皮肤分枝杆菌感染快速诊断和药敏试验方法的研究和应用
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摘要
近年来随着免疫抑制剂应用的增多、器官移植手术普及以及免疫相关性疾病如艾滋病的流行,分枝杆菌感染与日俱增。皮肤分枝杆菌感染的临床表现缺乏特异性,往往需要借助实验室检查,传统的检查方法包括分枝杆菌镜检、培养和组织病理。但是由于目前的实验室检查方法敏感性较低、耗时长、特异性较差,容易造成漏诊和误诊。PCR技术能够快速、特异、敏感地进行分子诊断,逐渐被广泛应用于分枝杆菌检测,然而PCR技术在此领域应用也有待改进。近来出现了一种新的核酸扩增技术,即环介导等温扩增(loop-mediated isothermal amplification, LAMP),它采用独特的引物设计实现等温条件下的核酸快速扩增。与传统PCR技术相比,该方法操作简单,快速灵敏,并且不需要特殊的仪器设备,尤其适合在基层医疗单位推广。
随着结核耐药菌株的逐渐增多,以及大多数非结核分枝杆菌(NTM)对常用的抗结核药物呈高度耐药,根据分枝杆菌药敏结果选择治疗方案,对指导临床治疗具有重大意义。传统药敏试验(绝对浓度法和比例法)操作繁杂、费时较长且稳定性差,很难满足临床需要。而2003年美国临床实验室标准化委员会(CLSI)制定的《分枝杆菌药敏试验通过标准》(CLSI M24-A)采用标准化的液基微量稀释法进行药敏试验,具有操作相对简便、不需要特殊设备、准确性高及重复性好的特点,可以筛选有效的新型抗菌药物。
海鱼分枝杆菌是一种见光产色的慢生型分枝杆菌,存在于淡水或咸水中,可导致多种鱼类患病。人类在接触了养鱼池、在污染的水中捕鱼或游泳时易被感染,因此常被描述为游泳池肉芽肿或养鱼池肉芽肿。虽然海鱼分枝杆菌感染常有文献报道,但是关于对其爆发感染的研究甚少。
本研究：①根据CLSI M24-A标准,在我们实验室建立起针对结核分枝杆菌、慢生型NTM和快生型NTM的液基微量稀释法,与国际标准水平接轨；并进一步采用该方法对我所近年来皮肤标本中分离出的结核菌和NTMI临床株进行药敏分析；同时评价常见分枝杆菌对氨苯砜和氯法齐明的体外实验敏感性。②建立起检测结核分枝杆菌的LAMP方法,初步探讨其在皮肤结核组织标本中的应用价值。③对江苏海安某渔厂进行调查研究海鱼分枝杆菌皮肤感染的小爆发状况,并给予感染患者含克拉霉素的治疗方案,观察其临床疗效。
本研究论文全文分为三章：
第一章液基微量稀释法体外测定常见分枝杆菌的药物敏感性
目的评价结核和非结核分枝杆菌对抗菌药物的敏感性。方法根据美国临床和实验室标准协会推荐的M24-A液基微量稀释法,测定临床结核和非结核分枝杆菌对异烟肼(INH)、利福平(RFP)、乙胺丁醇(EMB)、链霉素(SM)、左氧氟沙星(LOFX)、阿米卡星(AMK)、多西环素(DCC)、克拉霉素(CTM)、磺胺甲基异噁唑(SMZ)、氨苯砜(DDS)和氯法齐明(CLO)等药物的敏感性,其中结核分枝杆菌的结果与传统绝对浓度法进行比较。同时测定常见分枝杆菌标准株对氨苯砜和氯法齐明的体外敏感性。结果8株临床结核分枝杆菌中,除1株菌对SM耐药外,各株菌对INH、RFP、EMB、SM、LOFX五种药物均敏感。3株源于临床的海鱼分枝杆菌中,除1株菌对DCC耐药外,各株菌对RFP、AMK、DCC、CTM、SMZ五种药物均敏感。1株临床性胞内分枝杆菌对CTM敏感。3株临床性瘰疠分枝杆菌都对DCC耐药,其中除1株菌同时对RFP和EMB耐药外,各株菌对INH、RFP、EMB、SM、AMK、CTM、SMZ七种药物均敏感。3株临床性脓肿分枝杆菌中,除了1株菌同时对CTM和CLO敏感外,各株菌对LOFX、AMK、DCC、CTM、SMZ、DDS、CLO七种药物均表现为中间耐药或耐药。常见分枝杆菌标准株均对DDS耐药,结核、海鱼和堪萨斯分枝杆菌标准株对CLO敏感。结论不同分枝杆菌对抗菌药物的敏感性存在差异。可以参考体外药敏结果指导临床用药选择。氯法齐明是一种治疗结核、海鱼和堪萨斯分枝杆菌感染有潜力的药物。
第二章环介导等温扩增技术检测结核分枝杆菌方法的建立与初步应用
目的建立环介导等温扩增检测结核分枝杆菌的方法。方法针对结核分枝杆菌复合群gyrB基因序列设计三对特征性引物,采用环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)对结核分枝杆菌进行65℃等温扩增,1h后分析结果。确定该方法的敏感性和特异性,并初步探讨其在皮肤结核组织标本中的应用。结果采用LAMP方法检测结核分枝杆菌,敏感度与PCR相当；与其他分枝杆菌无交叉反应,具有良好的特异性。5例培养阳性临床标本中,LAMP和PCR方法敏感度均为2／5。结论结核分枝杆菌环介导等温扩增技术检测方法,不仅快速、特异、敏感,而且简便价廉,适于在基层实验室使用。
第三章江苏某渔厂海鱼分枝杆菌感染爆发的调查研究
背景海鱼分枝杆菌是一种非结核光产色慢生型分枝杆菌,可导致人类皮肤感染。虽然海鱼分枝杆菌感染常有文献报道,但是关于其爆发感染的研究甚少。2008年11月我们在本院诊治了一例来自江苏海安市某渔厂的海鱼分枝杆菌皮肤感染患者,问诊得知该患者所在渔厂还有多例可疑类似患者。方法我们前往该渔厂进行现场调查,对疑似病例采集病史、体格检查,完善相关检查包括：胸片、常规组织病理学检查及特殊染色、PPD试验、溃疡分泌物涂片抗酸染色、真菌培养、分枝杆菌培养及分子鉴定。综合病史、既往治疗情况、临床表现及相关检查,对患者进行初步或最终诊断。然后给予患者克拉霉素单一治疗或联合复方磺胺甲基异恶唑治疗,并随访观察疗效。结果经调查发现,海鱼分枝杆菌感染病例18例,男13例,女5例,病程2-55月。66.7%(12／18)的患者有明确的被鱼刺伤或咬伤史。所有患者均为肢端皮肤损害,其中2例表现为孢子丝菌病样分布。受检的13例患者中,仅7例组织学表现为感染性肉芽肿。分泌物涂片抗酸染色阳性率为4／9。PPD试验阳性率为10／18。组织块分枝杆菌培养阳性率为3／8,培养物经鉴定为海鱼分枝杆菌。含克拉霉素的治疗方案治愈率为72.2%(13／18)。治疗过程中有4例出现治疗抵抗,均发生于曾经接受过局部皮质激素封闭治疗者。而未接受过局部封闭治疗的患者,均未出现治疗抵抗。结论在临床诊治实践中,皮肤海鱼分枝杆菌感染的诊断较为困难,需要综合病史、皮损、组织病理、病原学检查以及治疗反应来综合判断。其中尤以病史具有重要的提示作用,如经常接触鱼类或可能的疫水。含克拉霉素的治疗方案能使大多数局限于皮肤感染的患者成功治愈。皮损局部皮质激素封闭可能是治疗失败的危险因素。通过戴防水手套等防止劳动时外伤,可以预防海鱼分枝杆菌的感染。
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 method
Objective 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 tuberculosis
Objective 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, China
Background 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.
随着结核耐药菌株的逐渐增多,以及大多数非结核分枝杆菌(NTM)对常用的抗结核药物呈高度耐药,根据分枝杆菌药敏结果选择治疗方案,对指导临床治疗具有重大意义。传统药敏试验(绝对浓度法和比例法)操作繁杂、费时较长且稳定性差,很难满足临床需要。而2003年美国临床实验室标准化委员会(CLSI)制定的《分枝杆菌药敏试验通过标准》(CLSI M24-A)采用标准化的液基微量稀释法进行药敏试验,具有操作相对简便、不需要特殊设备、准确性高及重复性好的特点,可以筛选有效的新型抗菌药物。
海鱼分枝杆菌是一种见光产色的慢生型分枝杆菌,存在于淡水或咸水中,可导致多种鱼类患病。人类在接触了养鱼池、在污染的水中捕鱼或游泳时易被感染,因此常被描述为游泳池肉芽肿或养鱼池肉芽肿。虽然海鱼分枝杆菌感染常有文献报道,但是关于对其爆发感染的研究甚少。
本研究：①根据CLSI M24-A标准,在我们实验室建立起针对结核分枝杆菌、慢生型NTM和快生型NTM的液基微量稀释法,与国际标准水平接轨；并进一步采用该方法对我所近年来皮肤标本中分离出的结核菌和NTMI临床株进行药敏分析；同时评价常见分枝杆菌对氨苯砜和氯法齐明的体外实验敏感性。②建立起检测结核分枝杆菌的LAMP方法,初步探讨其在皮肤结核组织标本中的应用价值。③对江苏海安某渔厂进行调查研究海鱼分枝杆菌皮肤感染的小爆发状况,并给予感染患者含克拉霉素的治疗方案,观察其临床疗效。
本研究论文全文分为三章：
第一章液基微量稀释法体外测定常见分枝杆菌的药物敏感性
目的评价结核和非结核分枝杆菌对抗菌药物的敏感性。方法根据美国临床和实验室标准协会推荐的M24-A液基微量稀释法,测定临床结核和非结核分枝杆菌对异烟肼(INH)、利福平(RFP)、乙胺丁醇(EMB)、链霉素(SM)、左氧氟沙星(LOFX)、阿米卡星(AMK)、多西环素(DCC)、克拉霉素(CTM)、磺胺甲基异噁唑(SMZ)、氨苯砜(DDS)和氯法齐明(CLO)等药物的敏感性,其中结核分枝杆菌的结果与传统绝对浓度法进行比较。同时测定常见分枝杆菌标准株对氨苯砜和氯法齐明的体外敏感性。结果8株临床结核分枝杆菌中,除1株菌对SM耐药外,各株菌对INH、RFP、EMB、SM、LOFX五种药物均敏感。3株源于临床的海鱼分枝杆菌中,除1株菌对DCC耐药外,各株菌对RFP、AMK、DCC、CTM、SMZ五种药物均敏感。1株临床性胞内分枝杆菌对CTM敏感。3株临床性瘰疠分枝杆菌都对DCC耐药,其中除1株菌同时对RFP和EMB耐药外,各株菌对INH、RFP、EMB、SM、AMK、CTM、SMZ七种药物均敏感。3株临床性脓肿分枝杆菌中,除了1株菌同时对CTM和CLO敏感外,各株菌对LOFX、AMK、DCC、CTM、SMZ、DDS、CLO七种药物均表现为中间耐药或耐药。常见分枝杆菌标准株均对DDS耐药,结核、海鱼和堪萨斯分枝杆菌标准株对CLO敏感。结论不同分枝杆菌对抗菌药物的敏感性存在差异。可以参考体外药敏结果指导临床用药选择。氯法齐明是一种治疗结核、海鱼和堪萨斯分枝杆菌感染有潜力的药物。
第二章环介导等温扩增技术检测结核分枝杆菌方法的建立与初步应用
目的建立环介导等温扩增检测结核分枝杆菌的方法。方法针对结核分枝杆菌复合群gyrB基因序列设计三对特征性引物,采用环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)对结核分枝杆菌进行65℃等温扩增,1h后分析结果。确定该方法的敏感性和特异性,并初步探讨其在皮肤结核组织标本中的应用。结果采用LAMP方法检测结核分枝杆菌,敏感度与PCR相当；与其他分枝杆菌无交叉反应,具有良好的特异性。5例培养阳性临床标本中,LAMP和PCR方法敏感度均为2／5。结论结核分枝杆菌环介导等温扩增技术检测方法,不仅快速、特异、敏感,而且简便价廉,适于在基层实验室使用。
第三章江苏某渔厂海鱼分枝杆菌感染爆发的调查研究
背景海鱼分枝杆菌是一种非结核光产色慢生型分枝杆菌,可导致人类皮肤感染。虽然海鱼分枝杆菌感染常有文献报道,但是关于其爆发感染的研究甚少。2008年11月我们在本院诊治了一例来自江苏海安市某渔厂的海鱼分枝杆菌皮肤感染患者,问诊得知该患者所在渔厂还有多例可疑类似患者。方法我们前往该渔厂进行现场调查,对疑似病例采集病史、体格检查,完善相关检查包括：胸片、常规组织病理学检查及特殊染色、PPD试验、溃疡分泌物涂片抗酸染色、真菌培养、分枝杆菌培养及分子鉴定。综合病史、既往治疗情况、临床表现及相关检查,对患者进行初步或最终诊断。然后给予患者克拉霉素单一治疗或联合复方磺胺甲基异恶唑治疗,并随访观察疗效。结果经调查发现,海鱼分枝杆菌感染病例18例,男13例,女5例,病程2-55月。66.7%(12／18)的患者有明确的被鱼刺伤或咬伤史。所有患者均为肢端皮肤损害,其中2例表现为孢子丝菌病样分布。受检的13例患者中,仅7例组织学表现为感染性肉芽肿。分泌物涂片抗酸染色阳性率为4／9。PPD试验阳性率为10／18。组织块分枝杆菌培养阳性率为3／8,培养物经鉴定为海鱼分枝杆菌。含克拉霉素的治疗方案治愈率为72.2%(13／18)。治疗过程中有4例出现治疗抵抗,均发生于曾经接受过局部皮质激素封闭治疗者。而未接受过局部封闭治疗的患者,均未出现治疗抵抗。结论在临床诊治实践中,皮肤海鱼分枝杆菌感染的诊断较为困难,需要综合病史、皮损、组织病理、病原学检查以及治疗反应来综合判断。其中尤以病史具有重要的提示作用,如经常接触鱼类或可能的疫水。含克拉霉素的治疗方案能使大多数局限于皮肤感染的患者成功治愈。皮损局部皮质激素封闭可能是治疗失败的危险因素。通过戴防水手套等防止劳动时外伤,可以预防海鱼分枝杆菌的感染。
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 method
Objective 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 tuberculosis
Objective 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, China
Background 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.
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[1]Negi SS, Anand R, Pasha ST, et al. Diagnostic potential of IS6110,38kDa,65kDa and 85B sequence-based polymerase chain reaction in the diagnosis of Mycobacterium tuberculosis in clinical samples[J]. Indian J Med Microbiol,2007, 25(1):43-49.
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[3]Tan SH, Tan BH, Goh CL, et al. Detection of Mycobacterium tuberculosis DNA using polymerase chain reaction in cutaneous tuberculosis and tuberculids[J]. Int J Dermatol,1999,38(2):122-127.
[4]Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Res,2000,28(12):E63.
[5]Iwamoto T, Sonobe T, Hayashi K. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples[J]. J Clin Microbiol,2003,41(6):2616-2622.
[6]Mori T, Sakatani M, Yamagishi F, et al. Specific detection of tuberculosis infection: an interferon-gamma-based assay using new antigens[J]. Am J Respir Crit Care Med,2004,170(1):59-64.
[7]Tanaka R, Matsuura H, Kobashi Y, et al. Clinical utility of an interferon-gamma-based assay for mycobacterial detection in papulonecrotic tuberculid[J]. Br J Dermatol,2007,156(1):169-171.
[8]Angus J, Roberts C, Kulkarni K, et al. Usefulness of the QuantiFERON test in the confirmation of latent tuberculosis in association with erythema induratum[J]. Br J Dermatol,2007,157(6):1293-1294.
[9]Aggarwal P, Singal A, Bhattacharya SN, et al. Comparison of the radiometric BACTEC 460 TB culture system and Lowenstein-Jensen medium for the isolation of mycobacteria in cutaneous tuberculosis and their drug susceptibility pattern[J]. Int J Dermatol,2008,47(7):681-687.
[10]Szeredi L, Glavits R, Tenk M, et al. Application of anti-BCG antibody for rapid immunohistochemical detection of bacteria, fungi and protozoa in formalin-fixed paraffin-embedded tissue samples[J]. Acta Vet Hung,2008,56(1):89-99.
[11]Goel MM, Budhwar P. Immunohistochemical localization of mycobacterium tuberculosis complex antigen with antibody to 38 kDa antigen versus Ziehl Neelsen staining in tissue granulomas of extrapulmonary tuberculosis[J]. Indian J Tuberc, 2007,54(1):24-29.
[12]Mustafa T, Wiker HG, Mfinanga SG, et al. Immunohistochemistry using a Mycobacterium tuberculosis complex specific antibody for improved diagnosis of tuberculous lymphadenitis[J]. Mod Pathol,2006,19(12):1606-1614.
[13]Padmavathy L, Rao LL, Ramanadhan, et al. Mycobacterial antigen in tissues in diagnosis of cutaneous tuberculosis[J]. Indian J Tuberc,2005,52:31-35.
[14]Kathuria P, Agarwal K, Koranne RV. The role of fine-needle aspiration cytology and Ziehl Neelsen staining in the diagnosis of cutaneous tuberculosis[J]. Diagn Cytopathol,2006,34(12):826-829.
[15]NCCLS. Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes; Approved Standard. NCCLS document M24-A [ISBN 1-56238-500-3]. NCCLS,940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA,2003.
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