Studies on Acid-fast Bacillus Detected in Non-leprosy Human Skin Tissues

Abstract

Nishimura et al.1)2) reported that acid-fast bacilli were present in the skin tissue of apparently healthy mice without giving to any symptom. We have also detected abundant acid-fast bacilli in the skin of healthy guinea pig, rabbit and monkey, which could not grow on a medium. The present paper described the results of our attempts to detect acid-fast bacilli in human skin tissues of non-leprosy cases. Some characterizations of the organism collected from one case are also presented. Collection Methods of Acid fast Bacilli from Specimens
Since it was difficult to detect bacilli in the specimens smeared from the skin section, we employed two methods for collection of the bacilli modifying the methods devised by Kar et al.4) and Dharmendra et al.5) According to Kar's method a small amount of 2 per cent of acetic acid was added to 0.1 to 0.5g of the minced skin tissue in order to make the tissue loose. The acid-fast bacilli were extracted with chloroform from the loosened tissue according to the Dharmendra's method. After addition of 20ml of chloroform, the mixture was homogenized for a minute in a Waring blender. Homogenizing with chloroform was repeated five times and the chloroform layers were pooled. Chloroform was evaporated to dryness. The residue was washed with ethyl ether, which was removed by centrifugation at 2, 000 rpm for 2 minutes. The sediment was suspended in a drop of distilled water, one drop put on a slide glass, after dryness the smear was stained with Ziehl-Neelsen stain. For another method of bacilli collection, 5g of skin tissue were minced and divided into five equal portions. To each portion were added 5ml of 2 per cent of acetic acid to minimize the contamination of tissue components and the mixture was homogenized in a Waring blender. The homogenate was stirred well with about 80ml of chloroform and the chloroform layer was transferred into an Erlenmyer flask. The chloroform layers from five portions were pooled and left standing overnight in a refrigerator. Since tissue particles contaminated in chloroform extracts attached by this procedure on the wall of flask, the clear chloroform extracts were obtained. The extracts were treated as mentioned above. Summary and Discussion
1) Attempts were made to isolate acid-fast bacilli from the specimens of ap-parently healthy skin of the human body, which were taken out from non-leprosy patients in the cases of surgical operations or in autopsies. The isolation was performed by the acetic acid-chloroform-ether collection method. The acid-fast bacilli were detected in 80 out of total 105 specimens tested. The detection was irrespective of age. The abundant bacilli were detected in 9 per cent of the positive cases. In the most abundant case, 5*106 bacilli were observed in 5g of the specimen.
2) In a specimen the aggregated bacilli were observed within the Subpapillary layer of the skin, though no sign of inflammation such as infiltration of the inflammatory cells was observed.
3) The specimens were emulsified and inoculated on neutral or acidic egg slants and incubated at both 33°C and 37°C. So far no acid-fast bacilli have grown on the slants.
4) Lepromin test was performed with the antigen prepared either from the isolated acid-fast bacilli or from the human leprosy bacilli. Both antigens gave negative results to the carriers of the isolated acid-fast bacilli and to the lepromatous patients. The tuberculoid leprosy patients reacted to the human leprosy lepromin, but not to the antigen prepared from the isolated acid-fast bacilli.
Figueredo et al.8) reported that acid-fast bacilli were detected in 31 out of 48 individuals who were apparently healthy but had some contacts with leprosy patients. Although he has not characterized the acid-fast bacilli, they suggested that those positive individuals would have been the healthy carriers of leprosy bacilli.