Characterization of the phylogenetic distribution and chromosomal insertion sites of five IS6110 elements in Mycobacterium tuberculosis: non-random integration in the dnaA-dnaN region.

SETTING: Five IS6110 chromosomal insertion sites were characterized in the multidrug resistant Mycobacterium tuberculosis 'W' strain. OBJECTIVE: To use insertion site probes to study the phylogenetic distribution of IS6110 in the M. tuberculosis genome. DESIGN: A total of 722 M. tuberculosis isolates, previously genotyped using the standard IS6110 Southern blot hybridization methodology, were re-hybridized with the Region A insertion site probe and representative strains were further hybridized with the Region B and C probes. Strains were grouped on the basis of having IS6110 insertions in these different regions and their relatedness was further compared by sequencing the IS6110 insertion sites. RESULTS: The insertion site probes revealed that the collection of Chinese isolates previously grouped as the Beijing strain family shared IS6110 insertions in common with the W and other genotypic group 1 strains. Unexpectedly, we found that IS6110 integrated at least 10 independent times between the dnaA and dnaN genes encoding deoxyribonucleic acid replication proteins. CONCLUSIONS: IS6110 insertion site mapping is able to identify genetic relatedness among a collection of M. tuberculosis clinical strains representing the breadth of species diversity. The mapping data indicate that IS6110 insertion sites are not always random.     

Molecular epidemiology of tuberculosis in Slovenia: results of a one-year (2001) nation-wide study

Slovenia is a small Central European country with a population of 1.99 million and an incidence of tuberculosis (TB) of 18.6 per 100,000 inhabitants in 2001. In a prospective nation-wide, 1-y DNA fingerprinting study, the genetic diversity of 99.7% of all Mycobacterium tuberculosis isolates obtained from Slovenian patients with culture-verified TB in 2001 were assessed using a standardized IS6110 restriction fragment length polymorphism (RFLP) method. Among 306 M. tuberculosis isolates, 228 different IS6110 RFLP patterns were found. The number of IS6110 copies varied from 2 to 16 (9.2 copies per isolate on average). Only 2 isolates (0.7%) with less than 5 IS6110 copies were identified. Clustered M. tuberculosis isolates were detected in 116 (37.9%) patients. The degree of recent transmission in the 1-y period was 25%. The clustering rate decreased with age from 46.4% (age group under 35 y) to 19.5% (age group above 65 y). A history of alcohol abuse and homelessness was found to be associated with clustering of TB cases. In conclusion, a high clustering frequency was identified among Slovenian TB patients. The study increased our understanding of important risk factors and routes of TB transmission in Slovenia.     

Cellular immune responses to Mycobacterium tuberculosis in a patient with Takayasu's arteritis.

INTRODUCTION: Takayasu's arteritis (TA) is a disease of unknown aetiology, characterized histologically by an inflammatory cell infiltrate that affects all layers of the arterial wall. Its association with tuberculosis (TB) was described 50 years ago, based on the presence of Langhan's giant cells and granulomas similar to those found in tuberculous lesions. The presence of TB in patients with TA well as been reported in several studies as well as a positive tuberculous response, but these associations could be fortuitous in countries where TB is endemic. Recent studies have shown that patients with TA have a heightened humoral response to mycobacterial antigens including the 65 kDa fraction, a heat shock protein (HSP) that has also been found to be expressed in the arterial wall of patients with TA. The purpose of this study was to determine lymphoproliferative response and interferon-gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) stimulated by live Mycobacterium tuberculosis (Mtb) H37Rv and a panel of mycobacterial antigens, in the hope of contributing to a better understanding of the cellular immune responses to Tuberculosis in Takayasu's arteritis. MATERIAL AND METHODS: Standard lymphoproliferation tests and IFN-gamma determination (ELISA) were performed in a 47-year old black man who fulfilled criteria for TA and 10 healthy controls, BCG vaccinated, Mantoux positive. The following were used: Mtb H37Rv, Purified Protein Derivative (PPD), purified 30 kDa, recombinant M. bovis BCG 10 kDa, 38 kDa, 65 kDa, 70 kDa, Short Term-Culture Filtrate Proteins (ST-CFP), Mid Term-Culture Filtrate Proteins (MT-CFP) obtained from H37Rv and phytohemaglutinin (PHA) as mitogen for positive control. RESULTS: PBMC from the patient with TA when compared to the mean values of the 10 healthy donors showed decreased proliferation in response to all antigens, with the exception of 65 kDa. The TA patient showed a similar pattern of IFN-gamma production to that obtained with control donors, with the exception of higher IFN-gamma production in response to ST-CFP and MT-CFP. CONCLUSIONS: We have shown reactivity of peripheral lymphocytes to HSP 65 kDa and a trend towards higher production of IFN-gamma in response to ST-CFP and MT-CFP in a patient with TA. These facts, together with the already established heightened humoral response, strengthens the association between TB and TA. However, HSP 65 kDa is not specific to TB and we conclude that similar studies using lymphocytes obtained from the arterial wall of TA patients may help to clarify the role of mycobacterial infection in Takayasu's arteritis.     

Mycolic acids and ancient DNA confirm an osteological diagnosis of tuberculosis

SETTING: The underlying trends in the past epidemiology of tuberculosis (TB) are obscure, requiring recourse to the archaeological record. It would therefore be of value to develop methods for reliable TB diagnosis in ancient populations. OBJECTIVE: To test the capability of two biomarkers, Mycobacterium tuberculosis complex mycolic acids and a DNA target (IS6110), for confirming an osteological diagnosis of TB in medieval individuals, based on the presence of Pott's disease and/or rib lesions. DESIGN: Osteological examination of three archaeological individuals (Medieval: approximately 1000 years old) revealed a Pott's disease case, one with no changes consistent with TB and one with rib lesions. Rib samples from these individuals were examined for the presence of Mycobacterium tuberculosis complex mycolic acids and mycobacterial DNA. RESULTS: Mycobacterium tuberculosis complex mycolic acids and the DNA target were detected in the Pott's disease case, whilst mycolic acids (insufficient for confirmation) alone were detected in the rib lesion case. CONCLUSIONS: Biomarkers provide a sensitive tool to detect ancient TB. Mycobacterium tuberculosis DNA is not distributed homogeneously, making multiple sampling essential. Mycolic acids seem more reliable for ancient TB diagnosis than IS6110. The demonstrated stability of mycolic acids show that they may be of value in tracing the palaeoepidemiology of tuberculosis back into antiquity.     

The dynamics of repeated elements: applications to the epidemiology of tuberculosis

We propose a stepwise mutation model to describe the dynamics of DNA fingerprint variation in Mycobacterium tuberculosis. The genome of M. tuberculosis carries insertion sequences (IS6110) that are relatively stable over time periods of months but have an observable transposition rate over longer time scales. Variability in copy number and genomic location of (IS6110) can be harnessed to generate a DNA fingerprint for each strain, by digesting the genome with a restriction enzyme and using a portion of the element as a probe for Southern blots. The number of bands found for a given genome approximates the number of copies of IS6110 it carries. A large data set of such fingerprints from tuberculosis (TB) cases in San Francisco provides an observed distribution of IS6110 copy number. Implementation of the model through deterministic and stochastic simulation indicates some general features of IS/TB dynamics. By comparing observations with outcomes of the model, we conclude that the IS/TB system is very heterogeneous and far from equilibrium. We find that the transposition parameters have a much stronger effect than the epidemic parameters on copy number distribution.     

Evaluation of 24-locus MIRU-VNTR in extrapulmonary specimens: study from a tertiary centre in Mumbai

Genotyping of Mycobacterium tuberculosis isolates is a useful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. There is a lack of information on the discriminatory power of standard 24-locus mycobacterial interspersed repetitive unit (MIRU) - variable number tandem repeats (VNTR) in India, which has the highest tuberculosis (TB) burden worldwide. Therefore, we assessed its utility on 69 M.?tuberculosis (MTB) isolates from patients with extrapulmonary tuberculosis, in comparison to standard insertion sequence (IS) 6110-Restriction fragment length polymorphism (RFLP) fingerprinting and spoligotyping. IS6110-RFLP (HGDI, 0.9987) identified a single cluster of 3 (4.3%) single-copy IS6110 isolates. Spoligotyping showed 69.5% clustering (HGDI, 0.8857). In contrast, MIRU-VNTR analysis identified 69 (100%) unique strains (HGDI, 1.0000). Within the study limits, this observed high discriminatory power suggests that 24-locus MIRU-VNTR genotyping could potentially be used to study long-term transmission of MTB infection in Mumbai. Moreover, high congruence between the MIRU-VNTR-based and spoligotyping-based strain groupings suggests that CAS, EAI and Beijing are the predominant strain lineages in the Mumbai TB patient population. The Beijing lineage isolates were found to be more significantly associated with multi-drug resistance (p?相似文献   

Evaluation of tuberculosis transmission in Tehran: using RFLP and spoligotyping methods

OBJECTIVES: To evaluate the DNA polymorphism among Mycobacterium tuberculosis (MTB) strains isolated from new smear positive tuberculosis (TB) patients residing in Tehran capital city of Iran, during the year 2001. METHODS: IS6110-restriction fragment length polymorphism (RFLP) and spoligotyping analyses were performed on 129 M. tuberculosis strains. Additional patient's information was collected for further epidemiological analyses. Patients whose isolates had identical RFLP and spoligotyping patterns were considered a cluster. RESULTS: The results show that the IS6110 were polymorphic and the strains with 8 or 9 IS6110 copy number were more frequently defected (42%). Out of 129 available isolates, 56 (43%) belonged to clusters and 72 (57%) did not. The risk factors like age, sex, family history or close contact and intravenous drug abuse were associated with clustering. Whereas, unemployment (61%) and poor living conditions (83%) contributed to diseases development in both groups. Spoligotyping of M. tuberculosis strains resulted in 46 different patterns, out of which 38 patterns were unique and reported for the first time. We found one M. tuberculosis strains with a pattern characteristic of the Beijing family. CONCLUSION: In the studied time period both reactivation (57%) and recent transmission (43%) were contributing to annual new TB cases in Tehran.     

New era in molecular epidemiology of tuberculosis in Japan

Molecular epidemiology of tuberculosis (TB) is a science to study TB transmission dynamics and to enhance our understanding of the epidemiology of TB by utilizing molecular typing methods as an adjunct to classical epidemiological approach. Before the era of molecular epidemiology, it was quite difficult to ascertain the source of the infections since M. tuberculosis is spread by air-borne droplets of respiratory secretions expelled by an infectious person to a susceptible host and it can remain latent as an asymptomatic infection for years. Now a day, our understanding of TB transmission dynamics has been refined by genotyping of M. tuberculosis strains. The methods of molecular epidemiology, especially IS6110 RFLP of M. tuberculosis, were first introduced to outbreak investigations and then gradually been expanded its application to population-based study in Japan. IS6110 RFLP is obviously a powerful tool for strain differentiation of M. tuberculosis but its labor-intensiveness limits the achievable throughput and makes it less useful for long-term prospective studies. Recently, apart from IS6110 RFLP, DNA amplification-based method, i.e., variable number of tandem repeats (VNTR) has appeared as a substitute for or adjunct to the IS6110 RFLP. In this symposium, we have invited four opinion leaders in molecular epidemiology of TB from different fields: Mycobacterium reference center, basic science, clinical practice, and public health practice. We, as the chairpersons of this symposium, hope that this symposium would trigger the development of molecular epidemiological network of TB in Japan. 1. Achievement and problem of molecular epidemiologic study with IS6110-RFLP analyses of tuberculosis in Okinawa: Shinji MAEDA (Mycobacterium Reference Center, The Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association) The long-term RFLP analyses of tuberculosis in Okinawa showed that endemic M. tuberculosis might be present. This is one of the achievements of our project study. On the other hand, for more effective examination of contact persons, information of molecular epidemiology should be used actively. Therefore because the analysis report needs to be sent back quickly, the PCR-based VNTR method should substitute for the RFLP analysis. 2. Basic knowledge and application of Variable Numbers of Tandem Repeats: Kei NISHIMORI (Department of epidemiology, National Institute of Animal Health) Genomic loci of Variable Numbers of Tandem Repeats (VNTR loci) in Mycobacterium tuberculosis complex and Mycobacterium avium, the history of analysis of VNTR loci, the hypothetical mechanisms of increase or decrease of number of repeats, the structures of the loci, and the necessity of standardizing the VNTR typing were introduced. 3. Clinical application of VNTR: Tomoshige MATSUMOTO, and Hiromi ANO (Department of Clinical Research and Development, Osaka Prefectural Hospital Organization Osaka Prefectural Medical Center for Respiratory and Allergic Diseases) Tuberculosis genotyping was first introduced to outbreak investigations and population-based studies. The advent of Variable Numbers of Tandem Repeats (VNTR) can be applied to clinical fields of not only Mycobacterium tuberculosis but also of Mycobacterium avium. In Osaka Prefectural Medical Center for Respiratory and Allergic Diseases, clinical application of VNTR was first introduced in Japan to determine whether Mycobacterium tuberculosis or avium disease was caused by reactivation or reinfection when relapsed. We showed some examples about usefulness of the clinical application of VNTR. 4. Molecular epidemiology of tuberculosis to improve TB prevention and control activities: Tomotada IWAMOTO (Department of microbiology, Kobe Institute of Health), Riyo FUJIYAMA, Noriko TANAKA, Yasuto KAWAKAMI (Kobe City Public Health Center), Chika SHIRAI (Hyogo-ku Health and Welfare Department, Kobe) M. tuberculosis isolates in Kobe have been characterized as: a) Beijing family strains are highly prevalent (77%), b) two major MIRU profiles in Beijing family were found, one is globally pandemic genotype and the other is locally prevalent strains, c) six strains belonged to T3-Osaka family, and d) Manila family strains made cluster consisting of 3 strains. Kobe VNTR Database which consists of 12-loci MIRU and 9 additional VNTR loci has been developed. The basis for the selection of these supplemental 9 VNTR loci and the application of VNTR database in TB control program were introduced.     

IS6110 restriction fragment length polymorphism of Mycobacterium tuberculosis isolates from an area of Casablanca, Morocco.

Using IS6110 RFLP, 61 isolates recovered from new cases of pulmonary tuberculosis (TB) were compared from September to December 1999 in Casablanca, Morocco, a city with a high incidence of TB. The majority of the isolates (92%) harboured 6-14 copies of IS6110. The minimal fraction of patients in groups of recently acquired infection is 13.1%. This preliminary study showed that IS6110 RFLP is a suitable method for finger-printing Mycobacterium tuberculosis in Casablanca. The unexpectedly low level of recent transmission of TB found in this study deserves further studies involving higher numbers of isolates recovered during a longer recruitment period.     

吉林市结核分支杆菌IS6110 DNA指纹图谱特征分析

目的　分析吉林市结核分支杆菌IS6110 RFLP图谱特征。方法 对53株结核分支杆菌分离株进行IS6110基因分型,得到IS6110 RFLP图谱;按菌株指纹特征的同源性高低予以分组,并进行分析。结果 吉林市结核分支杆菌IS6110 RFLP图谱特征为：(1)IS6110拷贝数目平均为13个;(2)A、B两组同源性很高,具有“北京基因型”特征,C组多态性较强;(3)IS6110 RFLP图谱特征分布无地域差异;(4)耐药菌株与敏感菌株图谱特征无明显差异,但初治耐药菌株成簇率较高。结论 吉林市结核分支杆菌IS6110RFLP图谱特征以“北京基因型”为主;但还具一些独有的特征。     

Development and evaluation of a multiplex polymerase chain reaction for the detection of Mycobacterium tuberculosis from pulmonary specimens

Abstract Background: The diagnosis of pulmonary tuberculosis is still a major challenge. Using a polymerase chain reaction (PCR), one can detect Mycobacterium tuberculosis in clinical samples within a few hours. However, single gene targets may result in false negativity due to the absence of target DNA in some M. tuberculosis isolates. The objective of this study was to develop and evaluate a multiplex PCR (M-PCR) using IS6110 and devR primers for the detection of M. tuberculosis in sputum samples. Methods: Sputum samples were collected from: (1) 200 confirmed cases of tuberculosis; (2) 100 suspected cases of tuberculosis diagnosed on the basis of clinical and radiological findings; (3) 200 non-tubercular patients suffering from respiratory diseases other than tuberculosis, in whom tuberculosis had been excluded. All 500 sputum samples were subjected to PCR using IS6110 primers, and M-PCR using IS6110 and devR primers; results were compared with conventional techniques. Results: It was found that M-PCR was 97.5% successful in detecting the presence of tuberculosis in the confirmed tuberculosis group as compared to 84.5% by IS6110-based PCR. In the suspected tuberculosis group, M-PCR could detect 45% of cases as compared to 40% by IS6110-based PCR. Overall, the specificities of both the PCR and M-PCR were found to be 96.5%. Conclusions: This study demonstrated that the M-PCR assay is more sensitive than the IS6110-based PCR for the detection of M. tuberculosis in sputum specimens and could be applied in situations of highly suspected tuberculosis when all others tests including IS6110 PCR are negative.     

结核分枝杆菌IS6110序列在石蜡组织中的检测、克隆与测序

目的：探讨套式-聚合酶链反应（Nested-PCR)检测石蜡组织中结核分枝杆菌的特异性和敏感性。方法：采用套式-PCR检测石蜡包埋组织中结核菌复合体特异插入序列IS6110，并对部分标本的PCR产物进行克隆和测序。结果：31例结核标本石蜡组织检出结核菌DNA共28例，套式-PCR的敏感度为90.3%,特异度为100%。阳性预测值为100%。随机选取两例PCR产物没是序结果与结核菌标准株H37Rv同源性分别为97%和95.3%。结论：套式-PCR检测常规石蜡包埋组织中结核菌IS6110序列具有特异性强和敏感性高的特点，可应用于临床诊断，尤其是对那些常规苏木精-伊红染色和抗酸染色无法确诊的病例更具意义。     

The IS6110 repetitive DNA element of Mycobacterium tuberculosis is not detected in exhaled breath condensate of patients with active pulmonary tuberculosis

BACKGROUND: A large tertiary referral hospital in inner-city Chicago. OBJECTIVES: To determine whether the IS6110 repetitive DNA element of Mycobacterium tuberculosis is detected in exhaled breath condensate of patients with newly diagnosed active pulmonary tuberculosis. METHODS: Ten hospitalized patients with positive Ziehl-Neelson-stained sputum smears were studied. Concurrent sputum cultures for mycobacteria were performed as well. Exhaled breath condensate was collected from each patient within 6 days of initiating antituberculosis chemotherapy (median 1.5 days). These samples were analyzed by polymerase chain reaction (PCR) using primers designed to amplify the IS6110 DNA fragment of M. tuberculosis. Exogenous M. tuberculosis DNA was added to exhaled breath condensate samples to detect PCR inhibitors. Concurrent cultures of exhaled breath condensate for mycobacteria were performed. RESULTS:M. tuberculosis was identified in 9 of 10 sputum cultures. One isolate was identified as Mycobacterium kansasii. The IS6110 repetitive DNA element of M. tuberculosis was not detected in any of the 10 exhaled breath condensate samples. Exogenous M. tuberculosis DNA added to these samples elicited the characteristic band pattern of M. tuberculosis on agarose gel electrophoresis. No PCR inhibitors were detected. Cultures of exhaled breath condensate showed no growth of mycobacteria. CONCLUSIONS: The IS6110 repetitive DNA element of M. tuberculosis is not detected in exhaled breath condensate of patients with newly diagnosed active pulmonary tuberculosis.     

Prospective evaluation of in-house polymerase chain reaction for diagnosis of mycobacterial diseases in patients with HIV infection and lung infiltrates.

SETTING: Rapid diagnosis of tuberculosis (TB) in AIDS is critical for optimal treatment to reduce mycobacterial dissemination, HIV-1 replication and mortality. The inadequate sensitivity of Ziehl-Neelsen staining and its inability to distinguish atypical mycobacteria delays accurate diagnosis. OBJECTIVE: To evaluate the polymerase chain reaction (PCR) for diagnosis of TB in bronchoalveolar lavage (BAL), blood and extra-pulmonary samples from patients with AIDS and pulmonary infiltrates. DESIGN: Specimens from 103 HIV-1-infected patients were prospectively analysed using bacteriological methods and IS6110-PCR. Smear-positive samples were also tested using 16S ribosomal-DNA-PCR to identify Mycobacterium avium complex (MAC) infections. Gold standard diagnosis relied on positive cultures or treatment outcome. RESULTS: Thirty-four patients exhibited TB, one TB and MAC and four MAC. The sensitivity of IS6110-PCR was 100% in smear-positive samples, 81.8% in smear-negative BAL, 66.7% in extra-pulmonary samples and 42.9% in blood. Its specificity was 97.1% in BAL and 100% in extra-pulmonary and blood specimens. The 16S rDNA-PCR identified M. avium from all smear-positive samples that grew MAC. CONCLUSIONS: IS6110-PCR proved useful in evaluating episodes with probable clinical diagnosis of pulmonary or mixed TB and negative smears, whereas 16S rDNA-PCR would be helpful for prompt differential diagnosis of MAC in smear-positive specimens.     

AmpliSensor—聚合酶链反应定量检测肺结核患者外周血结 …

目的 探讨ＡｍｐｌｉＳｅｎｓｏｒ－聚合酶链反应定量检测外周血中结核分支杆菌ＤＮＡ在肺结核的应用价值。方法 采用ＱｌＡａｍｐ和ＡｃｕＰｕｒｅ法提取，制备全血中模板ＴＢ－ＤＮＡ，应用ＡｍｐｌｉＳｅｎｓｏｒ－ＰＣＲ定量检测，并与ＩＳ６１１０－单管巢式聚合酶链反应（ＳＮ－ＰＣＲ）作比较。结果２００例肺结核患者的血液标本中，两种方法测得结核分支杆菌ＤＮＡ的阳性率分别为６０．５％、６３．５％。８５例非结核肺病     

Multidrug-resistant strains of Mycobacterium tuberculosis isolated from patients in Tehran belong to a genetically distinct cluster

Restriction fragment length polymorphism (RFLP) analysis was used to study the molecular epidemiology of tuberculosis (TB) in certain areas of Tehran. 120 isolates of Mycobacterium tuberculosis, including drug-resistant strains (n = 23), were analysed using polymorphic GC-rich sequence (PGRS) and IS6110 probes. There was considerable diversity among the strains cultured from patients from certain areas. The results of RFLP showed that multidrug resistant (MDR) isolates of M. tuberculosis in Tehran belong to a group of strains with low copies of IS6110 and PGRS. The degree of clustering was higher for the drug-resistant strains than for the susceptible ones (65% vs 20%). Based on the demographic data and results of RFLP, it appears that recent transmissions of TB from old patients have occurred in Tehran. However, drug-resistant TB in the city is mainly caused by strains that look different from those cultured from such patients. The majority of MDR isolates (85%) in this study contained a low copy number of IS6110 and PGRS in RFLP, and were mostly recovered from immigrants and refugees.     

rpoB mutations as an epidemiologic marker in rifampin-resistant Mycobacterium tuberculosis.

SETTING: Cases of rifampin-resistant Mycobacterium tuberculosis from the prison population in Madrid and from the general population in Spain. OBJECTIVE: To identify the rpoB mutations associated with resistance to rifampin and to investigate rpoB genotyping as an epidemiological marker in rifampin-resistant M. tuberculosis. DESIGN: Twenty-nine rifampin-resistant clinical isolates of M. tuberculosis, 15 obtained from the prison population in Madrid and 14 from the general population in Spain, were characterized by sequence analysis of the 81-bp core region of the rpoB gene and IS6110 DNA fingerprinting. RESULTS: All the isolates had mutations in rpoB, with those in codon 531 accounting for 41% of the total. Twenty-three (79%) isolates were highly resistant to rifampin (minimum inhibitory concentration > or = 64 mg/L). Nineteen different IS6110 fingerprints were observed: one was shared by seven isolates, one by three, two by two, and 15 were unique. Two IS6110 clusters could be divided into subclusters on the basis of rpoB analysis. Epidemiologic links were identified among patients whose isolates had identical IS6110 patterns and rpoB genotypes, but not between those with identical IS6110 patterns and different rpoB genotypes. CONCLUSION: Characterization of rpoB mutations can provide information about susceptibility to rifampin and be a useful epidemiological tool for discrimination of rifampin-resistant strains of M. tuberculosis with identical IS6110 fingerprints.     

Specificity of insertion sequence-based PCR assays for mycobacterium tuberculosis complex.

OBJECTIVE: To determine the specificity of different insertion sequence-targeted polymerase chain reaction (PCR) tests for Mycobacterium tuberculosis complex. DESIGN: One M. bovis BCG strain, two M. tuberculosis strains and ten species of mycobacteria other than tuberculosis (MOTT) were tested by three PCR assays based on the repetitive elements IS6110, IS1081 and IS990 under variable amplification conditions (different temperatures of primer annealing and numbers of reaction cycles). RESULTS: DNA amplifications based on the three insertion sequences yielded fragments of expected sizes only in DNA from M. tuberculosis complex strains when the tests were conducted at high stringency (65 degrees C). At the annealing temperature of 60 degrees C the PCR assay with IS6110-specific primers yielded a 245 bp fragment also in nine MOTT strains tested. This could result from previously reported homology between non-tuberculous mycobacteria and a central region of IS6110. Amplification assays based on IS1081 and IS990 gave false-positive results in some MOTT isolates only under very low stringency (55 degrees C), which could be due to non-specific priming of the target DNA at that temperature. CONCLUSION: Repetitive elements IS1081 and IS990 may represent a more reliable alternative to the more widely used IS6110 PCR target for tuberculosis diagnosis.     

Simultaneous use of two PCR systems targeting IS6110 and MPB64 for confirmation of diagnosis of tuberculous lymphadenitis

PCR has emerged as a powerful technique for detection of various pathogens including Mycobacterium tuberculosis. In present study, eighty one samples of lymph node biopsies from clinically suspected cases of tuberculous lymphadenitis were examined for AFB, culture on L?wenstein Jensen medium and simultaneous use of two PCRs targeting IS6110 and MPB64. Positivity with M. tuberculosis culture and AFB was 13.6% and 28.4% respectively. All samples culture positive for nontuberculous mycobacteria were negative by both PCR systems. Higher proportion of positive results were observed with PCR targeting IS6110 by which 56 of 81 (69.1%) samples showed positive results as compared to PCR targeting MPB64 by which 39 of 81 (48.2 %) samples showed positive results. When combined, 63 out of 81 (77.8%) samples were detected positive for M. tuberculosis DNA. However, 7/81 (8.6 %) samples remained negative by IS6110 but positive by MPB64 method. Thus our data suggest that the use of one additional PCR (other than IS6110 system) can reduce false negativity of PCR results in the samples harboring zero copy of IS6110 element which is known to exist in Indian population.     

Papulonecrotic tuberculide and stenosis of the abdominal aorta

Papulonecrotic tuberculide (PNT) is a rare form of skin tuberculosis affecting predominantly young adults, with a history of immunity to Mycobacterium tuberculosis. We report a case of a young Caucasian female with PNT who was also documented to have a stenotic segment in the abdominal aorta. The difficulty in clarifying and treating the primary disease and the association between a tuberculous infection and Takayasu's arteritis are discussed.