我国西北地区蜱土拉弗菌感染的检测与基因分型研究

目的了解我国西北部分地区蜱土拉弗菌自然感染状况及基因型别特征。方法采用布旗法收集蜱标本,鉴定种属后采用巢式PCR检测土拉弗菌的fopA基因,阳性标本用型特异性引物鉴定细菌种,通过两短串联重复序列(SSTR9、SSTR16)位点扩增进行基因型分析,用生物学软件对序列进行比较。结果共捕获11个蜱种、2 460只蜱标本,PCR扩增土拉弗菌fopA基因7个蜱种75只阳性,总阳性率为3.05%,不同蜱种阳性率差异有统计学意义(χ2=20.91,P<0.01),以日本血蜱的阳性率最高,为17.39%。所有阳性标本均属于B亚种。75份阳性蜱标本在SSTR9区共有9种不同的基因型,其中拷贝数为9的基因型在阳性蜱标本中占40.00%(30/75);不同地区的阳性标本在SSTR9区基因型别存在明显交叉,而在SSTR16区有完全相同的序列及重复序列的拷贝数。结论我国西北部分地区存在蜱土拉弗菌感染,且细菌的基因型丰富、复杂。     

利用多重PCR方法快速鉴定结核分枝杆菌北京基因型菌株

目的 建立结核分枝杆菌北京基因型菌株快速鉴定方法 ,研究该家族在人群中的流行传播的规律和特点。 方法 采用间隔区寡核苷酸（spoligotyping）分型与多重PCR方法 分别对临床分离的112株结核分枝杆菌进行北京基因型菌株鉴定。 结果在分析的112株结核分枝杆菌中,107株为北京基因型,多重PCR鉴定结果与Spoligotyping分型符合率为100%。 结论 与spoligotyping分型相比,多重PCR技术可以快速地区分北京基因型与非北京基因型株,且方法 简单,更适于基层试验室中对北京基因型结核分枝杆菌在人群中的流行进行大规模监测。     

血红素氧合酶1基因启动子区(GT)n重复序列多态性与广东潮汕人群冠心病易感性的关系

目的 探讨血红素氧合酶1基因启动子区(GT)n重复序列多态性与广东潮汕人群冠心病易感性的关系.方法 采用荧光标记PCR和毛细管电泳相结合技术检测血红素氧合酶1基因启动子区(GT)n重复序列多态性在300例冠心病患者以及性别、年龄相匹配的182例对照者中等位基因和基因型频率分布差异,重复次数n≤25为S型等位基因,n＞25为L型等位基因.Logistics回归分析基因型与吸烟、高血压、糖尿病、高血脂、家族史等冠心病危险因素间的交互作用.结果 等位基因和基因型频率在冠心病组与对照组中分布差异无显著性,基因型与吸烟(OR为1.790,95%CI为1.110-2.886)、高血压(OR为1.552,95%CI为1.045-2.304)、糖尿病(OR为1.727,95%CI为1.018-2.928)交互作用增加个体冠心病患病风险,SL+LL基因型与吸烟作用最为显著(OR为2.517, 95%CI为1.206-5.253).结论 血红素氧合酶1基因启动子区(GT)n重复序列多态性可作为潮汕人群伴危险因素个体冠心病易感风险评估的分子标记.     

1953～2000年新疆温宿县麻风病流行资料分析

目的探讨新疆温宿县麻风病的传入和流行史.方法采用流行病学调查法对麻风高流行区的80岁以上老人进行流行史调查和对麻风散发地区病人进行发病年代等追溯调查.结果累计发生63例,其中阿热力乡占82.54%,发病最早者为1949年;其他乡的病人均为阿热力乡感染后而迁入的.结论 20世纪40年代末以来温宿县麻风病在阿热力乡、托乎拉乡和包孜东乡有不同程度的流行.     

MLVA用于新疆部分地区结核分枝杆菌基因分型的初步研究

目的探讨结核菌株进行多位点可变数目串联重复序列分析(the multiple locus VNTR analysis,MLVA)。方法选择2010年至2011年新疆维吾尔自治区第五次结核病流行病学抽样调查资料,采用经典24位点方法进行基因分型。并采用Bio Numerics5.0数据库进行基因聚类分析。将结核分枝杆菌原始株,取一菌环溶于400μl TE中悬菌,80℃1 h灭活,12 000 r/min离心10 min,弃上清,600μl TE重新悬菌,进行多位点串联重复序列分析。结果新疆99株结核分枝杆菌分为2个基因群:分别为基因群Ⅰ和基因群Ⅱ,基因群Ⅰ66株(66.7%),基因株Ⅱ33株(33.3%);基因群Ⅰ是北京家族,基因群Ⅰ的66株结核分枝杆菌有65种不同的基因型,有2株结核分枝杆菌属于同一簇,成簇率为1.5%,基因群II33株结核分枝杆菌菌株的MLVA图谱不同,成簇率为0。结论新疆结核分枝杆菌菌株存在明显的基因多态性,以北京基因型菌株为主,同时还存在一定比例的非北京基因型,应加强对主要流行菌株流行的监控及管理。     

IS6110-PCR分型方法用于安徽、湖南、江苏三省结核病分子流行病学的初步研究

目的了解安徽、湖南、江苏省结核分支杆菌的基因型状况及其分子流行病学规律。方法以结核分枝杆菌插入序列IS6110序列为模板,设计一对特异外向引物,建立一种结核分枝杆菌的快速分子生物学分型方法——IS6110PCR分型方法,进行三省结核病分子流行病学研究。结果根据IS6110PCR指纹图谱,191株安徽、湖南和江苏三省的结核分枝杆菌菌株可被分成6个主要的基因型(Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ和Ⅵ),江苏省的主要基因型为I和V型,安徽省的主要基因型为III型,湖南省的主要基因型为I和II型,V型菌株全部为江苏省菌株,IV型菌株全部为湖南省菌株。结论三省之间结核分支杆菌的基因型存在明显的差异,各自存在着独立的流行菌型,其真实原因有待进一步研究。     

天津地区临床分离株结核分枝杆菌分子流行病学研究

目的探讨天津地区结核分枝杆菌临床分离株分子流行病学特征。方法连续收集天津市海河医院2005年8月16日～11月25日就诊病人痰培养阳性的结核分枝杆菌100株，采用间隔区寡核苷酸分型（Spoligotyping）和数目可变串联重复序列（VNTR）两种方法进行基因分型，并运用软件对二者的结果进行分析。依据北京分化支的定义。运用多重和实时定量PCR方法将其区分为W菌/典型北京家族菌株和非典型北京菌株，x^2检验分析两种亚群与患者年龄和耐药性之间的联系。结果排除污染菌株，共对96株结核分枝杆菌临床分离株进行两种方法的基因分型，Spoligotyping结果为91．7％为北京基因型（含3株类北京基因型）结核分枝杆菌（88／96）。VNTR分型可将北京基因型分为60种基因型。在北京分化支结核分枝杆菌中，W菌/典型北京家族菌株占93．2％（82／88）。两种北京分化支亚群与患者年龄和耐药性没有显著性统计学差异（P〉0.05）。结论天津地区结核病患者临床分离的结核分枝杆菌中，北京基因型呈现较为明显的优势。VNTR的分辨率明显高于Spoligotyping。北京分化支的两种亚群在天津地区临床结核病患者中均存在流行，但以W菌/典型北京家族菌株为主。     

分枝杆菌分子鉴定中常用的靶基因序列的研究进展

分枝杆菌种类繁多,至2000年底已发现有128种分枝杆菌,包括结核分枝杆菌复合群（MTBC,包括结核分枝杆菌、牛分枝杆菌、非洲分枝杆菌、田鼠分枝杆菌）,麻风分枝杆菌以及其他非结核分枝杆菌（nontuberculous mycobacteria,NTM）。近年来,非结核分枝杆菌感染有逐渐增加的趋势,据1990年、2000年第二次全国结核病流行病学抽样调查资料显示我国NTM的培养分离率由4.9%上升至11.1%。我院从1998—2004年NTM的感染占分枝杆菌属培养阳性率的1.5%上升至3.0%^[1]。     

DNA数目可变串联重复序列用于结核分枝杆菌分型研究

目的采用数目可变串联重复序列(VNTR)分型方法对安徽省的52株结核分枝杆菌进行分子分型研究,探讨该方法用于结核分枝杆菌分型的作用.方法设计引物,采用PCR和琼脂糖凝胶电泳技术对结核分枝杆菌13个VNTR位点进行检测,并通过BioNumerics 3.0软件进行DNA指纹图谱多态性分析.结果 52株结核分枝杆菌可分4个类别,其中88.5%菌株属于一个型,其他3个型所占比例很小,分别为5.8%、3.8%和1.9%.结论来自安徽的结核分枝杆菌存在主要的流行型,VNTR分型技术简便、快速,是较好的结核分枝杆菌分型方法.     

初治涂阳肺结核患者血清结核抗体IgG应用价值的再探讨

目的探讨初治肺结核患者血清结核抗体(TB-Ab)IgG检测的临床应用价值和其影响因素。方法回顾性调查1 911例初治涂阳肺结核患者血清TB-Ab IgG阳性率,并与同期涂阴患者健康人群进行对照;观察血清TB-Ab IgG阳性率与病灶范围大小,有无空洞,是否排菌及排菌量多少的关系,将所得数据作统计学处理;同时观察血清TB-Ab IgG与结核纯蛋白衍化物皮肤试验(PPD)二者之间有无相关性。结果初治涂阳肺结核患者血清结核抗体阳性率仅53.3%,初治涂阴者为20.5%,健康人群血清结核抗体阳性率0.4%;初治涂阳患者血清TB-Ab IgG的阳性率与病灶范围大小,空洞有无及排菌量多少均有密切关系。结论血清TB-Ab IgG检测在肺结核患者中的特异性和敏感性值得商榷,其临床应用和诊断价值有待进一步完善和提高。     

Transmission of leprosy: a study of skin and nasal secretions of household contacts of leprosy patients using PCR

It is generally held that dissemination of Mycobacterium leprae is from nasal mucosa and not through the skin of infected patients. In this study, we evaluated M. leprae in the unbroken skin and nasal secretions of multibacillary (MB) leprosy patients and their contacts. Specimens were examined by direct microscopy and polymerase chain reaction (PCR) for M. leprae DNA. Results showed that 60% of untreated MB leprosy patients examined histologically had acid-fast bacilli in the keratin layer. By PCR studies it was found that 80% of the patients had M. leprae DNA in skin washings and 60% had M. leprae DNA on swabs obtained from the nasal mucosa. Ninety-three contacts of the untreated MB cases were also tested for exposure to M. leprae by analyzing skin washings and nasal secretions by PCR. PCR analysis showed significant skin (17% positive) and nasal muscosal (4%) exposure in contacts before instituting treatment of the index cases. After 2 months of treating the index cases, all contacts tested were negative for M. leprae DNA. These data suggested that both skin and nasal epithelia of untreated MB leprosy patients contribute to the shedding of M. leprae into the environment and contacts of untreated MB cases are at risk for contact with M. leprae through both the nasal mucosa and exposed surfaces of their skin.     

Active surveillance of leprosy contacts in country with low prevalence rate

For advanced control of leprosy in Pakistan where the World Health Organization leprosy elimination goal was achieved in 1996, we conducted surveillance of Mycobacterium leprae-seropositive patients and their contacts and drug resistant strains of M. leprae.We measured anti-PGL-I antibody level in sera from leprosy patients and their contacts for early detection of M. leprae infection. Out of 34 leprosy patients undergoing treatment, 4 lepromatous leprosy patients were antibody positive, and 6.8 to 23.7 percent of occupational or household contacts were seropositive. Furthermore, three cases (1.2%) had a high antibody titer. For surveillance of drug resistant strains of M. leprae, dapsone and rifampin were targeted. Four out of 18 polymerase chain reaction (PCR) positive samples had mutation in folP gene, and among 10 PCR positive samples, one had a mutation in the rpoB gene.These results indicate that serological analysis of patient contacts might be useful to find out high risk individuals, and there are M. leprae strains resistant to chemotherapeutic agents in Pakistan.     

An approach to understanding the transmission of Mycobacterium leprae using molecular and immunological methods: results from the MILEP2 study

BACKGROUND: The current strategy for leprosy control using case detection and treatment has greatly reduced the prevalence of leprosy, but has had no demonstrable effect on interrupting transmission. METHODS: Three leprosy endemic communities in India were recruited, examined, and followed up sequentially over 2 yrs using nasal swabs and saliva collections. The nasal swabs were tested by polymerase chain reaction for the presence of M. leprae and the saliva was assayed for anti-M. leprae IgA. FINDINGS: Only 1.6% of 2552 nasal swabs were PCR positive, and 68% of saliva samples were positive for ML-IgA. BCG and household contact status was associated with the mucosal immune response, but not with PCR positivity. PCR positivity did not persist and most PCR positive results were in the wet season. INTERPRETATION: The findings contribute to our understanding of the epidemiology of M. leprae and the possible periods of greatest likelihood of exposure and transmission.     

Serologic response to mycobacterial proteins in hansen's patients during multidrug treatment

Humoral immune responses were studied in 24 leprosy patients treated with multidrug therapy (MDT) and 16 contacts. The patients were monitored for 2 to 3 years with repeated determination of IgG antibody levels directed to different mycobacterial proteins (Mycobacterium tuberculosis, Mt70; M. bovis, Mb65; M. leprae, Ml36, 28, 18, 10 kDa, and the complete protein M. leprae extract, MLSA). All recombinant antigens were used at 5 micrograms/ml concentration and the complete soluble M. leprae extract at 2 micrograms/ml. The results shown in this study reveal a clear decline in IgG antibodies directed toward mycobacterial proteins in the 12 multibacillary (MB) patients when they were submitted to MDT. Initially we found strong reactivity toward complete cytosolic protein and M. leprae membrane protein. The most reactive recombinant proteins in MB patients were Ml10, Ml36, Mt70 kDa and, finally, Ml18 kDa when compared to the paucibacillary (PB) group. After treatment was completed all lepromatous and borderline lepromatous patients showed low or undetectable levels as compared with their initial values before starting treatment.     

Nasal mucosa and skin of smear-positive leprosy patients after 24 months of fixed duration MDT: histopathological and microbiological study

The skin and nasal mucosa of 10 lepromatous leprosy patients who had completed 24 doses of fixed duration multidrug therapy (MDT) but who continued to be skin-smear positive for acid-fast bacilli (AFB) were examined histopathologically. The nasal mucosa showed granuloma fractions that exceeded those seen in the skin specimens, signifying that activity in this region subsides much more gradually than the activity in the skin. Mouse foot pad studies done using T900r mice with an inoculum from the nasal mucosa biopsy specimens of these patients did not demonstrate any growth of Mycobacterium leprae, indicating that these bacilli were not viable. A skin specimen from one patient grew significant amounts of bacteria in the T900r mouse foot pad. These results show that 2 years of treatment with MDT would prevent dissemination of M. leprae from the nasal mucosa and, therefore, should preclude further transmission of the disease. It also indicates that viable bacteria might persist in the skin of patients, especially those with an initial bacterial index of > or = 4+ who have completed 24 doses of regular MDT. Therefore, a more cautious approach to administering only 12 doses of MDT to highly positive multibacillary patients is suggested.     

Susceptibility to leprosy may be conditioned by an interaction between the NRAMP1 promoter polymorphisms and the lepromin response

Controversial results have been achieved by attempting to associate the NRAMP1 gene with Mycobacterium leprae susceptibility as well as with the Mitsuda reaction, which represents a specific immune response to M. leprae. This study evaluated this association as well as the interaction of the polymorphism (GT)(n) in the promoter region of the NRAMP1 gene with a specific immune response to M. leprae measured by the intradermal Mitsuda test in leprosy patients and in non-consanguineous household contacts. The study aimed to evaluate the association of this gene polymorphism with resistance or susceptibility to the disease, and/or with clinical forms of the disease, in a population in an endemic area served by the State Reference Center in Sanitary Dermatology and Leprosy, Federal University of Uberlandia, MG, Brazil. Leprosy patients (90) were diagnosed according to Ridley and Jopling criteria and they grouped into multibacillary (MB) and paucibacillary (PB) patients. The control group consisted of 61 non-consanguineous contacts. NRAMP1 promoter genotypes were obtained through amplification by the polymerase chain reaction (PCR) followed by the detection through the low ionic-strength single strand conformational polymorphism (LIS-SSCP) electrophoretic technique. There were no significant differences in the allelic and genotypic frequencies for alleles 2, 3, and 4 in relation to the Mitsuda test among patients and household contacts, nor between those with MB and PB forms. However, individuals with a negative lepromin response associated with genotypes 22 and 23 presented a 7- and 8-fold greater chance of developing leprosy, respectively. Therefore, the NRAMP1 gene promoter polymorphism exhibited an interaction with the lepromin response, suggesting that allele 2 of the NRAMP1 promoter is an independent genetic factor that predisposes cells to enable pathogen survival, probably due to its low efficiency in iron transport. However, establishment of the infection and disease development may be conditioned by other immunological and genetic factors.     

Lymphocyte transformation test in healthy contacts of patients with leprosy. II. Influence of consanguinity with the patient, sex, and age.

The study was carried out in the Gurage area of Ethiopia, where 53 household contacts of lepromatous patients, 37 household contacts of tuberculoid patients, and 91 control persons were examined with the lymphocyte transformation test (LTT) for their responses to whole and sonicated antigen preparation from M. leprae to BCG, M. avium, M. gordonae and phytohemagglutinin. The potential influence of host factors, namely the state of consanguinity with the leprosy patient, sex and age on the LTT responses was evaluated. In the 35 household contacts of "active," i.e., highly bacilliferous, lepromatous patients, consanguinity with a lepromatous patient was not associated with a significant depression of the LTT responses to M. leprae antigens. Male household contacts of active lepromatous patients showed significantly greater LTT responses to M. leprae antigens than female household contacts. Possible confounding factors for this finding are discussed. Sensitization of M. leprae antigens was present already in a high proportion of the 6 to 14 year old household contacts of active lepromatous patients, which was the youngest age group examined in our study. No significant results were found in any of the other patient contact groups with regard to the host factors examined.     

Antibodies to a synthetic analog of phenolic glycolipid-I of Mycobacterium leprae in healthy household contacts of patients with leprosy

Fifty-four household contacts of lepromatous patients, 39 household contacts of tuberculoid patients, and 99 control persons were examined with an enzyme-linked immunosorbent assay for their antibody responses to phenolic glycolipid-I (PGL-I) of Mycobacterium leprae using a synthetic analog (PGL-ISA) with the same terminal sugar epitope, namely, O-(3, 6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(alpha-L-rhamnopyranosyl )-(1----9)-oxynonanoyl-BSA. This study was conducted in the Gurage area of Ethiopia in 15 households with a leprosy patient and 15 matched control households. Household contacts with more than 1 year of exposure to a lepromatous patient had antibodies to PGL-ISA significantly more often (19 of 34 persons) than did household contacts with less than 1 year of exposure to a lepromatous patient (4 of 20 persons), household contacts of tuberculoid patients (8 of 39 persons), and persons without exposure to leprosy in the household (33 of 99 persons). No significant association was found between the prevalence of antibodies to PGL-ISA in the household contacts and disease activity in the lepromatous index patients at the time of examination; nor was there a significant association between antibody responses and age or sex of the contacts. The increased prevalence of antibodies to M. leprae antigen in healthy persons with more than 1 year of contact with a lepromatous patient provides further evidence that subclinical infection in leprosy is common, and is related to the type of leprosy in the index patient. The fact that antibodies to PGL-ISA were detected in one third of the persons without household exposure to leprosy emphasizes the necessity to always include comparable controls from the same endemic area in studies of leprosy contacts.     

Detection of Mycobacterium leprae DNA for 36kDa protein in urine from leprosy patients: a preliminary report

We have searched for Mycobacterium leprae DNA for 36kDa protein in urine using a M. leprae specific PCR technique. A limited number of 16 patients (of which 11 belonged to lepromatous leprosy and five to tuberculoid leprosy) and eight healthy individuals were included for the present study. The number of urine samples positive by PCR were 36.4% (4/11) in lepromatous patients and 40% (2/5) in tuberculoid patients. None of the samples from healthy individuals was positive. To our knowledge, the results indicate, for the first time, the presence of M. leprae DNA in urine from leprosy patients. Another important finding obtained out of the study is that amongst treated patients 66.6% (4/6) were positive whereas amongst untreated only 20% (2/10) were positive. From the present indicative data it appears that treatment improves the PCR results with urine as a sample. Thus, the approach could prove to be useful for monitoring the treatment response of individual patients and needs to be further evaluated with a large number of patients.     

Antibody responses to the 18-kDa protein of Mycobacterium leprae in leprosy and tuberculosis patients.

The 18-kDa protein of Mycobacterium leprae, as recognized by the monoclonal antibody L5, has a restricted species distribution, being confined to M. leprae and M. habana. We have developed a solid-phase ELISA using purified, recombinant M. leprae 18-kDa protein and compared the serological responses of Nepali leprosy and tuberculosis patients and endemic control subjects to the protein and the M. leprae phenolic glycolipid-I (PGL-I). Few control subjects had anti-18-kDa antibodies. A small proportion of paucibacillary (PB) leprosy and 42% of multibacillary (MB) leprosy patients had IgG anti-M. leprae antibodies. A similar proportion (47%) of Nepali tuberculosis (TB) patients were seropositive, and IgG anti-18-kDa antibody levels were significantly higher in MB and TB patients than in control subjects. By comparison, IgM anti-PGL-I antibodies were detected in 88% of MB leprosy patients and only 7% of TB patients. The possible reasons for the 18-kDa protein seroreactivity in TB patients are discussed, and the anti-18-kDa assay is compared with other antibody assays for protein and nonprotein antigens of M. leprae. It is concluded that the sensitivity and specificity of the anti-M. leprae 18-kDa ELISA are insufficient for the assay to be of clinical utility in leprosy patients.