New posology of potassium iodide for the treatment of cutaneous sporotrichosis: study of efficacy and safety in 102 patients.

BACKGROUND: The first therapeutic choice for the treatment of cutaneous sporotrichosis is oral itraconazole; however, the increase in cases of zoonotic transmission outbreak necessitates a search for effective and safe treatment alternatives. OBJECTIVE: To evaluate a new potassium iodide (KI) posology as an alternative for the treatment of limited cutaneous forms of sporotrichosis. METHODS: One hundred and two patients with sporotrichosis diagnosed by isolation of Sporothrix sp. were included and were divided into 2 groups that received different doses of KI: group A received the conventional dose, and group B received the reduced dose. The cure criteria were based on clinical and serological data. RESULTS: Seventy-nine patients (77.4%) reached clinical cure: 70.6% and 84.3% of groups A and B respectively. Sixteen patients (15.6%) were lost during follow-up, and seven changed drug therapy: five in group A and two in group B. The incidence of adverse events was similar for both groups (64.7%): predominantly metallic taste (44%), followed by mild gastrointestinal intolerance and acneiform eruption (10.7% each). No serious adverse events occurred, and there were no recurrences. Analysis of the results showed no statistically significant difference between groups (P = 0.9255). The improvement in serologic titres was significant in both treatment groups. CONCLUSION: Through statistical analysis, the usual posology was not shown to be superior to the one proposed in this study. Serology for sporotrichosis may be used as a valuable tool in the clinical monitoring of these patients.

Glycogen granules in resting and inflammatory rainbow trout phagocytes--an ultrastructural study.

The ultrastructural image of glycogen granules in the cytoplasm of rainbow trout phagocytes in sections stained by the conventional lead or uranyl-lead stains is highly dependent on fixation conditions, the granules being visible only when adequate fixation protocols are used. Morphometry of samples processed for the detection of peroxidase or esterase activities (to specifically label neutrophils and macrophages, respectively), and simultaneously stained for the specific detection of glycogen, showed that inflammatory peritoneal neutrophils were richer in glycogen granules than resting neutrophils. This increase in glycogen content occurs after the migration from the haematopoietic tissues and peripheral blood to the inflamed foci. Glycogen granules could not be found in resting peritoneal macrophages but were found in inflammatory macrophages. The macrophage granules occurred in smaller amounts than in neutrophils, and consisted of granules identical to those of neutrophils together with significantly smaller granules. No evidence for the utilization of glycogen by neutrophils phagocytosing bacteria within the peritoneal cavity was found.

Acquisition of autoimmunity genes by New Zealand mice is associated with natural resistance to infection by mycobacteria.

New Zealand (NZ) mouse strains comprise both autoimmune and non-autoimmune animals: NZ black (NZB) mice and the F1 hybrid (NZB/W) of NZB and NZ white (NZW) mice show spontaneous autoimmune disease by 6 months of age and die before the first year of age from renal disease, while NZW mice do not show autoimmune disorders. We investigated whether the autoimmunity-prone NZ animals (NZB and NZB/W) differ from the non-autoimmune NZW mice in susceptibility/resistance to mycobacterial infection. The three groups of NZ mice were infected by intraperitoneal inoculation of 10(8) colony forming units (cfu) of Mycobacterium avium. The M. avium infection was induced in 3-month-old mice (i.e., before NZB and NZB/W mice develop autoimmune disease) and studied for 4 months. Infected NZB and NZB/W mice showed evidence of renal disease at 2 and 4 months of infection (but not at 1 month). The non-autoimmune NZW mice were found to be susceptible to M. avium since they allowed massive proliferation (4-5 log growth) of the bacilli in liver and spleen. In contrast, both groups of autoimmunity-prone mice (NZB and NZB/W) were resistant to M. avium since their mycobacterial loads remained below the value of the initial inoculum. We conclude that in NZ mice the acquisition of autoimmunity genes is associated with expression of natural resistance to mycobacterial infection. This is consistent with the view that autoimmunity genes may have been evolutionarily selected because of their association with increased resistance of the host to infections by intracellular parasites.

Live but not heat-killed mycobacteria cause rapid chemotaxis of large numbers of eosinophils in vivo and are ingested by the attracted granulocytes.

We studied leukocyte chemotaxis triggered by a local injection of mycobacteria (Mycobacterium avium and M. smegmatis) in BALB/c and C57BL/6 mice. Our experimental model consisted of the induction of a subcutaneous air pouch in the dorsal area of mice and inoculation 6 days later of 10(8) CFU of myocobacteria. Inflammatory exudates were harvested from the air pouch cavities 15, 30, and 45 min after the injection of the inocula. Injection of the microorganisms resulted in the migration of an elevated number of eosinophilic granulocytes into the inflammatory cavities. At 30 min after the inoculation of the mycobacteria, the air pouches contained between (3.9 +/- 0.3) x 10(5) (M. avium) and (3.3 +/- 0.3) x 10(5) (M. smegmatis) eosinophils, corresponding to more than one-third (41.4 to 38.3%) of the leukocytes present in the inflammatory cavities. Less than one-half of the eosinophils were attracted to the air pouches when the same number of heat-killed mycobacteria were inoculated [(1.3 +/- 0.2) x 10(5) cells for M. avium and (1.5 +/- 0.2) x 10(5) cells for M. smegmatis]. Injection of gram-negative bacteria (Escherichia coli), of latex beads, or of casein resulted in the attraction of inflammatory eosinophils in numbers that were comparable to those attracted by the heat-killed mycobacteria. Our data document the fact that live mycobacteria exert a rapid chemotactic effect on eosinophils. We therefore postulate that mycobacteria either contain or induce the production of an eosinophilotactic factor. Because this chemotactic effect occurs during the acute inflammatory response to mycobacteria, it cannot be due to the formation of immune complexes (a major infection-associated chemotactic factor for eosinophils). The attracted eosinophils had an important role in the local phagocytosis of mycobacteria, as indicated by our finding, derived from thin-section electron microscopy quantifications, that at 30 min after M. avium inoculation the inflammatory exudates contained (2.2 +/- 0.5) x 10(5) mycobacterium-bearing eosinophils (corresponding to 57% of the total eosinophils), as compared with (2.1 +/- 0.1) x 10(5) neutrophils and (1.5 +/- 0.2) x 10(5) macrophages with ingested bacilli. We conclude that mycobacteria induce the attraction of eosinophils to inflammatory sites and that these granulocytes have the capacity to phagocytize these bacilli in situ.

New data on the ultrastructure of the membrane of Mycobacterium leprae.

In previous reports on the ultrastructure of Mycobacterium leprae, we described the occurrence of symmetric membranes in normal-looking bacilli from fresh or frozen samples primarily fixed with aldehydes. In those reports we admitted that such a symmetric profile, which is not found in the other normal mycobacteria, would not represent the structure of the normal membrane of the leprosy bacillus. We, therefore, re-analyzed the ultrastructure of the membrane of M. leprae. In the present work the micromorphology of the M. leprae membrane was studied by transmission electronmicroscopy after the fixation of fresh samples by OsO4 plus calcium followed by glutaraldehyde plus formaldehyde and calcium followed by uranyl acetate. The study of samples from two patients with lepromatous (LL) leprosy, three armadillos with natural leprosy, and one nude mouse with experimental leprosy showed that normal-looking bacilli present in lead-stained sections had asymmetric membranes with a thickness of 6.49 +/- 0.36 nm. These membranes showed periodic acid-Schiff (PAS)-positive components exclusively located in the outer half of the bilayer. We demonstrated that the symmetric profile of the M. leprae membrane described in our previous reports corresponds, as admitted in those reports, to an abnormal membrane structure. Such an abnormality was now found to result from the use of primary fixation with aldehydes or of samples stored frozen before fixation. These results indicate that, although ultrastructurally similar to that of the other mycobacteria, the membrane of M. leprae has a peculiar sensitivity to fixation by aldehydes. Such a characteristic, which was not found in M. lepraemurium, M. aurum, M. avium, and M. tuberculosis H37Ra, must reflect a unique membrane molecular structure, which is presently unknown.

Effects of freezing and thawing on the viability and the ultrastructure of in vivo grown mycobacteria.

The influence of different frequencies of freezing-thawing cycles on the viability of in vivo grown mycobacteria was investigated. Pieces of armadillo tissues naturally or experimentally infected with Mycobacterium leprae were analyzed. The viability of M. leprae was determined by mouse foot pad titration. The viability of cultivable mycobacteria, sometimes present in armadillo tissues, was determined by culture. Electron-microscopic studies were performed on fresh or frozen-thawed armadillo tissues with natural leprosy and on livers of C57BL/6 mice experimentally infected with M. avium or M. lepraemurium. We found that the percentage of viable M. leprae bacilli is identical for naturally infected and experimentally infected tissues, frozen and thawed once. When the tissues were subjected to a second freezing-thawing cycle, a considerable loss of viability was observed (65%-97%). A third freezing-thawing cycle was lethal for most of the M. leprae cells, and after four freezing-thawing cycles no viable bacilli were found. The cultivable mycobacteria present in some armadillo tissues were found to be more resistant than M. leprae to freezing-thawing since these mycobacteria could still be cultivated after four freezing-thawing cycles. The results of the electron-microscopy study support the conclusion that M. leprae is more sensitive to freezing-thawing than the cultivable mycobacteria and show that the cytoplasmic membrane appears to be the target for the lethal action of freezing-thawing on mycobacterial cells. These results emphasize the importance of avoiding repeated thawing and refreezing of M. leprae-infected tissues when viable M. leprae cells need to be studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved Thiéry staining for the ultrastructural detection of polysaccharides.

The use of silver vitellinate instead of silver proteinate in the Thiéry's staining for the ultrastructural detection of polysaccharides has advantages which derive from the lesser reactivity and higher stability of silver vitellinate solutions, allowing the obtention of a finer staining of periodic acid-Schiff (PAS)-positive materials with a cleaner background. The proposed method is particularly useful for the staining of PAS-positive cellular components at high resolution.

In vivo killing and degradation of Mycobacterium aurum within mouse peritoneal macrophages.

We studied the in vivo killing and degradation of Mycobacterium aurum, a nonpathogenic, acid-fast bacillus, within macrophages after inoculation into the peritoneal cavity of CD-1 mice. The degradative process could be divided in five successive steps that were characterized on ultrastructural and cytochemical grounds and the relative contributions of which were determined by quantitative electron microscopy of samples taken at different times. The main ultrastructural alterations observed during the degradative process were ribosome disaggregation, coagulation of the cytoplasmic matrix, and change in the membrane profile from asymmetric to symmetric, with loss of the polysaccharide components from the outer layer, followed by membrane solubilization and intracellular clearing, followed by digestion of the innermost (peptidoglycan) layer of the cell wall, and at the end of the process, disorganization and collapse of the remaining layers of the cell wall. The correlation between viability and morphology indicated that the first ultrastructural signs of viability loss are cytoplasmic coagulation, change in the membrane geometry, and disappearance of ribosomes. The labeling of lysosomes of peritoneal macrophages with ferritin or by the cytochemical demonstration of inorganic trimetaphosphatase showed that fusion of lysosomes with phagosomes containing mycobacteria occurs in the phagocytes in the mouse peritoneal cavity and is already extensive as soon as 1 h after the inoculation of the bacilli.

Ultrastructure of bacilli and the bacillary origin of the macrophagic inclusions in Whipple's disease.

An electron microscopic and cytochemical study of the Whipple bacillus in jejunal biopsies from three untreated patients was made using fixation procedures developed for the satisfactory preservation of bacterial ultrastructure. The envelopes of the normal-looking bacilli present free in the lamina propria consisted of the following layers. (i) A cytoplasmic membrane with a triple-layered profile and a mean thickness (peak-to-peak distance) of 6.08 nm. (ii) A thick (20 nm) cell wall containing peptidoglycan; the wall had a hitherto undescribed inner layer that contained polysaccharides, possibly teichoic acids. (iii) Surrounding the cell wall, a surface membrane with a symmetric profile and a mean peak-to-peak distance of 4.74 nm. The ultrastructural pattern of the Whipple bacillus wall corresponds to that of Gram-positive bacteria, but with an additional surface membrane. This membrane is different from the outer membrane of Gram-negative bacteria because it has a symmetric profile, is thinner and has no periodic acid-Schiff (PAS)-positive components. Normal-looking bacilli were seen very rarely inside jejunal macrophages, but degenerating bacteria were abundant in these phagocytes. Electron microscopy and ultrastructural cytochemistry of Whipple bacilli inside jejunal macrophages of the three untreated patients showed that the degenerative process is a sequence that leads to the loss of bacillary forms and to the accumulation of bacterial remnants resistant to degradation by the macrophage. These remnants correspond to the innermost, polysaccharide-containing portion of the bacillus wall. The progressive accumulation of these PAS-positive wall remnants is the origin of the intramacrophagic inclusions that are important in the histological diagnosis of Whipple's disease. The reported results indicate that in the three patients studied, the Whipple bacillus multiplies extracellularly, the bacteria that are phagocytosed by macrophages being degraded.

Correlation viability/morphology in Mycobacterium leprae.

The present study regards the correlation between the percent of viable M. leprae (as determined by the mouse foot pad technique) and the quantitative ultrastructural analysis of M. leprae cells in 6 armadillo's samples and 1 nude mouse foot pad. The quantitative ultrastructural study of 3 LL patients and 1 M. leprae-infected nude mouse was correlated to the Morphological Index. The results show that most M. leprae cells with continuous undeformed cell walls, continuous symmetric membranes, ribosomes and fibrilar nucleoids are viable bacilli. Some cells with the above ultrastructural pattern may be dead bacilli that did not yet enter the macrophage-induced degradative process that results in the disposal of the bacteria. Our results also show that degenerating M. leprae cells largely predominate in most samples studied. This means that, even in the absence of anti-leprosy treatment, dead M. leprae cells accumulate in the host's tissues. This point has to be taken into account in the calculation of the generation time of M. leprae in vivo, the dynamics of the leprosy bacillus in susceptible hosts being influenced by the simultaneous occurrence of growth, death and degradation. Since known facts in regards to the physiology of bacterial membranes make it difficult to accept the PAS-symmetric membrane of viable M. leprae as the membrane of growing bacilli, a search of M. leprae cells with asymmetric membranes was undertaken in appropriate samples from nude mice. Several M. leprae cells with normal ultrastructure and Thiéry-asymmetric membranes were found in the foot pads of one mouse. Although this observation must be confirmed in another sample, it suggests that M. leprae would not be an exception to the general concept that the membranes of all growing Gram-positive bacteria have PAS-positive components located only in the outer layer. The M. leprae cells that have normal ultrastructure and symmetric membranes and that are viable would represent some sort of resting cells, that is, living but not growing bacteria. All the, 11, 263 individual bacillary profiles scored in the ultrastructural study included in the present study exhibited the micromorphological characteristics of acid-fast bacteria.

Ultrastructural characterization of normal and damaged membranes of Mycobacterium leprae and of cultivable mycobacteria.

Microdensitometry showed that the membrane profiles of normal cultivable mycobacteria were very asymmetric (outer layer denser and thicker than the inner layer), while the profiles of normal-looking M. leprae in lepromatous patients, in experimentally infected armadillos and in nude mice were approximately symmetric; moreover, the membrane of M. leprae was thicker than that of cultivable species. Using two cytochemical methods for the ultrastructural detection of periodic acid-Schiff (PAS)-positive molecules (the Thiéry procedure, and staining with phosphotungstic acid at low pH) we found that the membrane of cultivable mycobacteria, growing in vitro or in vivo, had PAS-positive components exclusively in the outer layer, while the normal-looking M. leprae in patients and in armadillos had membranes with PAS-positive components in both layers. The membranes of damaged cultivable mycobacteria, in vivo or in vitro, and of damaged M. leprae, in patients or armadillos, were PAS-negative.

Electron microscopic study of Mycobacterium leprae membrane.

We report the results of the study by transmission electron microscopy of normal Mycobacterium leprae in the tissues of experimentally infected armadillos. Several fixation procedures were used and compared to those previously employed in the study of M. leprae in lepromatous leprosy patients. The results show that the ultrastructure of M. leprae is identical in both hosts. The demonstration of a symmetric membrane profile in M. leprae in armadillos confirms our previous results. This characteristic of the M. leprae membrane is peculiar in that it is not shared by any of the easily cultivable species of mycobacteria we have studied so far.

The interpretation of the ultrastructure of mycobacterial cells in transmission electron microscopy of ultrathin sections.

In the present report the authors discuss several aspects of the ultrastructure of mycobacterial cells as seen by transmission electron microscopy of ultrathin sections that are relevant in the characterization of normal versus altered bacteria. The importance of the use of adequate fixation conditions is stressed and illustrated with examples showing that normal, but inadequately fixed, mycobacterial cells may exhibit micromorphological alterations similar to those typical of cells affected in several situations such as autolysis, heterolysis, and antibacterial treatments.

Ultrastructural alterations of mycobacterium leprae in skin biopsies of untreated and treated lepromatous patients.

1. -- Mycobacterium leprae cells under a process of progressive disaggregation are present in the skin of both treated and untreated patients. 2. -- The ultrastructural alterations observed during the degenerative process seem to be qualitatively similar in treated and untreated patients. 3. -- The proportion of altered M. leprae cells increases during the treatment, mainly with rifampicin and rifampicin + clofazimine + diaminodiphenyl sulfone. 4. -- The cell wall of M. leprae is the last bacterial structure to disappear during the degenerative process.

Ultrastructure of Mycobacterium leprae and other acid-fast bacteria as influenced by fixation conditions.

A procedure using aldehydes, OsO4, Ca++ and uranyl acetate was selected for study of the fixation of Mycobacterium leprae in skin biopsies of leprosy patients. The ultrastructural pattern of recognized normal M. leprae cells fixed by the above procedure was characterized, and was found to be similar to that of other acid-fast bacteria fixed by the same procedure, except for the geometry of the membrane profile. Under such fixation conditions, this profile is always asymmetric in in vitro-cultured normal Nocardia asteroides, M. aurum and M. tuberculosis, whereas in skin biopsies no M. leprae cells with asymmetric membranes have been found so far. The implications of this observation for the interpretation of the ultrastructure of damaged M. leprae cells found in skin biopsies are discussed.

Effects of local anesthetics on bacterial cells.

The membrane effects of chlorpromazine, nupercain, tetracain, and procain were studied using Bacillus cereus, B. megaterium, B. subtilis, and Streptococcus faecalis, protoplasts from S. faecalis, and isolated membranes from B. subtilis. Chlorpromazin, nupercain, and tetracain produced characteristic micromorphological alterations after treatment for 5 to 30 min at pH 7.0 and 20 degrees C; the membrane staining pattern changed from asymmetric to symmetric, complex mesosome-like structures appeared, and membrane fractures and solubilization occurred. Procain at concentrations up to 100 mM did not induce detectable alterations. Protoplasts were quickly lysed by 10 mM tetracain. A rapid and extensive leakage of K+ was induced by chlorpromazin, nupercain, and tetracain. Procain (100 mM) induced a slight K+ leakage. The membrane respiratory activity of intact B. cereus cells (as measured by the triphenyl tetrazolium reduction) and the succinic dehydrogenase activity of B. subtilis isolated membranes were found to be inhibited by the four local anesthetics. The concentrations that produced 50% inhibition of those activities are correlated with the hydrophobicities of the anesthetic molecules.