Numbers Of Acid-fast Bacilli Research Articles

Inhibition of the polymerase chain reaction by sputum samples from tuberculosis patients after processing using a silica-guanidiniumthiocyanate DNA isolation procedure.

With the objective to evaluate PCR-mediated detection of Mycobacterium tuberculosis DNA as a diagnostic procedure for diagnosis of tuberculosis in individuals attending ambulatory services in Primary Health Units of the City Tuberculosis Program in Rio de Janeiro, Brazil, their sputum samples were collected and treated with a DNA extraction procedure using silica-guanidiniumthiocyanate. This procedure has been described to be highly efficient for extraction of different kind of nucleic acids from bacteria and clinical samples. Upon comparing PCR results with the number of acid-fast bacilli, no direct relation was observed between the number of bacilli present in the sample and PCR positivity. Part of the processed samples was therefore spiked with pure DNA of M. tuberculosis and inhibition of the PCR reaction was verified in 22 out of 36 (61%) of the samples, demonstrating that the extraction procedure as originally described should not be used for PCR analysis of sputum samples.

Fine-needle aspiration cytology of postvaccinial disseminated bacillus Calmette-Guerin infection.

Nine patients with primary immunodeficiency who received bacillus Calmette-Guerin (BCG) vaccine at birth developed disseminated BCG lesions and presented clinically with generalized skin rash and skin nodules. Fine-needle aspiration biopsy of the skin nodules and/or enlarged lymph nodes was performed in all patients. The most common cytologic pattern encountered was cellular smears showing a large number of histiocytes with abundant streaked cytoplasm in a background of neutrophils and debris. No granulomas were noted. Ziehl-Neelsen (ZN) stain for acid-fast bacilli showed a large number of these bacilli within the cytoplasm of the histiocytes, and extracellularly. This pattern was seen in 6 patients. The cytologic smears from 3 patients showed epithelioid granulomas in a background of neutrophils and debris. ZN stain for acid-fast bacilli showed fewer numbers of these bacilli compared to the first cytologic pattern. In conclusion, the most common cytologic pattern of postvaccinial disseminated BCG lesions in immunocompromised patients is a large number of histiocytes with abundant streaked cytoplasm in a background of neutrophils and debris. No epithelioid granulomas are seen in this pattern. A less frequent pattern is also encountered which shows epithelioid granuloma in a neutrophilic background. In both cytologic patterns, ZN stain for acid-fast bacilli is positive. However, in the first and most common pattern, the number of acid-fast bacilli is much larger than that seen in the second pattern. The different cytologic patterns might be related to the status of immunity of patients at the time of biopsy.

Effectiveness of BCG vaccination in protecting possums against bovine tuberculosis

Three groups of eight possums were vaccinated with BCG Pasteur by the subcutaneous, intratracheal or intragastric routes, with a fourth group serving as unvaccinated controls. Forty-two days after the start of vaccination, five possums from each group were challenged intratracheally with virulent Mycobacterium bovis. The vaccination by the subcutaneous or intratracheal routes resulted in a marked reduction in the severity of disease compared with the unvaccinated animals or the animals vaccinated intragastrically. The severity of the disease was assessed by changes in bodyweight, pathological changes in the lungs and bronchial nodes and the number of acid-fast bacilli in the lesions. Before the challenge, lymphocyte blastogenic responses to bovine ppd were observed in the eight animals vaccinated subcutaneously and in two of the animals vaccinated intratracheally.

Evaluation of polymerase chain reaction amplification of Mycobacterium leprae-specific repetitive sequence in biopsy specimens from leprosy patients.

Biopsy specimens were obtained from 102 leprosy patients before chemotherapy and examined by polymerase chain reaction (PCR) using the primers amplifying the 372-bp DNA of a repetitive sequence of Mycobacterium leprae. The PCR results were then compared with bacterial indices (BI) of slit-skin smears and biopsy specimens. The intensities of DNA bands were in general correlated with the numbers of acid-fast bacilli, and even a sample with only one organism gave a PCR positive result. Ten 5-micron sections from each frozen tissue sample were pooled and processed for DNA preparation. PCR was positive for 11 (73.3%) of 15 biopsy specimens with BI of 0 determined for the paraffin sections from the same biopsy samples. PCR also gave positive results for 84 (96.6%) of 87 BI positive biopsy samples. Although the difference in overall results between the two methods was not statistically significant, PCR seemed to have an advantage over microscopic examination in detecting M. leprae in biopsy specimens negative for acid-fast bacilli. Further evaluation of PCR using more specimens from leprosy patients who are bacteriologically negative is warranted to ensure PCR's advantage over the conventional microscopic examination for the diagnosis of leprosy.

Mycobacterium paratuberculosis Monoassociated Nude Mice as a Paratuberculosis Model

In this study, a paratuberculosis (Johne's disease) model was developed by intragastrically dosing gnotobiotic athymic nude mice with Mycobacterium paratuberculosis. The mice infrequently shed bacilli from their intestinal tracts during the first 4 months after inoculation. Following this time, increasing numbers of M. paratuberculosis (greater than 4.0 log10 bacilli per fecal pellet by 40 weeks) were recovered from the feces of the 12 mice that remained in the isolator. A similar pattern of recovery of M. paratuberculosis was obtained from the ileum, cecum, colon, and liver. Histopathologic lesions and acid-fast bacilli were rare during the first 4 months of infection and then, with time, increased in prevalence and severity. Mice maintained for 7 months or longer exhibited severe granulomatous inflammation and large numbers of acid-fast bacilli in the gastrointestinal tract and liver (up to 10(8) log10 colony forming units per gram wet weight). Five mice maintained for 7 months or more developed clinical signs consistent with those seen in paratuberculosis (weight loss, chronic diarrhea); three of these mice eventually died or became moribund and were euthanatized. M. paratuberculosis monoassociated mice released increased levels of tumor necrosis factor activity into their sera, as compared to uninfected control mice, when they were injected with bacterial lipopolysaccharide. The clinical signs, fecal shedding of M. paratuberculosis, granuloma formation, and progressive bacillary multiplication observed with these mice are consistent with naturally occurring M. paratuberculosis infection of ruminants (Johne's disease). This model will be useful for future studies of immunoregulation and antimicrobial therapy of paratuberculosis.

Efficacy of a cell-mediated reaction to the purified protein derivative of tuberculin in the disposal of Mycobacterium leprae from human skin.

The purpose of this study was to evaluate the effects of a delayed-type cell-mediated immune response to Mycobacterium tuberculosis antigen on the Mycobacterium leprae load in the skin of leprosy patients. Twelve patients with the lepromatous form of leprosy have been injected intradermally with 5 units of the purified protein derivative of tuberculin (PPD). Ten individuals responded with areas of induration ranging from 12 to 21 mm in diameter, and two were unresponsive (less than 10 mm). Twenty-one days thereafter, the injected and control sites were biopsied, and the histology, number of acid-fast bacilli, nature and phenotype of the emigrant cells, and ultrastructural characteristics of the lesions were evaluated. Eight of the 10 responding patients showed reductions in the number of acid-fast bacilli by factors ranging from 5 to 10,000. Two responders and both nonresponders exhibited no discernible decline in the number of organisms. The reduction in bacillary load was correlated with an intense mononuclear cell infiltrate, the maintenance of a high CD4+ T-cell/CD8+ T-cell ratio, the formation of granulomata, and the extensive destruction of previously parasitized macrophages.

Bacillary growth, interleukin 2 production defect, and specific antibody secretion governed by different genetical factors in mice infected subcutaneously with Mycobacterium lepraemurium

Different mouse strains were infected Subcutaneously in the footpad with 10 7 Mycobacterium lepraemurium (MLM). At various stages of the infection, the number of acid-fast bacilli (AFB) in different organs, spleen cell interleukin 2 production, and specific IgM and IgG serum antibodies to MLM sonicate were assessed. Strains were separable into two distinct groups depending on the number of AFB recovered from the different organs, without any obvious influence of the Bcg gene. Thus C57BL/6, DBA/2, (C57BL/6 × DBA/2)F 1 and C3H/Pas mice belonged to the high resistance group and DBA/1, BALB/c, and CBA strains to the low resistance group. Interleukin 2 production was depressed only in C57BL/6 and C3H/Pas mice. Anti-MLM antibody response also markedly varied according to strains, in terms of antibody titers, Ig class distribution, and species specificity, but with a different genetic pattern from that observed for MLM growth Control.

Strain variation of lymphokine production and specific antibody secretion in mice infected with Mycobacterium lepraemurium

Mice from strains showing either phenotypical expression of Bcg gene (C57BL/6, BALB/c, DBA/1, and (C57BL/6 X DBA/2)/F 1, CBA, A/J, DBA/2) were infected intravenously with 10 7 Mycobacterium lepraemurium (MLM). The number of acid-fast bacilli (AFB) within the spleens, the ability of spleen cells to produce in vitro interleukin 1 and 2, and the serum levels of specific anti-MLM antibodies were assessed 3 months later. The number of AFB recovered from the spleens of various strains followed the strain distribution of genetically controlled innate resistance established for Mycobacterium bovis infection. A decrease of interleukin 2 production by spleen cells could be detected in C57BL/6, DBA/1, DBA/2 and (C57BL/6 X DBA/2)F 1 mice only. The level of anti-MLM antibodies was found to be higher in C57BL/6, BALB/c and A/J mice than in the other strains tested. Thus no evidence appeared of a direct influence of the Bcg gene on lymphokine production and antibody secretion.

Experimental tuberculosis in red deer (Cervus elaphus)

This study was designed to investigate experimental Mycobacterium bovis infection of red deer (Cervus elaphus). Three intravenously inoculated deer (dose 10 microg-1000 microg) developed miliary tuberculosis of the lungs and all died within 28 days of being infected. No clinical illnesses were observed in four subcutaneously (dose 1 microg-1000 microg) and three intratracheally (dose 10 microg-100 microg) inoculated deer. At the conclusion of the experiment six weeks post inoculation, these seven animals reacted to 2 mg/ml of bovine purified protein derivative. The principal lesions in the intravenously inoculated deer were in the lungs which had multiple foci of necrosis containing very large numbers of acid fast bacilli. A gradation of changes was seen in the subcutaneously inoculated deer. The animal receiving the 1 microg dose only had lesions at the injection site and the draining prescapular lymph node. Deer receiving higher doses also had histopathological changes in the lungs and liver. Microscopic changes in the intratracheally infected animals were restricted to the thoracic cavity. The ability of the deer to controlled infection was related to the route of inoculation.

Attenuation of Drug-treated Murine Leprous Bacilli to Mice

Previously, the author reported the papers that the globi inoculated sites were observed to be optimum condition, meaning that when non-drug treated leprous bacilli were injected mice which had the natural resistance to human leprous suppressed. The above results have suggested that the drug-treated leprous bacilli would have lower virulence. As a consequence I then made the following two investigations used the murine leprous as a model experiment.a. The inoculation test on mice was performed used the murine leprous bacilli obtained from the drug-treated leprous mice (INH-, or RFP-group) injected with Hawaiian strain place of human leprosy. All the control group mice showed murine leprous disease within the forth month. The appearance of the nodules the INH-group mice showed two months, and the RFP-group mice showed three months later than that of the control mice.b. The inoculation test on mice was used the murine leprous bacilli, grown Ogawa Yolk medium containing streptomycin 50mcg/ml. The SM-resistant bacilli (SMR) of Hawaiian strain was selection on the yolk medium containing SM from 0.1 to 10 mcg/ml. The SM-R thus obtained the medium containing SM at 0.1mcg/ml was inoculated into a medium containing a higher cocentration of SM. The final SM concentration media containing 50mcg/ml. I have also tried to obtain other drug-resistant bacilli, for INH, RFP and DDS. However, the isolation of these drug-resistant bacilli all proved unsuccessful. The inoculation size each SM-R and control mice prepared was 103, 104, and 105, respectively. The numbers of acid-fast bacilli were counted the staining bacilli by Ziehl-Neelsen stain like the foot-pad method. In all mice inoculated with the SM-R bacilli the nodules were not palpable at the site of inoculation during eleven months after the injection. In the control mice, which had been inoculated with murine leprous bacilli the nodules were observed five months after the injection. Two mice SM-R group were observed small nodules as autopsy eleven months after the injection.It can be said that the both drug-treated murine leprous bacilli, in vivo and in vitro , were attenuated to mice. When experimental transmission of human leprosy into mice is studied, it would seem better to use non-treated leprous bacilli.

Experimental murine leprosy: growth of Mycobacterium lepraemurium in C3H and C57/BL mice after footpad inoculation.

Forty-three female C57/BL and C3H mice were inoculated with 2.7 X 10(6) Mycobacterium lepraemurium into each hind footpad. The foot thickness and the number of acid-fast bacilli in the footpad and popliteal and inquinal lymph nodes were recorded. In addition the morphological index and the mean bacillary length were determined in the footpad and in the popliteal lymph node. The bacilli multiplied in both strains during the first 4 weeks after inoculation. After that time no further increase in acid-fast bacilli was observed in the C57/BL strain; the bacilli became elongated and the morphological index decreased. These changes were preceded by a local swelling of the footpad due to the onset of an immune reaction. Thus, under the present conditions, C57/BL mice were able to resist experimental infection with M. lepraemurium by developing an immune response. In C3H mice no indication of an immune reaction was detected, and the bacilli continued to multiply throughout the observation period. The mouse footpad model seems to provide an excellent basis for the use of experimental murine leprosy to study immunity to mycobacterial infections. Certain aspects of the present model are discussed in relation to the mouse footpad model as used in the study of M. leprae infection in mice.

Application of Nyka's method for the staining of mycobacteria in leprous skin sections.

Biopsy specimens from 20 leprosy patients have been studied. Adjacent sections were in each case both stained by the ordinary Ziehl‐Neelsen method and after oxidation with periodic acid (Nyka's method). Sections from tuberculoid, indeterminate and lepromatous lesions all revealed an increased number of acid fast bacilli after oxidation. In ten examinations of tuberculoid and indeterminate cases the increase observed was in average four‐fold. It is concluded that there is a considerable higher number of bacilli present in leprous tissue than revealed by the ordinary Ziehl‐Neelsen method.

Experimental study on the mechanisms of delayed hypersensitivity reaction, with special reference to emigration and function of mononuclear cells.

Delayed hypersensitivity reaction is characterized by Infiltration of mononuclear cells (macrophages and lymphocytes). The mechanisms of emigration and function of mononuclear cells in local skin lesions were studied in experimental tuberculosis. A chemotactlc activity for mononuclear cells was found in the extract of tuberculin skin sites, and proteases active at pH 3, 6, and 9 were found in the extract and partially purified using ammonium sulfate and column chromatography. Relationship between chemotactlc activity and protease activities was discussed. Similar protease activities were found in the extract of sensitized lymph node cells, and proteases active at pH 6 and 9 were increased when homo‐genate of sensitized lymph node cells was Incubated with PPD for 3 hours at 37°C. It appears that PPD could concern to release protease from sensitized lymphocytes and involve in delayed hypersensitivity reactions. The role of mononuclear cells in tuberculous skin lesions were studied in the sense of bacteriocidal activity for tubercle bacilli. Certain macrophages in the skin lesions were produced locally by cell division. Macrophages in granulation tissue around necrotic foci were specifically matured and strongly active for/9‐galactosidase, and the number of acid fast bacilli in macrophages and the intensity of staining for /3‐galactosidase were reversely correlated. This was also confirmed in irradiated rabbits with 400 rads of whole body radiation. These findings suggest that high levels of lysosomal enzymes are involved in destruction of bacilli.

Growth of Mycobacterium lepraemurium in diffusion chambers containing human embryonic skin cells and in cell-free chambers.

Summary: Mycobacterium lepraemurium grew in diffusion chambers containing human embryonic skin cells maintained in mice, guinea pigs and Petri plate monolayer cultures of human embryonic skin cells. Growth also occurred in diffusion chambers without skin cells maintained in mice and guinea pigs. A 28·8-fold increase in the number of bacilli was obtained when chambers containing skin cells were incubated in mice for 50 days and a 14·2-fold increase when incubated in guinea pigs. Significantly, chambers without skin cells gave a 12·2-fold increase in the number of acid-fast bacilli when maintained in mice for 50 days but only a 5·9-fold increase when maintained in guinea pigs for 50 days.

Multiplication of Mycobacterium leprae in the mouse foot-pad. 1. Thermoresistance of Mycobacterium leprae

By the aid of the mouse foot-pad passage technic as a means of evaluation for the multiplication of Mycobacterium leprae, observation was made on the survival of this organism at different temperatures. Strain B2409, isolated by Dr. Shepard and maintained at the author's laboratory, and strain Yamamura, isolated at the same laboratory, were employed as sources of M. leprae. The procedures used for inoculation, harvesting, and quantitative evaluation were essentially the same as those described by Shepard.Hanks' balanced salt solution (BSS) was used for preparing a bacillary suspension and its dilutions. The mice used were of the CF # 1 strain.Storage of bacillary suspension at 5°C: A suspension containing 1.6×105 acid-fast bacilli (strain B 2409) per ml in BSS was stored in a refrigerator at 5°C over a period from 24 hours to 2 weeks. At certain intervals of time during storage, 0.03ml of bacillary suspension, containing 4.8×103 organisms, was inoculated into each mouse at the left hind foot-pad. Harvesting was made on the 120th, 180th, 300th. and 365th day after inoculation. The number of acid-fast bacilli in the foot-pad was counted by the microspot method.There was no significant change in viability of the organisms among the samples at 24, 48, 72, and 96 hours of storage at 5°C. A considerable reduction in their viability, however, was observed in the sample at 7 days of storage.Storage of bacillary suspension at -25°C: A suspension of M. leprae (strain Yamamura) containing 4.8×103 bacilli per 0.03ml was stored in a deep freezer at -25°C over a period from 24 hours to 12 weeks. Then it was inoculated into mice at the foot-pad. The counting of bacilli was performed in the same way as mentioned above. The viability of M. leprae was not significantly affected by freezing and storage at -25°C within 12 weeks.Inactivation of the organism at 60°C: A suspension of M. leprae (strain B2409) containing 4.8×103per 0.03ml was kept in a water bath at 60°C for 5 to 60 minutes. Then 0.03ml of this suspension was inoculated into mice at the foot-pad. As a result, the viability of M. leprae was reduced gradually by the prolongation of time for treatment at 60°C, and completely lost by heating for 30 minutes.

The Experimental Infection of Rabbits with Duval's Chromogenic Acid-Fast Bacillus from Human Leprosy

lated from the lesion of human leprosy. Claims have been made by some that experimental transmission of the disease was possible, while others have reported failures. It is an established fact that the experimental production of the leprous lesion has been accomplished in certain laboratory animals with acidfast cultures from human leprosy and an emulsion of leproma rich in Hansen bacilli. However, there is no claim that a progressive type of infection has occurred in the experimental animal. Nor is there any mention of difficulty in recovering from the experimental lesion the culture that was employed, which point in itself would support specific pathogenesis. Nicolle' inoculated 2 bonnet monkeys subcutaneously with ground tissue from a human patient with generalized nodular leprosy. He noted that a nodule appeared at the site of injection. Histological study revealed small areas of lymphocytes, mononuclear cells, and moderate numbers of acid-fast bacilli. Reenstjerna2 inoculated monkeys with suspension of ground lepromata from patients exhibiting nodular leprosy. A small, round, hard nodule was produced at each inoculated site. The nodules finally disappeared though the author does not state how long they persisted. Kedrowski,3 who cultivated a non acidfast diphtheroid from 4 cases of human leprosy, states that when this bacillus was injected into rabbits it became acidfast after a residence of several weeks in