Association of CD209 and CD209L polymorphisms with tuberculosis infection in a Northeastern Brazilian population.

Tuberculosis (TB) caused by Mycobacterium tuberculosis, is major cause of morbidity and mortality worldwide. So far, many candidate genes have been investigated for their possible association with TB. Dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3) grabbing non-integrin (DC-SIGN) and Liver/lymph node-specific intercellular adhesion molecule-grabbing non-integrin (L-SIGN), encoded by CD209 and CD209L genes respectively, are known for binding to M. tuberculosis on human dendritic cells and macrophages. We screened 4 single nucleotide polymorphisms (SNPs) in the promoter region of CD209, namely -939G>A (rs735240), -871A>G (rs735239), -336A>G (rs4804803) and -139G>A (rs2287886) and tandem repeat polymorphisms in exon 4 of CD209 and CD209L genes looking for association with TB in a Northeastern Brazilian population (295 subjects, 131 TB patients and 164 healthy controls). The -139G>A and -939G>A SNPs were associated with susceptibility to TB, and in particular with pulmonary and extra-pulmonary forms respectively. The -871A>G and -336A>G SNPs were associated, the first with protection to both pulmonary and extra-pulmonary TB, the latter only with the pulmonary form. An association between GGAG haplotype and protection to TB infection was also found. Also tandem repeat polymorphism in CD209L exon 4 was associated with TB infection. This study provides evidence of an association between CD209 and CD209L polymorphisms and TB development in a Brazilian population, suggesting that variations in these genes may influence the protection and susceptibility to infection caused by M. tuberculosis.

HIV-Induced Thymic Insufficiency and Aging-Related Immunosenescence on Immune Reconstitution in ART-Treated Patients.

The mechanisms underlying unsatisfactory immune reconstitution in HIV-1 positive patients under ART have not been fully elucidated, even after years of investigation. Thus, this study aimed to assess the correlation between age and thymic production profile, and its influence on inadequate immunological recovery. Here, 44 ART-treated patients with undetectable plasma HIV-1 load (<40 copies/mL) were classified as 31 immunological responders (IR) and 13 immunological non-responders (INR), according to their CD4+ T-cell count after 18 months of ART. The thymic function was assessed by identifying recent thymic emigrants (RTEs) CD4+ T cells (CD4+/CD45RA+CD31+) in PBMCs using flow cytometry. Clinical data were also analyzed from medical records. The INR group showed a higher age at ART initiation (41 ± 3.0) compared to the IR (33.7 ± 2.1) group (p = 0.041). Evaluating RTE CD4+ T-cells, we observed a lower percentage in the INR group (19.5 ± 6.3) compared to the IR group (29.9 ± 11.5) (p = 0.012). There was a strong negative correlation between age at ART initiation and RTE CD4+ T-cells in INRs (r = -0.784, p = 0.004). Our study has highlighted the thymic insufficiency and aging-related immunosenescence with unsatisfactory immunological recovery during ART in HIV-1 positive patients.

In vitro bioevaluation and docking study of dihydrosphingosine and ethambutol analogues against sensitive and multi-drug resistant Mycobacterium tuberculosis.

Tuberculosis remains a major public health problem and one of the top ten causes of death worldwide. The alarming increase in multidrug-resistant and extensively resistant variants (MDR, pre-XDR, and XDR) makes the disease more difficult to treat and control. New drugs that act against MDR/XDR strains are needed for programs to contain this major epidemic. The present study aimed to evaluate new compounds related to dihydro-sphingosine and ethambutol against sensitive and pre-XDR Mycobacterium strains, as well as to characterize the pharmacological activity through in vitro and in silico approaches in mmpL3 protein. Of the 48 compounds analyzed, 11 demonstrated good to moderate activity on sensitive and MDR Mycobacterium tuberculosis (Mtb), with a Minimum Inhibitory Concentration (MIC) ranging from 1.5 to 8 µM. They presented 2 to 14 times greater potency of activity when compared to ethambutol in pre-XDR strain, and demonstrated a selectivity index varying between 2.21 and 82.17. The substance 12b when combined with rifampicin, showed a synergistic effect (FICI = 0.5) on sensitive and MDR Mtb. It has also been shown to have a concentration-dependent intracellular bactericidal effect, and a time-dependent bactericidal effect in M. smegmatis and pre-XDR M. tuberculosis. The binding mode of the compounds in its cavity was identified through molecular docking and using a predicted structural model of mmpL3. Finally, we observed by transmission electron microscopy the induction of damage to the cell wall integrity of M. tuberculosis treated with the substance 12b. With these findings, we demonstrate the potential of a 2-aminoalkanol derivative to be a prototype substance and candidate for further optimization of molecular structure and anti-tubercular activity in preclinical studies.

Performance of IS6110-LAMP assay for detection of Mycobacterium tuberculosis complex in blood and urine samples from patients with extrapulmonary tuberculosis.

BACKGROUND: The diagnosis of extrapulmonary tuberculosis (EPTB) shows numerous difficulties because of non-specific symptomatology and low sensitivity of conventional methods. Loop-mediated isothermal amplification (LAMP) is a fast and low-cost technique, which can amplify under isothermal conditions an amount of target DNA copies into approximately a billion copies. OBJECTIVE: The present study aimed to evaluate a IS6110-LAMP system for Mycobacterium tuberculosis detection in blood and urine samples from patients with EPTB. METHODS: The collected samples (n = 122) were stratified in two groups: Group EPTB - patient samples with confirmed EPTB (n = 61); Group non-TB - patient samples without TB (n = 61). The urine samples underwent decontamination, and the components of blood samples were separated (plasma and PBMC). DNA extractions were performed in all biological samples followed by IS6110-LAMP assay technique. The detection limit was evaluated through dilution curves (1:10) using Mtb reference strain (H37Rv) genomic DNA. FINDINGS: The detection limit of IS6110-LAMP was 10 fg/µL (∼10-20 bacilli/µL). The IS6110-LAMP technique sensitivity and specificity were 95.65 % and 79.25 %, respectively, with a general kappa agreement index of 0.762. MAIN CONCLUSIONS: Based on these results, IS6110-LAMP test showed considerable diagnostic parameters, being able to aid in the speed and accuracy of the final EPTB diagnosis.

Identification of nontuberculous mycobacteria species by multiplex real-time PCR with high-resolution melting.

INTRODUCTION: Nontuberculous mycobacteria (NTM) species, as human pathogens, are increasing in the world, as is the difficulty of accurately identifying them. Differential diagnosis, especially between the M. tuberculosis complex and NTM species, and the characterization of NTM species is important. This study aimed to evaluate the performance of a molecular system based on multiplex real-time PCR with high-resolution melting (HRM) for the identification and differentiation of NTM species of clinical importance of an endemic area for tuberculosis in northeastern Brazil. METHODS: The technical protocol of the molecular system was based on multiplex real-time PCR-HRM, and evaluated the sensitivity and specificity of the detection of NTM species in mycobacterial clinical isolates from the studied region. The gold standard method was specific gene sequencing. RESULTS: The sensitivity and specificity of multiplex real-time PCR-HRM modified for differentiation between NTM and M. tuberculosis were 90% and 100%, respectively. The PCR-HRM sensitivities for the characterization of NTM species (M. kansasii, M. abscesses, M. avium, and M. fortuitum) were 94.59%, 80%, 57.14%, and 54%, respectively. CONCLUSIONS: The multiplex real-time PCR-HRM modified assay has the potential to rapidly and efficiently identify nontuberculous mycobacteria of clinical importance, which is crucial for immediate implementation of the appropriate therapy and thus avoiding complications and sequelae in patients.

TST conversions and systemic interferon-gamma increase after methotrexate introduction in psoriasis patients.

BACKGROUND: Tuberculosis screening in psoriasis patients is complex due to the immunological alterations associated with psoriasis, the presence of comorbidities, and the effect of immunosuppressive treatment. However, it is not established whether the results of screening tests are affected by these factors in psoriasis patients. OBJECTIVES: To determine whether there is a change in the results of the tuberculin skin test (TST) or the interferon-gamma release assay (IGRA) in psoriasis patients living in tuberculosis (TB)-endemic area after 12 weeks of methotrexate (MTX) treatment and to investigate the association of the test results with clinical and inflammatory markers. METHODS: Forty-five patients were selected for a prospective single-arm self-controlled study and followed for at least 18 months. The TST, IGRA, Psoriasis Area and Severity Index (PASI), and inflammatory factors (erythrocyte sedimentation rate (ESR), C-reactive protein, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha levels), were determined before and after 12 weeks of oral 15 mg per week MTX administration and compared. The associations between the IGRA and TST results were verified before and after treatment according to inflammatory factors and clinical characteristics (age, blood glucose, weight, body mass index, disease duration, and PASI). RESULTS: We collected data on 25 patients who completed the full course of therapy and the follow-up. None of the patients developed TB. TST positivity was significantly elevated at week 12 (25% baseline vs 44% at week 12, P < 0.037). Three IGRAs followed the TST conversions. There was no difference between TST and IGRA pre- or posttreatment. Serum IFN-γ increased significantly in week 12 (15.95 pg/ml baseline vs 18.82 pg/ml at week 12, P < 0.005) and tended to be higher among TST-positive patients (P = 0.072). The baseline IGRA was associated with a higher ESR (P = 0.038). None of the test results were associated with clinical characteristics. CONCLUSIONS: In addition to the classic booster effect, TST conversions in patients using MTX can occur due to an increase in IFN-γ. However, it is not possible to exclude true TST conversions. Therefore, other diagnostic methods, like IGRA or chest tomography, should be used when the TST has intermediate results.

Performance of the IS6110-TaqMan® assay in the diagnosis of extrapulmonary tuberculosis from different biological samples.

INTRODUCTION: This study evaluated the performance of the IS6110-TaqMan® assay in different types of biological samples and tissues for laboratory diagnosis of extrapulmonary tuberculosis. METHODS: 143 biological samples and tissues from patients with suspected extrapulmonary tuberculosis from the health services of Recife/Pernambuco/Brazil were evaluated with the IS6110-TaqMan® assay. RESULTS: The sensitivities of the IS6110-TaqMan® assay calculated for blood, urine, both blood and urine samples, tissue biopsies, extrapulmonary body fluid samples, and all samples from patients calculated together were 55.9%, 33.3%, 68.8%, 43.8%, 29.6%, and 73.7%, respectively, and the specificities were 80%, 100%, 78.6%, 100%, 100%, and 84.2%, respectively. CONCLUSIONS: The accuracy of qPCR was high in various clinical sample types. The analysis of more than one type of clinical sample collected from the same patient with extrapulmonary tuberculosis enhances the diagnostic power of the IS6110-TaqMan® assay when compared with the use of only one clinical sample.

DEFB1 gene polymorphisms and tuberculosis in a Northeastern Brazilian population.

ß-Defensin-1, an antimicrobial peptide encoded by the DEFB1 gene, is known to play an important role in lung mucosal immunity. In our association study we analyzed three DEFB1 functional polymorphisms -52G>A (rs1799946), -44C>G (rs1800972) and -20G>A (rs11362) in 92 tuberculosis patients and 286 healthy controls, both from Northeast Brazil: no association was found between the studied DEFB1 polymorphisms and the disease. However we cannot exclude that this lack of association could be due to the low number of subjects analyzed, as suggested by the low statistical power achieved for the three analyzed SNPs (values between 0.16 and 0.50).

Identification of nontuberculous mycobacteria species by multiplex real-time PCR with high-resolution melting

Abstract INTRODUCTION: Nontuberculous mycobacteria (NTM) species, as human pathogens, are increasing in the world, as is the difficulty of accurately identifying them. Differential diagnosis, especially between the M. tuberculosis complex and NTM species, and the characterization of NTM species is important. This study aimed to evaluate the performance of a molecular system based on multiplex real-time PCR with high-resolution melting (HRM) for the identification and differentiation of NTM species of clinical importance of an endemic area for tuberculosis in northeastern Brazil. METHODS: The technical protocol of the molecular system was based on multiplex real-time PCR-HRM, and evaluated the sensitivity and specificity of the detection of NTM species in mycobacterial clinical isolates from the studied region. The gold standard method was specific gene sequencing. RESULTS: The sensitivity and specificity of multiplex real-time PCR-HRM modified for differentiation between NTM and M. tuberculosis were 90% and 100%, respectively. The PCR-HRM sensitivities for the characterization of NTM species (M. kansasii, M. abscesses, M. avium, and M. fortuitum) were 94.59%, 80%, 57.14%, and 54%, respectively. CONCLUSIONS The multiplex real-time PCR-HRM modified assay has the potential to rapidly and efficiently identify nontuberculous mycobacteria of clinical importance, which is crucial for immediate implementation of the appropriate therapy and thus avoiding complications and sequelae in patients.

Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine.

INTRODUCTION: Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. METHODS: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). RESULTS: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). CONCLUSIONS: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.

Osteoarticular tuberculosis in an HIV-positive patient: a case report.

The authors report a case of a 38-year-old HIV-positive woman, with subcutaneous nodules on the thoracic region with 3 months of evolution. Clinical, laboratory, and epidemiological features were evaluated and associated with apparent damage to the T11-T12 vertebrae, identification by imaging tests, positivity in a polymerase chain reaction-based test, and reactivity to the Mantoux tuberculin skin test (PPD-RT 23). The patient was diagnosed with osteoarticular tuberculosis and received treatment for a year, and clinical cure was achieved.

Rapid detection and differentiation of mycobacterial species using a multiplex PCR system.

INTRODUCTION: The early diagnosis of mycobacterial infections is a critical step for initiating treatment and curing the patient. Molecular analytical methods have led to considerable improvements in the speed and accuracy of mycobacteria detection. METHODS: The purpose of this study was to evaluate a multiplex polymerase chain reaction system using mycobacterial strains as an auxiliary tool in the differential diagnosis of tuberculosis and diseases caused by nontuberculous mycobacteria (NTM) RESULTS: Forty mycobacterial strains isolated from pulmonary and extrapulmonary origin specimens from 37 patients diagnosed with tuberculosis were processed. Using phenotypic and biochemical characteristics of the 40 mycobacteria isolated in LJ medium, 57.5% (n=23) were characterized as the Mycobacterium tuberculosis complex (MTBC) and 20% (n=8) as nontuberculous mycobacteria (NTM), with 22.5% (n=9) of the results being inconclusive. When the results of the phenotypic and biochemical tests in 30 strains of mycobacteria were compared with the results of the multiplex PCR, there was 100% concordance in the identification of the MTBC and NTM species, respectively. A total of 32.5% (n=13) of the samples in multiplex PCR exhibited a molecular pattern consistent with NTM, thus disagreeing with the final diagnosis from the attending physician. CONCLUSIONS: Multiplex PCR can be used as a differential method for determining TB infections caused by NTM a valuable tool in reducing the time necessary to make clinical diagnoses and begin treatment. It is also useful for identifying species that were previously not identifiable using conventional biochemical and phenotypic techniques.

Bone tuberculosis: a case report on child.

The authors report a case of a 12-year-old child with a complaint of pain and deformity in the lower thoracic region that had lasted for two years. Clinical, epidemiological and laboratory characteristics associated with images of apparent damage in the T9-T10 and T11-T12 vertebrae taken by radiography of the thoracic spine and nuclear magnetic resonance in addition to the positivity of the molecular test based on the polymerase chain reaction, led to tuberculous spondylitis being diagnosed and specific therapy was started. Culture of vertebral biopsy was positive for Mycobacterium tuberculosis after thirty days.

Performance of the IS6110-TaqMan® assay in the diagnosis of extrapulmonary tuberculosis from different biological samples

Abstract INTRODUCTION: This study evaluated the performance of the IS6110-TaqMan® assay in different types of biological samples and tissues for laboratory diagnosis of extrapulmonary tuberculosis. METHODS: 143 biological samples and tissues from patients with suspected extrapulmonary tuberculosis from the health services of Recife/Pernambuco/Brazil were evaluated with the IS6110-TaqMan® assay. RESULTS: The sensitivities of the IS6110-TaqMan® assay calculated for blood, urine, both blood and urine samples, tissue biopsies, extrapulmonary body fluid samples, and all samples from patients calculated together were 55.9%, 33.3%, 68.8%, 43.8%, 29.6%, and 73.7%, respectively, and the specificities were 80%, 100%, 78.6%, 100%, 100%, and 84.2%, respectively. CONCLUSIONS The accuracy of qPCR was high in various clinical sample types. The analysis of more than one type of clinical sample collected from the same patient with extrapulmonary tuberculosis enhances the diagnostic power of the IS6110-TaqMan® assay when compared with the use of only one clinical sample.

Detection of Mycobacterium tuberculosis complex by nested polymerase chain reaction in pulmonary and extrapulmonary specimens.

OBJECTIVE: To compare the performance of nested polymerase chain reaction (NPCR) with that of cultures in the detection of the Mycobacterium tuberculosis complex in pulmonary and extrapulmonary specimens. METHODS: We analyzed 20 and 78 pulmonary and extrapulmonary specimens, respectively, of 67 hospitalized patients suspected of having tuberculosis. An automated microbial system was used for the identification of Mycobacterium spp. cultures, and M. tuberculosis IS6110 was used as the target sequence in the NPCR. The kappa statistic was used in order to assess the level of agreement among the results. RESULTS: Among the 67 patients, 6 and 5, respectively, were diagnosed with pulmonary and extrapulmonary tuberculosis, and the NPCR was positive in all of the cases. Among the 98 clinical specimens, smear microscopy, culture, and NPCR were positive in 6.00%, 8.16%, and 13.26%, respectively. Comparing the results of NPCR with those of cultures (the gold standard), we found that NPCR had a sensitivity and specificity of 100% and 83%, respectively, in pulmonary specimens, compared with 83% and 96%, respectively, in extrapulmonary specimens, with good concordance between the tests (kappa, 0.50 and 0.6867, respectively). CONCLUSIONS: Although NPCR proved to be a very useful tool for the detection of M. tuberculosis complex, clinical, epidemiological, and other laboratory data should also be considered in the diagnosis and treatment of pulmonary and extrapulmonary tuberculosis.

The performance of an in-house nested-PCR technique for pleural tuberculosis diagnoses.

INTRODUCTION: This study evaluated the performance of an in-house nested-PCR system for the detection of the Mycobacterium tuberculosis complex in pleural fluid, blood and urine samples from pleural effusion tuberculosis patients by health services physicians in Pernambuco, Brazil. METHODS: A prospective double-blind study with 37 hospitalized patients of both sexes, aged over 15, was used to investigate the diagnosis of pleural effusion. The criteria used to define the cases included the demonstration of bacillus in biological samples by smear or culture or by a granulomatous finding in the histopathological examination, associated with an evident response to specific treatments to each clinical situation. Pleural fluid, blood and urine samples were collected and subjected to routine tests and the nested PCR technique to assess for M. tuberculosis amplification. RESULTS: In total, 37 pleural effusion patients took part in the study, of whom 19 (51.3%) had tubercular etiologies and 18 (48.7%) had etiologies from other causes. When the pleural fluid, blood and/or urine sample in-house nested-PCR sensitivities were evaluated simultaneously, the results were positive regardless of the biological specimen (the sensitivity was 84.2%); however, when the blood and/or urine samples were analyzed together, the sensitivity was 72.2%. When the pleural fluid samples were evaluated alone, the sensitivity was only 33.3%. CONCLUSIONS: The performance of the diagnostic pleural tuberculosis nested-PCR was directly related to the diversity of the samples collected from the same patient. Additionally, this study may identify a need to prioritize non-invasive blood and urine collection for this diagnosis.

MBL2 gene polymorphisms and susceptibility to tuberculosis in a northeastern Brazilian population.

The innate immune system represents the first line of host defense against pathogens. Genetics factors regulating the immune responses play a role in the susceptibility to infectious diseases, such as tuberculosis (TB). We analyzed MBL2 promoter and exon 1 functional single nucleotide polymorphisms (SNPs) in a group of 155TB patients and 148 healthy controls in order to evaluate their influence on the onset of infection and TB development. There was no association between MBL2 -550 HL promoter polymorphisms and susceptibility to develop TB, but heterozygous -221 Y/X genotype was significantly more frequent in pulmonary TB patients than controls. Moreover, MBL2 exon 1 O allele, was significantly associated with susceptibility to TB development in general (p=0.023, OR=1.61, 95% CI 1.05-2.49) and pulmonary TB (p=0.0008, OR=2.16, 95% CI 1.35-3.46); C allele at codon 57, as well as A/C genotype, were significantly more frequent in TB patients than in controls. Our results indicate that MBL2 polymorphisms, especially at codon 57, could be considered as risk factors for TB development.

DEFB1 gene polymorphisms and tuberculosis in a Northeastern Brazilian population

Abstract β-Defensin-1, an antimicrobial peptide encoded by the DEFB1 gene, is known to play an important role in lung mucosal immunity. In our association study we analyzed three DEFB1 functional polymorphisms -52G>A (rs1799946), -44C>G (rs1800972) and -20G>A (rs11362) in 92 tuberculosis patients and 286 healthy controls, both from Northeast Brazil: no association was found between the studied DEFB1 polymorphisms and the disease. However we cannot exclude that this lack of association could be due to the low number of subjects analyzed, as suggested by the low statistical power achieved for the three analyzed SNPs (values between 0.16 and 0.50).

Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine

Abstract: INTRODUCTION : Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. METHODS: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). RESULTS: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). CONCLUSIONS: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.

[Differentiation of micobacteria by multiplex PCR]. / Diferenciação de micobactérias por PCR multiplex.

This study aimed to optimize a method based on the polymerase chain reaction - multiplex PCR - for differentiation of mycobacteria species of interest for public health. The multiplex PCR was based on simultaneous amplification of the hsp65 gene, which is present in all species of the Mycobacterium genus, the dnaJ gene, which is present only in Mycobacterium tuberculosis and Mycobacterium avium and the IS6110 insertion sequence, which is present in the Mycobacterium tuberculosis complex, generating amplicons of 165 bp, 365 bp and 541 bp, respectively. The detection limit was 1 fg for the hsp65 target, 100 pg for dnaJ and 0.1 fg for IS6110. The multiplex PCR detected down to 100 pg of DNA of Mycobacterium tuberculosis. The system was shown to be specific and sensitive for detection of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. The results obtained using reference strains of mycobacteria showed that multiplex PCR may be a fast, sensitive and specific tool for differentiation of mycobacteria.