나환자 족저궤양의 미생물학적 연구

Abstract

Chronic ulcers which develop on the sole of the feet or the lower legs constitute one of the most common complications in leprosy patients. Medical professions and paramedical personnels, whether directly involved in the treatment of leprosy cases or not, have become familiar with this troublesome and distressing complication. At the same time it has been a general experience that violent infection, general sepsis and tetanus resulting from the chronic ulcers are extremely rare in spite of long persistence and intractability of the ulcers.

In 1959, Price firsts introduced the term "plantar ulcer" and defined it as a chronic ulceration of the anesthetic sole of the foot, situated in well-defined areas overlaying a bony prominence, resistant to local or systemic therapy and characterized by a marked tendency to recurrence. Price's definition (1959a) of plantar ulcers was the result of careful empiric studies. In recent years, application of the knowledge gained in the diseases with neural damage, and careful studies of the static and dynamic forces in the normal and diseased foot have resulted in great advances in our understanding of the etiology and natural history of plantar ulcers in leprosy (Price, 1959a; 1959b: 1959c: 1960a; 1960b: 1961: 1962: 1963: 1964a: 1964b,: Anderson, 1961; Ross, 1962; Anderson, 1964)

Although the incidence of plantar ulecrs among leprosy patients shown come variation with differences in the locality, country and the investigator, it is generally accepted that a considerable fraction of leprosy patients suffers from such chronic ulcers. Price (1964a) observed plantar ulcers in ratios varying from 6 to 16 per 100 feet in leprosy patients under treatment. Furthermore, Price's observation in Nigeria indicated that incidence of plantar ulcers in leprosy patients was 8 to 12 times greater than the incidence in case of foot drop which resulted from other causes than leprosy, and 20 to 40 times greater the incidence of plantar ulcers in the patients with Charcot's progressive neuropathic muscular atrophy.

In spite of the fact that following the development of plantar ulcers microbial invaders from the unclean surrounding environment have free access of the lesions and there is likelihood that sooner or later the ulcers should become infected, surprisingly the occurrence of violent secondary infections of such ulcers is rather rare and be far no satisfactory explanation has been put forward far such an

unusual host-parasite relationship.

The rarity of pyogenic infections of plantar ulcers might be a possible explanation for the low occurrence of generalized secondary infections in leprosy.

Andersen (1961) pointed out that the relative absence of infection in these open ulcer lesions exposed to dust and dirt wart remarkable and that it might he taken as a negative proof of the traumatic origin of plantar ulceration in leprosy patients.

In the past only a few reports have been made on the microbiological study of plantar ulcers in leprosy patients. Murata (1912) observed a remarkable effect with antistreptococcal serum therapy for patients with streptococcal infections of the ulcers, of which precise location was net indicated. Bashima (1943) carried out mouse toxicity tests with staphylococci isolated from "leprous" ulcer. Later, Kojima (1953) introduced the term "perforating ulcer" in reference the deep penetrating leprous ulcers and found Mycobacterium leprae present in the lesions accompanied by sequestered formation of bone. Price (1964a, b) reported that the extent of secondary infections of plantar ulcers with pyogenic organisms could influence the duration of ulcer healing. Recently, Goodwin and Wood(1970) conducted a bacteriological study of plantar ulcers of Ethiopian leprosy patients and showed that the range of pathogens isolated was what would be expected from infected wounds.

In attempts to elucidate the natural history of secondary infections of plantar ulcers in leprosy patients and to provide possible explanations about the rarity of such secondary infection of plantar ulcers in leprosy patients in the absence of

chemotherapeutic drugs, studies were made with plantar ulcers in leprosy patients on 1) identification and characterization of microbial infections in the ulcer lesions, 2) effects of wide use of chemotherapeutic agents, particularly of antibiotics on the microbial organisms in the ulcers of leprosy patients who had

been under prolonged medication with sulfone drugs such as DDS, 3) histopathological and biochemical characteristics of the ulcer lesion, and 4) possible toxic effect of the ulcer tissues on the microbial organisms.

Materials and Methods

A. Materials

1. Leprosy patients : Among the in-patients at Yong-ho Leprosy Hospital and the domiciliary leprosy cases treated at the World Vision Special Skin Clinic, a total of 71 patients with plantar ulcer or lower leg ulcer were included in this study. Of 71 leprosy patients included, 54 were lepromatous type and 17 tuberculoid type: 41 were male and 30 female; grouped by age, 4 were in their second decade, 7 in their third decade, 14 in the fourth decade, 32 in the fifth decade and 14 were in the sixth decade or older (Table 1). Location of ulcers was as follows; base of 2nd, 3rd, 4th and 5th toes; 25: ulcers of pad of toes: 4: heel: 8: lateral border of the foot: 7: base of big toe: 4: plantar arch: 2: and others: 6. (Table 2). Duration of the ulcers was as follows: less than 1 year was 12, one to 2 years: 22:

two to 3 years: 13 and over 3 years: 24 (Table 3).

2. Culture media and chemicals: For primary isolation and subculture of the microorganisms, blood agar and Sabouraud's dextrose agar plates were used.

Thioglycollate broth, Sabouraud's dextrose broth and Ogawa egg medium were used for enrichment cultures.

Chemicals used for biochemical tests, identification of the isolated microorganisms. and for amino acid determination of the ulcer tissues were the products of the E. M. Merck Company.

Antibiotic discs supplied by BBL and Difco Company were used for antibiotic susceptibility tests of the isolated organisms.

B. Methods

1. Isolation of microorganisms from the ulcer lesions

a. Bacteria

The culture specimen was inoculated into 2 tubes of thioglycollate broth and the growth after 24 to 48 hours of incubation at 37℃ was examined by preparing Gram stained and acid-fast stained slides.

Pure culture of the isolated organisms was obtained by inoculating the growth in the thioglycollate broth onto 2 plates of blood agar, one for aerobic and the other for anaerobic culture. The growth on the blood agar plates after 24 hours of

incubation at 37℃ was examined for colonial morphology and by Gram staining. Stock cultures of pure isolates were made on nutrient slants for biochemical and antibiotics susceptibility tests.

b. Acid-fast bacteria

The culture specimen was inoculated into 3 tubes of Ogawa egg slant and incubated at 37℃ up to 8 weeks. Colonial growth was examined weekly from the first week of incubation, and the growth was confirmed by microscopic examination of acid-fast stained preparations.

c. Fungi

The culture specimen was inoculated into Sabouraud's dextrose broth containing Actidione and incubated at 25℃ for 1 week. The growth wart examined far the presence of mycellium and or yeast-like organisms, and pure culture of the isolates was made on Sabouraud's dextrose agar medium. Identification of fungal growth was made by macroscopic observation of the growth, microscopic observation of slide culture preparations and biochemical teats.

2. Identification of the microbial isolates and antibiotics susceptibility test The microbial isolated from the ulcer lesions were identified by microscopic observation of Gram and acid-fast stained preparations, growth characteristics of each isolate and the biochemical tests (Table 4). For antibiotics susceptibility test, 22 antibiotics were included and Ericsson's disc agar diffusion methods was employed(Appendix 1). Minimal inhibitory concentartion of the antibiotics wart determined by tube serial dilution method.

3. Determination of amino acids distribution of the ulcer tissues

Biopsies tissue was rinsed with distilled water, ground in a tissue homogenizer and extracted with HCI-ethanol (pH 1.3) at 4℃ for 24 hours. The amino acids of the extract were separated with thin layer chromatography using multiple-developing method.

4. Histopathological observations

Biopsied tissues was fixed with 10% buffered formalin, paraffin-embedded, sectioned and stained with hematoxylin-eosin and acid-fast stain.

Conclusion

The status of microbial infections of the chronic ulcers developed on the sole and lower legs of leprosy patients was investigated microbiologically, and the results are summarized as fellows:

1. A total of 101 strains of microorganisms were isolated from plantar ulcers and lower leg ulcers of 71 leprosy patients. The isolated microorganisms consisted of 93 strains of bacteria and 8 of fungi. Neither anaerobic organisms nor acid-fast bacteria were isolated through the culture experiments.

2. Of the 93 strains of bacteria isolated, there were 47 strains (46.54%) of staphylococci, 22 strains (21.78%) of Gram-positive bacilli, 20 strains (19.8%) of Gram-negative bacilli, 2 strains of unidentified bacteria and 2 of Gram-negative cocci(1.98%), respectively. Fungal isolates consisted of 7 strains (6.98%) of monomorphic mold and 1 (0.99%) of a yeast-like organism.

3. Antibiotic susceptibility test indicated that bacterial isolates were rather highly susceptible to ampicillin, kanamycin and erythromycin, but most of the isolates were resistant to 15 antibiotics including penicillin, streptomycin, tetracycline and chloramphenicol. Among the antibiotic-resistant strains of bacteria isolated many of them appeared to be drug-dependent.

4. Determination of minimal inhibitory concentration (MIC) of antibiotics against the bacterial isolates revealed that growth of penicillin-resistant strains of bacterial isolates was not inhibited at a concentration of 200 IU of penicillin but the growth was completely inhibited at a concentration of 15 mcg of erythromycin per milliliter.

5. Determination of amino acids distribution showed that the ulcer tissues contained a smaller number of amino acids than normal tissues.

6. Histopathological examination of the ulcer lesions revealed fibrosis and a variable degree of vascular proliferation in the granulation tissue layer, marked intimal thickening of the walls of the proliferated blood vessels and a slight degree of inflammatory cell infiltration.

In conclusion, the following observations made in this study appear to provide substantial explanation for the rarity of violent fecal or systemic infection at or through chronic plantar ulcers in leprosy patients., 1) Since the most of bacterial isolates from the ulcer lesions exhibited rather high degree of resistance to many of the antibiotics tested, it can be assumed that such organisms are of lowered virulence and of diminished invasiveness, 2) Histopathological findings of the ulcer tissues are suggestive of insufficient circulation of blood and lymph both in and around the ulcer lesions, 3) The comparative paucity of amino acids distributed in ulcer tissues implies that the ulcer tissues provide an unfavorable niche for the growth of the microbial organisms encountered.