CANDIDIASIS AND CANDIDEMIA
By Philip Paul Hoekstra, III

ABSTRACT: Systemic distribution of the ubiquitous enteric fungus, Candida albicans, may require less opportunism than had previously been considered. High enteric levels of Candida albicans have been demonstrated to produce significant spill-over into the host's peripheral circulation. It has also been established that Candida albicans can shed its characteristic cell wall and may also vary its cell-surface immunogens. Such abilities may enable this microbe to camouflage its identity from the host's immunity and permit proliferation into the systemic circulation as non-transients.

By means of high-resolution optical microscopy, I have consistently observed significant numbers of an unfamiliar structure in vital peripheral blood specimens from candidiasis patients. The observed morphologic changes and evidence from nucleic acid co ntent, auto-fluorescence, and most specifically, immuno-fluorescence monoclonal antibody techniques have indicated the identity of these observed structures to be Candida albicans, existing as blastospores and cell-wall deficient colonies. It is surmise d that proliferation into peripheral circulation of this microbe, especially as a cell-wall deficient, accompanies enteric over-growth. Further, it is speculated that this systemic distribution may be augmented by the action of polyene antibiotics and i nnate enzyme activity.

The unicellular fungus, Candida albicans is probably among the first non-viral microorganisms an individual encounters, usually at birth, establishing a lifelong relationship as one of the most uniformly occurring of the common bowel flora. The natural proliferative and disruptive tendencies of any intimate microbe are countered by such host mechanisms as will serve to limit the scope and numbers of the microorganism. The interaction between Candida albicans and the human host requires an array of prot ective mechanisms to maintain a relatively benign steady state, termed commensalism. These mechanisms will be of various specifications and effectiveness as part of a generalized defensive strategy against microbial infection. Physical barrier, such a s mucosal membranes, and biochemical schemes, such as low gastric pH, proteolytic pancreatic enzymes, low redox potential, elevated body temperature, and inhibitory substances produced by competitive bowel flora provide considerable obstacles to enteric infection by fungi. Despite the assumed effectiveness of these physical and biochemical protective systems, a study by Krause and Matheis (2) demonstrated significant numbers of Candida albicans in the blood and urine of healthy human subjects, 2 hrs and 3 hrs respectively, after oral administration of this fungal microorganism. The successful introduction of this microbe into these intimate media testifies to the incomplete defense provided by exclusion mechanism alone. However, the blood and urine of these subjects were essentially cleared of Candida albicans within several hours and it was noted that these subjects did not experience any prolonged deleterious symptoms as a result of this ordeal (2).

Innate human immunity is, generally, potent in the ability to prevent infection by Candida albicans in its classical forms (3) as a blastospore or mycelia. In healthy patients, an effective response to the standard cell-surface antigens of Candida albic ans provides a sort of a benchmark by which to assess the status of a cellular-based immune competence (4). The rapid clearance of the microbe may indicate the effectiveness of this immunity in the healthy individual.

IMMUNE SUPPRESSION This fungal microorganism has demonstrated the capacity to invade many intimate body regions and has been associated with such disorders as endocarditis, pyelonephritis, and meningitis (5, 6). The limited pathogenic potential of Candi da Albicans may be realized as an opportunist contingent upon the loss of host defenses, specific as well as nonspecific (7). The manifestations and consequences of such an opportunistic overgrowth and dissemination of this microorganism will be largely dependent on the degree of the host's debilitation. Administration of therapeutic levels of common antibiotics will often result in overgrowth of Candida albicans in the bowel (8), probably due to susceptibility of competitive bacterial species (9). Th e study by Krause and Matheis indicated the probability of a significant spill-over of Candida albicans into the peripheral circulation blood in instances of enteric overgrowth (2). In addition, the short half life of the individual microbe in the perip heral circulation of the immune competent host is likely the consequence of cell-mediated immune activity. The determination of significant numbers of Candida albicans in the host's systemic circulation, not as transients, but rather as agents of infect ion is commonly thought to herald a significant loss of cell-mediated immunity (5). Such a systemic infection of this fungus is often encountered as the consequence of cytotoxic drug administration for malignancy. The lack of an effective response agai nst the characteristic cell-surface antigens of Candida albicans in its classical form may also indicate a profound state of immune paralysis (10). The ability of any microorganism to elicit a host's specific defense largely depends on the immunogenic p roperties of microbe's cell surface (11). Fungal cells surface characteristics are generally described as poorly immunogenic (3, 13). Perhaps this is attributable to an immunologic similarity with some of the host's endogenous cell-surface characterist ics(13).

Both standard laboratory strains of Candida albicans and those isolated from human subject patients have demonstrated an extraordinary variability of phenotypic properties within single strains (14). This variability occurred at a frequency considered t oo great (1.3 x 10X4) to attribute to genetic mutation and was speculated to represent a polygenetic switching (13). Cloned colonies produced reversible changes in such basic properties as macromorphology, fermentation and assimilation of sugars, the ab ility to reduce tetrazolium salt, and the tendency to produce mycelia (15). This capacity for extreme and reversible phenotypic variability may present the host with a diversity of cell surface immunogenic characteristics (16). Such an ability has been established in such pervasive and successful human pathogens as Salmonella and the trypanosomas (14) and may, thus, enable Candida albicans to evade much of the host's more specific immune mechanisms.

Unlike animal cells, bacteria and fungi are encompassed by a rather rigid cell wall, which provides protection from environmental extremes and a physical definition against as osmotic gradient as great as 15,200 Torr (17). A variety of factors inhospita ble to growth of the classical microorganisms may induce a variant, resistive state deficient in the cell wall (18). This altered state can be reliably induced by the action of therapeutic levels of many common antibiotics, especially those that interfe re with cell wall synthesis (18). The action of lysozyme, often associated with the phagocytic activity of cell-mediated immune response, is also known to induce cell-wall deficiency in Candida albicans and many other microorganisms (19). Shed of the c ell wall, microorganisms are known to remain viable and often have an enhanced dissemination compared to the parent forms (19). Cell-wall deficient (CWD) Candida albicans demonstrated the ability to pass 0.2 mm pore size filters (19). Such atypical mic robial growth, reported as early as 1925 (20), were, at one time, considered limited in medical relevance and thought mere laboratory curiosities. Considerable technical and procedural difficulties combined with a generic diminution of pathogenesis has resulted in a historic lack of appreciation of the widespread clinical implications of CWD forms (19, 21) .

The immunogenic characterization specific to species, indeed, a strain of bacteria or fungi is largely determined by the cell-surface antigenic composition of the cell wall (11, 17, 22). The cell-surface characteristics of a CWD microorganism are, typic ally, quite different and variable from those of classical forms (23). It is more likely, then, that innate immunological response against any classical microbial form would have significantly diminished effectiveness against a CWD variant. The associa tion of CWD microorganisms with significant human pathologies has been established in many diseases (24). Mattman has demonstrated growth of the pertinent organism as CWD forms in every instance of significant internal infection (25). Candida albicans and various Streptococcus, Proteus, and Clostridium species have frequently been demonstrated as CWD of pathologic potential (26, 27, 28, 29, 30). Nativelle and Deparis reported growth of CWD forms three times more frequently than classical microorganis ms from blood cultures of randomly selected hospital patients (31). Mattman et al., using a nonspecific technique, cultured for CWD forms in the peripheral blood specimens from noninfectious hospital patient admissions and determined microbial growth in 39%; 72% of that growth being CWD (25). From 67 randomly selected necropsies performed on individuals with no evidence of active infection, Doland and his co-workers successfully cultured growth of CWD bacteria and/or fungi, principally Candida albican s, from 58% of the 211 specimens (32). Doland et al. concluded such microorganisms to represent normal flora of the intestinal organs. Meinecke observed the multiple variations of CWD Candida albicans to resemble platelets, erythrocytes, or even leukoc ytes (33). Meinecke speculated CWD Candida albicans may appear frequently in vivo and be misidentified as endogenous cells of the host (33).

C.O. Truss described a clinical syndrome that is characterized by disorders involving the gastrointestinal tract and the nervous and reproductive systems (34). These disorders include symptoms of abdominal bloating, intestinal gas, indigestion, constipa tion or diarrhea, chemical sensitivities, food allergies, hypoglycemia, premenstrual tension, endometriosis, prostatitis, vaginitis, chronic cutaneous fungal infections including dermatophytids, inability to concentrate, frequent mood shifts, degraded me mory, fatigue/lethargy, depression, and desire for refined carbohydrates or yeast containing foods (34). Truss noted the medical histories of these patients often included frequent or chronic administration of broad-spectrum antibiotics, especially tetr acycline, corticosteroids, or progesterones (8, 24). Truss observed frequent amelioration of symptomatology by antimycotic treatment and attributed this syndrome to the toxic consequences secondary to enteric overgrowth of Candida albicans. Truss terme d this syndrome chronic candidiasis though the symptoms were clearly more systemic than mucocutaneous thrush and yet, these patients were not typically described as severely immune deficient. Objective determination as to the causation of this syndrome as more than a coincidental occurrence of these disorders has been elusive. The essentially universal occurrence of Candida albicans as a human enteric microbe and the casual role of humoral immunity as part of a specific defense from infection by this organism probably account for the reported unreliability of antibody analysis as an indicator of infection (35). Routine blood cultures and analysis for standard cell-wall immunogens of Candida albicans are reported typically negative and similarly unre liable as indicators of this syndrome (34). The validity of Candida albicans as fundamental to this disorder has been questioned as evidence of fungal overgrowth in the blood has been lacking. The polysystemic symptom pattern described in some patients by Truss does not seem to be entirely explained by the gastrointestinal overgrowth of Candida albicans, especially when the organism is found in almost all stool cultures.

The frequent symptomatic relief attained with antimycotic therapies and the rapid return of symptoms reported in many patients after discontinuation of the polyene antibiotics intrigued me sufficiently to investigate this syndrome for the elusive role hy pothesized of Candida albicans.

I chose to employ high-resolution optical microscopy using dark-field illumination to realize a high-contrast and richly detailed imagery free of artifact from vital preparations of patient's blood. I observed distinct vesicles approximately 2-4 mm in d iameter, often seen budding. I also noted a considerable number of structures apparently consisting of aggregated bodies. I considered these structures grossly similar to aggregated thrombocytes. However, in observing the fine detail and dynamic chang es, I discerned notable differences in these structures from aggregated thrombocytes. The individual bodies comprising these unfamiliar structures seemed less discrete and were observed to form complete vesicles that would, in turn, later seem to bud or form tube-like projections as great as 60 mm in length.

The possibility of these budding vesicles being fungal blastospores and the aggregate structures being CWD microorganism became increasingly intriguing. This possibility then prompted a project to attempt an identification of these structures and offer ed the ability to objectively characterize the candidiasis syndrome by their occurrence. Specimens of the peripheral blood of one thousand (1000) subjects determined as candidiasis patients by experienced clinical evaluation of symptoms were analyzed mi croscopically employing the single blind method. Concentrations of the vesicular and aggregate body forms were localized at 500X magnification. Fine detail of these structures and dynamic changes were then studies at 1250X magnification (36). Discernm ent of these structures was based on morphologic recognition and dynamic changes as characterized in the initial study. Three direct blood smears were made at the same time for these patients. One of these smears was prepared with the vital stain acrid ine orange for nucleic acid characterization (37). A second smear was left unstained, but studies for fluorescent phenomenon (26, 38). The third smear was stained with fluorescent tagged anti-candida antibody if the vital preparation demonstrated a gre at number of the vesicular or aggregate body structures(19, 37). Two hundred (200) replicates were made as standards for each staining technique except the immuno-fluorescent technique where only thirty-six were made. Two hundred and fifty additional s ubjects were also included in this investigation. These individuals were clinically characterized as free from the candidiasis syndrome but, often with other health disorders.
STATISTICAL ANALYSIS

The study by Krause and Matheis (2) indicates a significant enteric overgrowth of Candida albicans would probably result in a discernible spill-over of the blastospore into the peripheral blood (2). The observed budding vesicles of 2 to 4 mm diameter is consistent with the fungal organism (39). The observed production of mycelia of 1 to 1.5 mm diameter and up to 60 mm length would indicate the Reynolds-Braude Phenomenon that is considered reliably characteristic of Candida albicans (40). Various Myco bacterium species are known to also produce mycelia, however, the diameter of mycobacterial mycelia is considerably less than that observed in this study (41). Krause and Matheis study also indicated the transitory nature of Candida albicans blastospore s in the immune competent individual (2). Truss and the physicians that participated in this project often regard candidiasis patients as immunologically diminished buy, yet functionally competent (34).

The second structure though composed of aggregate bodies is considered by Mattman morphologically similar to know aggregates of CWD microorganisms. Both structures were observed to absorb the acridine orange vital stain and fluoresce a fiery orange indi cating abundant RNA content (26), dissimilar to thrombocytes. Both structures also retained the fluorescein tagged anti-candida antibody characterizing Candida albicans (8). The second structure was also observed to fluoresce a blue-green, characterist ic of CWD Candida albicans (26). The second structure was also consistently indistinct in morphology through all the techniques employed. The relatively rapid rate of vesicle formation, especially as seen in the most symptomatic patients is characteris tic of a spheroplast (42), a CWD form with incomplete amount of cell wall retained by the organism. I observed this second structure, indicated, then as CWD colonies of Candida albicans, as the most consistently characterizing factor discernible from th ese symptomatic individuals. This structure, may represent a non-transient population of Candida albicans at a previously unsuspected level of intimacy. The systemic nature of such a relationship could evade the directed immunologic defenses of an immu ne competent host due to the variable and uncharacteristic cell-surface properties of a CWD form of Candida albicans. The presence of CWD Candida albicans in the peripheral blood may not be as ominous or symptomatic as might be thought because of the re latively benign nature of this anthropophilic parasite and, perhaps more importantly, a diminished aggressiveness characteristic of most CWD forms.

IMMUNE SUPPRESSIVE NATURE OF CANDIDA

The presence of CWD Candida albicans in the peripheral blood of patients with significant enteric overgrowths, especially when administering polyene antibiotics may not be surprising in consideration of Mattman's report of CWD growth of the variant micro be in every instance of significant internal infection (25). In hopes of employing these observations is a reliable method of objectifying candidiasis, a clinical program is being established to correlate vital microscopic analysis with patient history, symptomatology, and efficacy of antimycotic therapy.

Dedicated to the boundless scientific resource I have found in Lida H. Mattman, the unfailing support of my Father, and the cooperation of my colleagues and patients in realizing this project. This research has been supported, in part, by LIVCELL ANALY SIS INC. and personal appreciation is extended to Jeffrey Katke, William Shaddle, and the many other people of LIVCELL dedicated to providing state-of-the-art technologic health assessment at a practical level. To all these people and others unmentioned , my humble thanks and appreciation for making this work possible.
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(C) Copyright 1984 by Philip Hoekstra III. All Rights Reserved. This article, nor any part of this article may be reproduced in any written, electronic, or verbal or any other manner whatsoever without the expressed, prior, and written approval of the author.