Skin biopsy and slit-skin smears from 46 leprosy patients and 4 nonleprosy patients were tested for the presence of Mycobacterium leprae by the polymerase chain reaction (PCR) using primers based on the sequence of the LSR/15 kD gene. The PCR was found to be specific and sensitive, with a detection level of 10 and 100 bacilli. PCR using skin biopsies gave a higher detection rate than did slit-skin smears, probably due to the higher density of bacilli in a 4-mm punch biopsy. Dot blot hybridization with radioactive probes was 10-fold more sensitive than the ethidium bromide staining. Eight patients who did not show acid-fast bacilli in tissues by the conventional methods were shown to have PCR-amplified M. leprae DNA. False-negative results were obtained in 3 cases even though formal evidence for tissue inhibitors was absent.
In these studies we evaluated the activity of low levels of five antimicrobials against Mycobacterium leprae-infected mice when administered singly and in all possible two- and three-drug combinations. Antibiotics studied were: dapsone (D) 0.0001% in the diet, rifampin (R) 20 mg/kg by gavage once monthly, minocycline (M) 0.004% in the diet, clarithromycin (C) 0.001% in the diet, and sparfloxacin (S) 5 mg/kg by gavage five times weekly. Singly each agent was found bacteriostatic (D + R) or partially bactericidal (M, C, and S) but not fully bactericidal. All 10 two-drug regimens were found at least bacteriostatic, 2 being "partially bactericidal" and 4 being "fully bactericidal." Of the 10 three-drug regimens, 9 were found "fully bactericidal" and the other "partially bactericidal." We conclude that combinations of antibiotics active against M. leprae are generally additive in combination.
In this work we describe the purification and characterization of armadillo immunoglobulins. The IgM was precipitated using low-strength ionic solution and further purified by filtration through Sephadex G-200. The IgG was obtained in pure form by precipitation of serum with ammonium sulfate and DEAE-cellulose ion exchange chromatography. The purity of these immunoglobulins was evaluated by polyacrylamide gel electrophoresis. The results showed 28-kDa light chains and 55-kDa and 70-kDa heavy chains for IgG and IgM, respectively. The rabbit antibodies against these molecules were used to prepare fluorescein (FITC) and peroxidase conjugates. The FITC conjugate was used to quantify IgM-bearing lymphocytes. An average of 17% of peripheral blood lymphocytes were sIgM+ from 14 healthy animals. Additionally, in the same animals we quantified lymphocytes with the capacity to form rosettes with sheep red-blood cells; the average for this marker was 10%. Also, the production of crossreacting antibodies to BCG was evaluated in healthy and Mycobacterium leprae-inoculated animals using the peroxidase conjugates. All animals with active infection recognized BCG antigens.
In this paper we report the purification and study of the immunogenic properties of the Mycobacterium leprae 18-kDa protein antigen produced and secreted by the yeast Saccharomyces cerevisiae, using an expression system we recently described [Biotech. Lett. 16 (1994) 1241-1246]. The 18-kDa protein was purified from the yeast culture media by precipitation, ion exchange chromatography (MonoQ) and exclusion size chromatography (Sephacryl S-100). The biological properties of the recombinant protein, previously irradiated with gamma rays, were assayed by immunization of mice. Humoral and cellular responses, monitored by antibody production and delayed-type hypersensitivity, respectively, were obtained. Furthermore, gamma-irradiation of the recombinant protein prior to the administration was shown to significantly potentiate the T-cell response. The data suggest that this irradiated recombinant antigen could be used in a more sensitive standardized skin test to monitor M. leprae infection.
In Weifang Prefecture, Shandong Province, and Wenshan Prefecture, Yunnan Province of China, leprosy was highly prevalent in the 1950s. Due to differences in geographical conditions and socioeconomic development, the decline in leprosy prevalence between 1955 and 1993 was 99.5% (10.1 to 0.05/10,000) in Weifang and 93.9% (19.7 to 1.2/10,000) in Wenshan. The decrease in the detection rate was 99.9% (35.2 to 0.05/10,000) in Weifang and 91.7% (69.9 to 5.8/10,000) in Wenshan. The decrease was more apparent in these two prefectures since the implementation of multidrug therapy (MDT) in 1986. Findings such as specific detection rates by age, sex and type, as well as the multibacillary, child, and deformity rates of patients detected since 1980 were studied. Using the detection and prevalence rates between 1980 and 1993, the number of patients until the year 2000 is extrapolated for these two prefectures.
Preparations of armadillo-derived Mycobacterium leprae used in vaccine trials were analyzed using a combination of morphological, chemical and immunological criteria. When compared to more recent preparations, vaccine lots prepared in 1984 and 1985 were found to contain fewer intact bacilli and lower amounts of M. leprae antigens. These differences may be characteristic of the original preparations, or alternatively, may have arisen during prolonged storage. The early vaccine lots were those used in the recently published Venezuela trial.
Multidrug therapy (MDT) recommended by the World Health Organization (WHO) had been administered in 1982 to a cohort of multibacillary (MB) leprosy patients. Treatment was administered for a minimum period of 2 years or until skin-smear negativity for acid-fast bacilli was achieved (whichever was later). Among 980 MB leprosy patients who completed treatment, 11 patients (1.1%) experienced lepra reactions during surveillance. Probable predictive factors are discussed. The incidence of lepra reaction seemed to be three times more common in borderline (BL) leprosy than in lepromatous (LL) leprosy. The majority of these events occurred during the first 3 years of surveillance. All of these episodes were treated with steroids without antileprosy chemotherapy. None of these patients was confirmed as experiencing a relapse during the subsequent period of surveillance.
The effect of the pH of a cell-free liquid medium on the activity of Mycobacterium leprae during incubation of the cells was investigated. As a parameter for evaluating the activity, the amount of adenosine triphosphate (ATP) extracted from the incubated cells collected by centrifugation was measured. The results demonstrate that the activity of M. leprae cells was maintained at a significant level for approximately 4 weeks at 30 degrees C in 0.05 M phosphate buffer containing 10% fetal calf serum at pH 7.0 compared to cells at other pHs tested, but activity was not preserved in phosphate buffer at pH 7.0 without serum and incubated at 37 degrees C. The maintenance of the activity under these conditions was prolonged somewhat by the addition of glycerin (2%) to the medium, and was definitely inhibited by rifampin but not by either penicillin or isoniazid. From the results reported here, it could be postulated that the optimal pH of cell-free media for the study of cultivation of M. leprae is 7.0.
