Microorganisms in blood and tumour tissue from patients with malignancies
of breast or genital tract
Atypical microbes in blood and
cancer tissue
By Dr Erik O. H. Enby,
MD
and Dr R.S. Chouhan, MD, Göteborg 1994.
Abstract
Objectives
This study aims at looking for microbial growth in neoplasms and
blood from patients with malignancies of breast or genital tract
and to compare possible findings in both with those of healthy blood.
Method
Tissue and blood from 120 patients suffering from carcinoma of cervix
and 50 patients suffering from breast cancer and in addition blood
from 100 healthy volunteers were studied using Nomarski’s
interference contrast microscopy.
Result
Spherical soap-bubble-like forms, granules that move at great speed
as well as long slender hyphae were observed. Some formations were
suspected to be so-called L-Forms, others showed a growth-pattern
similar to that of the pathogenic actinomycetes and fungi. These
were most resistant to different kinds of chemical or physical influences.
Conclusion
Cell wall deficient, pleomorphic forms of microbes are found in
both neoplasms and blood of cancer patients. They might be involved
in the disease process.
The pictures in the article and a video
of vital cancerous tissue were demonstrated at the World Congress
in Gynecology & Obstetrics - FIGO - in Montreal, Canada in 1994.
Introduction
The answer to what causes cancer remains most elusive. Another question
that may be as complex as the cancer cause, is the cancer provocation
and incitement. Among the list of causes, microbes remain probably
the oldest, but still a highly fascinating subject for investigations.
Studies on microorganisms in blood samples from both diseased and
subjectively healthy persons have previously been reported(1,
2, 3). While many viruses have been
found to be associated with tumour growth in both animals(4)
and human beings(5, 6), bacteria
and fungi are lesser known causative agents(7, 8,
9).
In the past decade a number of new and potentially significant microbes
have been added to the list of more than 20 species inhabiting the
human body that are significant pathogens in human disease(1).
Primary microbiological analyses of cancerous human tissue have
previously been conducted by us since these types of investigations
have only been sparsely reported before(10, 11).
The use of the interference contrast microscopy technique was initiated
in 1983 by one of the authors (E. E.), and investigations were primarily
focused on vital blood samples from both control groups and patients
diagnosed with cancer or other severe illnesses(12).
Nomarski´s interference contrast microscopy has made it possible
to study the interaction of the tissue and its invaders with the
least disturbance to the system under observation. While previously
reported studies generally were concentrated on investigations of
isolates, through blood- and tissue-cultures, the present study
is focused on a direct examination of freshly obtained blood and
tissue samples.
Materials
and Methods
Peripheral blood as well as tumour tissue from 120 women suffering
from carcinoma cervix and 50 women suffering from breast cancer
were studied over a period of two years. Blood from 100 healthy
female volunteers within the age group of 35 - 55 years served as
controls.
Peripheral blood was obtained by puncturing the finger tip of a patient
using an Autolet® finger pad puncture system after taking aseptic
precautions. Strong antiseptics were avoided before pricking the finger
to avoid affecting the fragile membranes of the erythrocytes. The
drop of blood was collected on a sterile glass slide and a sterile
coverslip was placed on the drop. The sample was allowed to spread
evenly by capillary action in the space between the slide and the
coverslip, care being taken to avoid application of pressure on the
coverslip. The specimen slide was placed on the microscope and immediately
studied.
Cancer tissue was freshly obtained from patients using aspiration
cytology, biopsy and from surgical specimen. In the case of surgical
specimen, tissue was taken for the present study as soon as the
tissue was removed from the body without waiting for the surgery
to be completed. Pieces of cancer tissue were taken from the centre
of the specimen to avoid contaminating the microflora as far as
possible. The tissue was placed on a sterile, dry glass slide and
a sterile coverslip was placed on the specimen. The specimen was
carefully pressed out to form a thin layer using an extra object
slide placed on the coverslip. Leitz´® immersion oil was
applied on the edges of the cover slip to prevent drying of the
specimen. This technique enables a study of the sample for more
than 100 hours under the microscope. The dynamic processes going
on in the specimen under study can consequently be followed in real
time. The initial microscopic examination was done immediately or
within a maximum of three hours from the time of obtaining the specimen.
The Microscope used was a Leitz´ Dialux 20® equipped with
a modified UK condenser for darkfield, lightfield and interference
contrast with a Plan- Floutar- objective, a binocular phototube FSA
and a 100 watt halogen lamp. All documentation was made using a Leitz´
Vario Orthomat® automatic camera and Kodak® film. Video micrography
was done using a Panasonic CD-20® Video camera attached to the
microscope and a Panasonic video recorder. Video images were processed
on line using a “Kramer®” active composite video processor
and viewed on a Sony Trinitron® colour video monitor. All observations
were initially made at 100xmagnification with the lightfield to obtain
an overview of the specimen and then followed by the use of high magnification
1200x utilizing interference contrast.
Results
Small spherical irregular moving particles, less than 3 µm
in diameter, were observed mainly in the plasma of blood from 68
of the 100 healthy individuals (Figure
1). In blood samples from patients, however, large microbe-like
formations showing different morphological appearances were observed
in the plasma together with the red and white blood cells (Figure
2).
Large colonies of small granules with sizes ranging from 0.5 to
3 µm were also found (Figure
3). These granules are quiescent when observed immediately after
the specimen has been sampled. However, after a few hours, they
eventually begin to move and subsequently, their activity increases
dramatically. The granules can then spread out on the whole glass
slide, and finally the whole blood sample is found teeming with
these forms. In addition to this uncontrolled activity, it has also
been observed that these granules dissolve and disappear or even
change their form and consequently develop into rod and dumbbell-like
formations. Often, free granules at the edge of a colony show motion.
After several hours, these granules disperse from the outer layers
and move out among the surrounding erythrocytes (Figure
4).
When the specimen was kept under constant observation for a period
of time it was found that the granules sometimes increased immensely
in number. This could be indicated on the slide during the first
week of observation. Two very special phenomena among these granules
were noticed: a) a fusion of two granules into a new blob and b)
a sudden explosion-like disappearance of these structures. Sometimes,
the granules also developed into worm-like forms which were extremely
active. In individuals with advanced cancer, large parts of the
blood cells appeared to become totally destroyed when they interacted
substantially with the granules (Figure
5). Long slender filamentous hypha-like structures could also
be observed in some samples (Figure
6).
Spherical granules in the range of 2 to 10 µm in diameter
were observed in cancer tissue (Figure
7). They were in constant motion in the fluid, but showed restricted
mobility in the more solid parts of the specimen (Figure
8).
Microscopic studies of tissue samples from advanced cases of cancer
also indicated much larger granules, sometimes attaining comparatively
immobile giant sizes (Figure 9).
Another observation utilizing the interference contrast microscopy
was filament-like structures crossing the solid parts of the specimen
in all directions (Figure 10
and figure 11) and they appeared
to be similar to those found in blood samples. In some instances,
the granules described earlier were found along the filaments, seemingly
connected to them. However, they did not show any mobility at all.
The quantitative appearance of these observed microbial formations
could reach close to 50% of the total volume of the tumour under
investigation.
Alternate freezing to -10° C and thawing of the tissue for ten
times or more did not alter the structure, quantity, mobility or
the activity of these granules. Heating the tissue in a domestic
microwave for 10 minutes using the power of 1200 watt did not seem
to hamper their ability to oscillate. Antibiotics, antifungal and
cytotoxic agents etc. failed to restrain the activity of these organisms.
Discussion
Microbes have been observed by many researchers in tumours and in
blood as mentioned earlier in this paper. In order to research this,
the interference contrast microscopy was selected by the authors
as an appropriate technique. It also allowed the specimen to be
observed for long durations without having to stain or fix it. This
facilitates real time observations of dynamic changes without interfering
the process or the substance.
Presence of tiny globular structures of less than 3 µm in
size in the blood of normal individuals corroborates Enderlein’s(2)
observations of similar granules, which he termed “symbionts”.
The findings that they keep changing forms, from bacteria-like,
to those of fungi and highly mobile worm-like forms, indicate a
capacity to metamorphose. If observations were based on fixed and
stained samples, the possibility for each stage or form to be interpreted
as an entirely different organism remains high.
Since the days of Béchamp, polymorphism in microbes, despite
many reports still remains an unpopular theme(2).
Zopf(13) in 1892 reported that “fission fungi,
probably with some exceptions, are able to pass through different
developmental stages”. Further, Winkler(14)
in 1899 wrote that “Currently bacteriology holds the belief
that each species of bacteria has only a certain very simple form
…and that it retains this form during its only mode of reproduction,
which is by division into two…the slight change of form which
happens during growth consists essentially of elongation, or shortening,
or a local swelling. More remarkable deviations in form which are
observed frequently are considered involution or degenerative”.
In contrast Winkler found that bacteria pass through stages with markedly
different morphology.
The very few researchers who continued to work in line with the microbial
cause for cancer at the beginning of the 20th century demonstrated
that microbes had a remarkable pleomorphological tendency. Bunting(15),
studying Hodgkin’s disease, isolated a pleomorphic organism
that produced a picture of morbus Hodgkin when inoculated into animals.
Mazet(16), again studying this disease, isolated
26 strains of pleomorphic aerobes. His isolates were similar to those
reported by the present authors in that they underwent transformations
from granule stages to actinomycetes and yeast-like forms. Attempts
to address the issue of malignancies facilitating microbes or caused
by microbes were made by Diller et al.(4, 17).
They demonstrated that microbes were required for development of tumours.
Diller’s bacterium is a slow growing pleomorphic form that resembles
both Corynebacterium and Mycobacteria. Nuzum’s coccus(18)
isolated from human breast cancer is yet another example of a pleomorphic
form that causes malignancies including primary epithelioma in man.
Because of their high degree of pleomorphism and relative absence
of cell walls, the organisms described were compared with “L-Forms”
reported by Klieneberger-Nobel(19), or a cell-wall
deficient microorganism as defined by Dienes(20,
21).
The organisms reported in our paper meet most of the criteria for
atypical bacteria laid by Charache(22) in several
ways:
a) Visualization of atypical forms directly from the clinical material.
b) Failure of these forms to grow under conditions which would readily
support growth of the parent organism (since routine blood culture
reported negative, the authors consider this criterion to have been
met).
c) Ability of these organisms to survive in an osmotically controlled
medium (the forms reported here have survived more than osmotic changes).
However, while Charache’s protoplasts are reported to disintegrate
when heated, the forms reported by the authors did not exhibit a similar
character.
d) The clinical course consistent with bacteriological findings (the
generally accepted view is that microbes are not expected to be found
in the blood of patients suffering from breast cancer or cervix cancer).
In carcinoma of the breast, where the growth has ulcerated, and in
case of carcinoma cervix, tumour tissue may show bacterial contaminants.
The authors have studied the organisms reported here in patients with
different stages or severity of the disease and found a consistent
pattern that may be considered as an appropriate clinical course.
Razin(23), studying the effect of various agents
on mycoplasma, bacterial protoplasts, spheroplasts, and L-Forms
reported that the mycoplasma and L-forms were much more resistant
to lysis by osmotic shock and to alternate freezing and thawing
than protoplasts and spheroplasts. The ability to survive alternate
freezing and thawing and various conditions by the organisms reported
in this paper makes them similar to stable L-Forms.
Indications throughout the course of investigation suggest that
these microorganisms undergo a development cycle. Löhnis(24)
in 1916 observed “The development of the bacteria is characterised
not by the irregular occurrence of more or less abnormal forms but
by the regular occurrence of many different forms and stages of
growth connected with each other by constant relations”. Perhaps
the phenomenon observed by the authors might subsequently be a case
of completing cycle or only another case of metamorphoses, which
is often found in biology.
It is surprising that many of the forms observed have been reported
in various organisms. Löhnis(25) reviewed 1309
articles written between 1838 and 1918 and described many of the stages
or forms:
• Globules or yeast-like forms in Clostridium by Ghon and Sachs
and Grassberger, in V.cholerae by Maasen.
• Discharging cysts or giant round forms by Winogradsky in Nitrosomonas
and in Pneumococcus by Artigalas.
• Filaments which branch or develop buds in B. radicoila by
Conn, in E. coli by Matzuschita, in Pasteurella pestis by Albrecht
and Ghon.
• Balloon forms with rhizoid sprouting in M. leprae by Lutz,
in V. cholera by Fischer and in S. rubrum by Meirowsky.
• Slender and long filaments or actinomyces-like stages in M.
tuberculosis by Metchnikoff, in M. leprae by Meirowsky, in Pseudomonas
by Marx and Carpano and many others in many different bacteria. It
is interesting to note that this stage have been seen in numerous
species by many researchers.
By comparing the morphology and appearances of the microorganisms
in samples from controls and cancer patients we have observed that
there is a strong correlation between the morphology of the microbes
and the present stage of cancer. This connection is not only found
for different cancers or for acute infections, but it is also detected
in samples from many chronic disorders such as multiple sclerosis,
lupus, asthma etc(12, 21).
Tumours could be a circumstance of a phenomenon representing a relationship
between a growth product (the tumour), a soil (the tissue) and a
factor causing a growth process (the infection), and growth can
only continue if there is a nutritive soil in close relation to
the growth product. We can maintain that nothing can grow in itself,
as a growth-product cannot function as a nutritive soil to itself
for its own growth. That could be an axiom. As growth throughout
nature always reduces the nourishment contents of a soil towards
poverty, the infectious growth process will gradually change and
impoverish the tissues entirely according to the dominance of a
growth process over a soil. Consequently, the capacity of a neoplasm
to grow would mean that it is something distinctly different from
the tissues within which it is growing. From a conceptual point
of view it would also be impossible to defend the opinion that it
is emanating from normal tissue cells that suddenly changed their
behaviour, since the concept of growth implies a soil, which is
something quite different from the growth product. The fact that
plenty of granular forms are observed in tumours and great colonies
of the same type in blood, allows for an assumption that the tumours
represent only a fraction of the whole disease process in the body.
Budding and multiplying locally, these granules may eventually create
tumours in and at the expense of a tissue as substrate, changing
its microstructure and metabolism and destroying it mechanically.
The tumours may then be a nodal point from where dissemination of
infectious granules out into the bodily fluids takes place, totally
in accordance with the fact that spreading implies a focus.
Patients with growth processes in their tissues similar to those described
in this paper, will often develop tumours, which then will become
a very obvious part of their symptom pictures. Surgical excision or
radiation here may be an equivalent of local treatment of a focus
of infection, thereby providing an explanation for the possibility
of recurrence. Chemotherapy, in spite of acting on the body as a whole,
may at times be ineffective. The microbe-like formations described
above might help to explain this because the initiation and continuation
of the malignant disease process might be due not only to so-called
cancer cells of eukaryotic origin, where chemotherapy is the therapy
of choice, but also to prokaryotic microbial activity, where this
therapy is less or not at all effective.
Since the authors are clinicians more than microbiologists they
wondered throughout the study as to why there is such a sparsity
of reports and information in current textbooks in microbiology
on pleomorphism and observations and findings such as those described
above. Perhaps Löhnis’ comment that ”standard
textbooks contain photographs of the variants, never noticed because
our eyes have been trained so very well not to see them”
is more true now than it was in 1921. Much needs to be done in evaluating
and analyzing further the behaviour, culturability and the biochemical
and immunological response of the forms observed. However, the authors
believe that, if brought to notice, more results can be obtained
from different centres, paving the way for a better understanding
of the scourge that is cancer.
Acknowledgement
Thanks are due to Q. Ingemar Ljungquist, to Dr. Lennart Sjölin,
CTH/GU, Gothenburg, for valuable help in preparing the manuscript,
to Tord Wallin of Cybex AB for his timely help with computers and
to Monica Bryant for her editing contributions.
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