Skin Mycobacterial Infection In The Study And Application Of Rapid Diagnostic And Susceptibility Testing Methods

With the growing number of the patients with AIDS and other immunosuppressive diseases, the incidence of mycobacteria infections has been increased in recent years. Cutaneous mycobacterium infection is still difficult to make diagnoses for dermatologists. Conventional diagnosis continues to rely on smear microscopy, culture and histopathology. These tests have known limitations. PCR technique, which is a rapid, specific, sensitive molecular method, has been increasingly widely used in detection of mycobacterium. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency, and rapidity. Due to its easy operation without sophisticated equipment, it will be simple enough to use in small-scale hospitals and clinical laboratories in developing countries.Because of increase in resistant strains of Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM) highly resistant to common anti-TB drugs, drug susceptibility testing is essential to guide clinical treatment. Traditional methods, including absolute concentration and proportion method, are time-consuming. Broth microdilution method in M24-A standard, which was developed by CLSI, is reliable, practical and easy to use in the clinical laboratory.Mycobacterium marinum is a slow-growing mycobacterium that causes disease in many fish species from fresh or salted water. Human infection follows contact with fishes or contaminated water and is often described as'swimming pool granuloma'or'fish tank granuloma'. M.marinum infection is well reported in the literature, but outbreak of infection has been seldom described.In this study:①According to CLSI M24-A standard, we established broth microdilution method in our laboratory and tested the susceptibility of M. tuberculosis and NTM isolated from skin samples. Also, we evaluated the susceptibility of clinical common mycobacteria to clofazimine and dapsone.②We established LAMP method to detect Mycobacterium tuberculosis. Also, we primarily studied the application of LAMP in tissue specimen from the patients with cutaneous tuberculosis.③We conducted an investigation for M.marinum infection from a fish-plant in Jiangsu Haian. The paper consists of three chapters. ChapterⅠDrug susceptibility testing of common mycobacteria by broth microdilution methodObjective To evaluate the susceptibility of common mycobacteria to antimicrobial agents. Methods Clinical Laboratory Standard Institute (CLSI) Broth microdilution method M24-A was applied to test the susceptibility of clinical strains of M. tuberculosis and NTM to isoniazid(INH), rifampin(RFP), ethambutol(EMB), streptomycin(SM), levofloxacin(LOFX), amikacin(AMK), doxycycline(DCC), clarithromycin(CTM), sulfamethoxine(SMZ), dapsone(DDS) and clofazimine(CLO). Also we tested the susceptibility of common mycobacterial standard strains to dapsone and clofazimine. Results 8 clinical strains of M.tuberculosis were sensitive to INH, RFP, EMB, SM and LOFX except one strain resistant to SM.3 strains of M.marinum were sensitive to RFP, AMK, DCC, CTM and SMZ except one strain resistant to DCC.1 strain of M.intracellulare was sensitive to CTM.3 strains of M.scrofulaceum were resistant to DCC, but sensitive to INH, RFP, EMB, SM, AMK, CTM and SMZ except one strain resistant to RFP and EMB.3 strains of M.abscessus were resistant to LOFX, AMK, DCC, CTM, SMZ, DDS and CLO except one strain sensitive to CTM and CLO. Common mycobacterial standard strains were resistant to dapsone. M.tuberculosis, M.marinum and M.kansasii were sensitive to clofazimine. Conclusion The sensitivity of various mycobacteria to antimicrobial agents is different. Drug susceptibility testing is essential to guide clinical treatment. Clofazimine appears to be an agent of potential efficacy against M.tuberculosis, M.marinum and M.kansasii.ChapterⅡDevelopment and application of Loop-mediated Isothermal Amplification (LAMP) to detect Mycobacterium tuberculosisObjective To establish a method of Loop-mediated isothermal amplification (LAMP) for detection of Mycobacterium tuberculosis. Methods The species-specific primers were designed by targeting the gyrB gene. LAMP was performed at 65℃, and the amplified DNA was detected after one hour. Then the sensitivity and specificity were evaluated. Also, we primarily studied the application of LAMP in tissue specimen from the patients with cutaneous tuberculosis. Results The LAMP reaction had a sensitivity equivalent to that of PCR. There was no crossing-reaction with other Mycobacterium. Both LAMP and PCR were positive in two of five culture-positive samples. Conclusion LAMP assay is rapid and simple to run, cost effective, sensitive and specific and has potential usefulness for application in clinical laboratory. ChapterⅢOutbreak of Mycobacterium marinum Infection, Jiangsu, ChinaBackground Mycobacterium marinum is a slow-growing mycobacterium that causes skin infection in humans. Although M.marinum infection is well reported in the literature, outbreak of infection has been seldom described and the treatment is not standardized. In November 2008, we detected an index patient with M.marinum infection from a fish-plant in Jiangsu Haian and prepared to conduct an investigation. Methods We went to the fish-plant and conducted an investigation for M.marinum infection. In suspected cases, complete data were collected including medical history, clinical manifestations, histologic feature, PPD test, microbiology, and response to treatment. Preliminary or final diagnosis was made. Therapeutic regimens, including clarithromycin monotherapy or combined treatment of clarithromycin and SMZco, were prescribed. Results A total of 18 outbreak patients with M.marinum infection were found. There were 13 men and 5 women. The duration of disease ranged from 2 to 55 months (mean 21.6 months).66.7%(12/18) of patients had previous history of being stabbed or bitten by fish. All patients showed only skin lesions,2 patients presented with sporotrichoid spread. Biopsies were performed for 13 patients,7 patients showed infective granulomas. AFB stain for secretion smears were positive in 4 of 9 patients with ulceration. PPD test was positive in 9 of all tested patients.3 of 8 patients had positive isolates on L-J medium which were identified as M. marinum.13 of 18 patients were cured finally with clarithromycin-containing regimens with cure rate of 72.2%, except two patients lost. We noted that four patients, who had received local corticosteroid injection, were resistant to therapy, while resistance didn't occur in patients who didn't receive local corticosteroid injection. Conclusions In clinical practice, the diagnosis of M.marinum skin infection is difficult and made on the basis of history, lesion, histologic feature, microbiology, and response to treatment. The history of contact with fish and aquaria plays an important role for diagnosis. Clarithromycin-containing regimens should be successful in most patients with infection limited to the skin. Local corticosteroid injection may be risk factor for treatment failure. Preventative strategies should be developed for fish related activity, such as wearing gloves.