Schwarz
and Baum 5 ), and the possibility of giant forms of H. capsulahmi, the need for
cultures, in addition to histopathologic studies, is more apparent, if one is to
make an accurate diagnosis in such instances.
SUMMARY
Large yeastlike cells, up to 20 microns in diameter, were observed in tissue
explants of organs containing Hisloplasma capsulahmi. Such cells develop rapidly
in explants and are numerous after incubation at 37 C. for 1 week. Subcultures
of such tissues reveal growth which is grossly and microscopically characteristic
of / / . capsulahmi.
The large cells seem to develop by enlargement of small (typical) yeastlike
cells; they are not formed from hyphae. Germinal tubes were observed arising
from some of the large cells.
In some instances, the large cells have a thick (double) wall, and these may be
confused with Blastomyces dermalilidis.
REFERENCES
1. CRUMKINIS, R. M., AND KESSEL, J. F.: Histoplasmosis (Darling) without splenomegaly.
Am. J. Trop. Mod., 11: 435-449, 1931.
2. DE MONBBEUN, W. A.: The cultivation and cultural characteristics of Darlings's Hisloplasma capsidalum. Am. J. Trop. Med., 14: 93-125, 1934.
3. HALEY, L. D.: Saprophytic form of Hisloplasma capsulatum in vivo. Yale J. Med., 24:
381-383, 1952.
4. HuMiMiiiEY, A. A.: Reticuloendothelial cytomycosis (histoplasmosis of Darling). Arch.
Int. Med., 65: 902-918, 1940.
5. SCHWARZ, J., AND BAUM, G. L.: Blastomycosis. Am. J. Clin. Path., 21: 999-1029, 1951.