Antimicrobial susceptibility testing of Mycobacterium leprae by non-radioactive bioluminescence assay was developed. Optimization of the assay conditions such as temperature and time for ATP extraction, bacteria dose, preparation of bacteria suspension and pH of culture medium was carried out using M. leprae Thai 53 strain. Samples of bacterial suspension of M. leprae were first treated with filamentous cell treatment reagent at room temperature for 30 minutes and ATP was extracted from the leprosy bacilli by heating at 60 degrees for five minutes. Luciferin luciferase was added to the extract after cooling to room temperature followed by measurement of relative light units (RLU) of each sample using a luminometer. The concentrations of the drugs used for the evaluation of antimicrobial activities of rifampin (RFP), clofazimine (CLF), ofloxacin (OFLX) and clarithromycin (CAM) were 0.125, 0.50, 2.0 and 8.0μg/ml respectively. Middlebrook 7H9 broth medium was used (pH6.6) as the basal medium and the bacilli were cultivated at 32°C for 0-14 days. ATP was extracted from 0.1ml of culture suspension and inhibition of the luminescent activity was calculated. The results were compared to that obtained by radio-active CO2 detection system, Buddemeyer method which is commonly used for measuring anti-M. leprae activity. There was a good correlation between the results obtained by ATP method on the tenth day of culture and the results obtained by Buddemeyer method on the seventh day of culture. ATP method may be useful for the determination of drug susceptibility of M. leprae.
Most of 148 newly registered leprosy patients in the past 10 years from 1995 to 2004 in Japan had received some examinations, such as histopathology test (85%), skin smear test (84%), estimation of PGL-I antibody titer (55%) or detection M. leprae with PCR method (41%). 17% of newly patients should receive wrong therapy, if they hadnot had aforesaid clinical examinations in addition to count skin lesions. Improvement of technical level at skin smear test should be required for accurate treatment program in Japan. In Japanese patients, the number has been decreasing year by year; epidemiological condition was different between in Okinawa prefecture and in others, about ratio of sex, kind of type and age group.
We present the situation of leprosy in Brazil, reporting about epidemiology, clinical criteria for classification, multidrugtherapy and special situations, as co-infection. This material was presented in the 79th Annual Meeting of Japanese Hansen's Disease Association in May 2006, during a discussion about the Japanese Guidelines for leprosy treatment.
Laboratory tests necessary for the diagnosis of leprosy have not been well introduced in general hospitals and clinical laboratories. Therefore, several tests have been performed in Leprosy Research Center, National Institute of Infectious Diseases since July, 1997, as a part of administrative examinations (tests done by request of Ministry of Health, Labour and Welfare). These examinations include histopathology, serum antibody titers (anti-PGL-I antibody), PCR test and bioactivity of anti-bacterial agents.
To develop the rapid and simple genomic diagnostic method, we analyzed the partial dnaA sequence of 27 mycobacterial species. The partial dnaA sequence could distinguish M. kansasii and M. gastri. Based on this region and RLEP sequence of M. leprae, we established the loop-mediated isothermal amplification method (LAMP) to detect each species. The LAMP method for M. kansasii and M. gastri, could detect 500 copies. Five copies of M. leprae genomic DNA could be detect in 30min. To simplify the sample processing, the LAMP assay was performed with FTA filter paper. M. leprae bacilli were applied on filter paper that lyses bacilli and bound DNA, eliminating sample centrifugation and extraction procedures. Assays of number standards showed reproducible detection rate 50 bacilli of M. leprae. Thus, The LAMP assay combined with FTA card has the advantages of rapid and simple detection and provides a practical, economical, and specific method for the diagnosis of M. leprae and NTM infection.
Antibiotic susceptibility test of Mycobacterium leprae still relies on the time consuming methods based on the growth of M. leprae in the mouse footpad. Thus, the establishment of a rapid, simple and reliable method for the detection of drug-resistant M. leprae is one of the most urgent subjects in the treatment of leprosy patients. Recently, many data on the mutation of specific genes correlating with drug resistance have been accumulated. Application of these data permit the establishment of new gene diagnostic methods for drug susceptibility test of leprosy. In this paper, the method using the low density oligonucleotide array that enables the detection of base substitutions involved in resistance against anti-leprosy drugs on a single platform was discussed. The low density oligonucleotide array described in this paper will open the new perspectives in terms of patient management for leprosy with low cost requirement.
らい菌に対する生体防御反応は、細胞性免疫が中心に営まれている。らい菌が生体内に感染すると、らい菌を貧食した抗原提示細胞の表面に、らい菌由来主要抗原がMHC抗原と共に発現する。 T細胞は、その主要抗原を認識して活性化する。本研究において、らい菌主要抗原の一つとして Major Membrane Protein (MMP)-IIが同定された。MMP-IIは樹状細胞およびマクロファージを活性化すると同時に、生体内でT細胞により認識されるタンパク抗原であった。MMP-II遺伝子の上流に結核菌Ag85A由来分泌シグナルを発現させBCGに組み込ませたリコンビナントBCGは、ナイーブT細胞を強く活性化した。ワクチンとしての必要条件を満たしていた。