• Mycobacterium tuberculosis
• Colonoscopy
• Diagnosis
• Endoscopic findings
• Histologic findings
• Immunohistochemistry
• Intestinal tuberculosis
• Real-time Polymerase Chain Reaction

• Humans
• Tuberculosis, Gastrointestinal/diagnosis
• Tuberculosis, Gastrointestinal/microbiology
• Peru
• Male
• Female
• Retrospective Studies
• Adult
• Real-Time Polymerase Chain Reaction
• Middle Aged
• Immunohistochemistry
• Aged
• Young Adult
• Granuloma/diagnosis
• Granuloma/microbiology
• Granuloma/pathology
• Mycobacterium tuberculosis/isolation & purification
• Mycobacterium tuberculosis/genetics
• Adolescent
• Sensitivity and Specificity

• Immunology
• Tuberculosis diagnosis
• Tuberculosis pathogenesis
• Tuberculosis treatment

• Humans
• Algorithms
• Culture Techniques
• Extensively Drug-Resistant Tuberculosis
• History, 15th Century
• History, 16th Century
• History, 17th Century
• History, 18th Century
• History, 19th Century
• History, 20th Century
• History, Ancient
• Interferon-gamma Release Tests
• Mycobacterium tuberculosis
• Nucleic Acid Amplification Techniques
• Polymerase Chain Reaction
• Tuberculin Test
• Tuberculosis/diagnosis
• Tuberculosis/epidemiology
• Tuberculosis/history
• Tuberculosis/immunology
• Tuberculosis, Multidrug-Resistant
• Tuberculosis, Pulmonary/diagnosis
• Tuberculosis, Pulmonary/epidemiology
• Tuberculosis, Pulmonary/history
• Tuberculosis, Pulmonary/immunology

Tuberculosis (TB) is ranked as the top killer among infectious diseases worldwide. Early and accurate diagnosis of the disease is crucial to end the global TB epidemic. The current commercially available molecular tests are still unaffordable by most TB affected communities. Herein, we developed a novel rapid and sensitive diagnostic method to detect the IS6110 sequence of Mycobacterium tuberculosis (M. tuberculosis) complex using PCR-magnetic bead ELISA. PCR amplification ofa 123 bp repetitive sequence of the IS6110 gene was performed by using digoxigenin (DIG) and biotin-labelled primers. Streptavidin-conjugated magnetic beads were used to collect the dual-labelled amplicons and subsequently, colourimetric detection was done by using horseradish peroxidase (HRP)-conjugated anti-DIG antibody. This method is able to detect M. tuberculosis DNA down to 0.5 fg per reaction within 3 hours. The sensitivity of IS6110 PCR detection by magnetic bead ELISA is 100 times higher than that of conventional agarose gel electrophoresis. The assay specificity was determined using a panel of DNA extracted from 10 common bacteria causing lower respiratory tract infections. No cross-reactivity was detected from those bacteria by IS6110 PCR-magnetic bead ELISA. Thus, the novel highly sensitive and specific, reduced assay time and simplicity of this PCR-magnetic bead ELISA for the detection of the specific gene of M. tuberculosis complex makes it an attractive diagnostic tool for large-scale screening of tuberculosis in standard clinical laboratories.

Tuberculosis (TB) is ranked as the top killer among infectious diseases worldwide.

The diagnosis of extrapulmonary tuberculosis is still a challenge because of its pauci-bacillary nature. The aim of the study was to evaluate the role of a multiplex PCR assay in the diagnosis of extrapulmonary tuberculosis and to compare the efficiency of two targets, IS6110 and MPB64 to detect Mycobacterium tuberculosis.

150 extrapulmonary samples (61 pus/aspirate, 46 tissue, 32 body fluids, and 11 urine) from clinically suspected cases of tuberculosis were included in the study. All the samples were subjected to direct fluorescent microscopy, TB culture (BacT/ALERT 3D, biomerieux, Durham, North Carolina, USA) and a Multiplexed Tandem PCR targeting two mycobacterial DNA sequences, IS6110 and MPB64. Master-Mix reagents and primers were prepared by AusDiagnostics Pvt. Ltd (Alexandria, New South Wales, Australia). The performance of the assay was assessed using a composite gold standard, which included clinical characteristics, microbiology smear as well as culture, histopathology, cytology, radiology, and response to antitubercular therapy.

20.3%, 23.6%, and 45.3% of specimens were positive by smear, culture, and PCR, respectively. The sensitivity and specificity of the multiplex PCR was 91.9% and 88.4%, respectively, using the composite gold standard. Positive and negative predictive values of the PCR were estimated as 85.1% and 93.8%, respectively. Higher positivity was observed with target IS6110 (44.6%) as compared to target MPB64 (18.9%). The sensitivities of IS6110 and MPB64 individual targets were 90.3% and 64.5%, respectively, and specificities were 88.4% and 97.7%, respectively.

PCR can play an important role in rapid and accurate diagnosis of extrapulmonary tuberculosis. IS6110 alone is an effective target in our part of the country.

• Extrapulmonary tuberculosis
• IS6110
• MPB64
• Multiplex PCR

• Antigens, Bacterial/genetics
• Bacteriological Techniques/methods
• Culture Media
• DNA, Bacterial/analysis
• Gene Amplification
• Humans
• Multiplex Polymerase Chain Reaction
• Mycobacterium tuberculosis/genetics
• Sensitivity and Specificity
• Tuberculosis/diagnosis

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.

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.

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).

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.

• IS6110 sequence
• Mycobacterium tuberculosis
• Nested polymerase chain reaction
• Pleural tuberculosis

• BACTEC culture
• Pott's disease
• Ziehl-Neelsen microscopy
• histopathological and molecular method
• tubercular spine

AIM: To investigate the utility of immunohistochemical (IHC) staining with an antibody to Mycobacterium tuberculosis (M. tuberculosis) for the diagnosis of intestinal tuberculosis (TB).

METHODS: We retrospectively identified 10 patients (4 males and 6 females; mean age = 65.1 ± 13.6 years) with intestinal TB. Clinical characteristics, including age, gender, underlying disease, and symptoms were obtained. Chest radiograph and laboratory tests, including sputum Ziehl-Neelsen (ZN) staining, M. tuberculosis culture, and sputum polymerase chain reaction (PCR) for tubercle bacilli DNA, as well as Tuberculin skin test (TST) and QuantiFERON-TB gold test (QFT), were examined. Colonoscopic records recorded on the basis of Sato’s classification were also reviewed, in addition to data from intestinal biopsies examined for histopathological findings, including hematoxylin and eosin staining, and ZN staining, as well as M. tuberculosis culture, and PCR for tubercle bacilli DNA. For the present study, archived formalin-fixed paraffin-embedded (FFPE) intestinal tissue samples were immunohistochemically stained using a commercially available species-specific monoclonal antibody to the 38-kDa antigen of the M. tuberculosis complex. These sections were also stained with the pan-macrophage marker CD68 antibody.

RESULTS: From the clinical data, we found that no patients were immunocompromised, and that the main symptoms were diarrhea and weight loss. Three patients displayed active pulmonary TB, six patients (60%) had a positive TST, and 4 patients (40%) had a positive QFT. Colonoscopic findings revealed that all patients had type 1 findings (linear ulcers in a circumferential arrangement or linear ulcers arranged circumferentially with mucosa showing multiple nodules), all of which were located in the right hemicolon and/or terminal ileum. Seven patients (70%) had concomitant healed lesions in the ileocecal area. No acid-fast bacilli were detected with ZN staining of the intestinal tissue samples, and both M. tuberculosis culture and PCR for tubercle bacilli DNA were negative in all samples. The histopathological data revealed that tuberculous granulomas were present in 4 cases (40%). IHC staining in archived FFPE samples with anti-M. tuberculosis monoclonal antibody revealed positive findings in 4 patients (40%); the same patients in which granulomas were detected by hematoxylin and eosin staining. M. tuberculosis antigens were found to be mostly intracellular, granular in pattern, and primarily located in the CD68⁺ macrophages of the granulomas.

CONCLUSION: IHC staining with a monoclonal antibody to M. tuberculosis may be an efficient and simple diagnostic tool in addition to classic examination methods for the diagnosis of intestinal TB.

• Colonoscopy
• Intestinal tuberculosis
• Immunohistochemistry
• Monoclonal antibody
• Mycobacterium tuberculosis

Worldwide, tuberculosis (TB) is a major public health problem and the rapid diagnosis and appropriate chemotherapy become the first priority and a serious challenge to improve TB treatment. In the objective of early TB diagnosis and rapid detection of Mycobacterium tuberculosis (MTB) in the clinical specimens, the utility of the Polymerase Chain Reaction (PCR) using the Insertion Sequence 6110 “IS6110" as target was compared to conventional methods.

Out of 305 patients with different clinical manifestations: suspected, new, drug relapse, drug failure and chronic cases were enrolled in this study and tested by mycobacteriological and PCR techniques for the investigation about the tubercle bacilli.

The results of the in house “IS6110" PCR showed a good sensitivity (92.4%) and high specificity (98.0%), the positive and negative predictive values were 96.4 % and 95.3 % respectively.

This study showed clearly that the PCR testing using the “IS6110" in the routine analysis is a potential tool for the rapid TB diagnosis, especially for critical cases and would be of great interest to help the clinician in the misdiagnosed critical cases by the traditional radiology.

• IS6110
• Mycobacterium tuberculosis
• PCR
• Tuberculosis

• IS6110
• Mycobacterium tuberculosis
• Phylogenetic analysis
• Tuberculosis diagnosis

Piezoelectric DNA-based biosensor technology was developed as a new method for detection of M. tuberculosis. This method consists of immobilizing a thiol-modified oligonucleotide probe on the gold electrode surface of a quartz crystal, using a self-assembled monolayer method. The advantage of this study is that a non-amplified genomic bacterial DNA target was used. Instead, the genomic DNA was digested by restriction enzyme to obtain DNA fragments containing the target sequence. The fabricated biosensor was evaluated through an examination of 200 samples. No cross hybridization were observed against M. avium complex and other microorganisms. This target DNA preparation, without PCR amplification, will reduce time, costs, and the tedious step of amplification.

• Mycobacterium tuberculosis
• non-amplified genomic DNA
• piezoelectric biosensor

Comparative genomic analysis of M. tuberculosis H37Rv and M. bovis BCG have shown that 16 RDs (Regions of Differences) are deleted in BCG and have shown six deletion regions in M. tuberculosis H37Rv. RD1, is present in M. tuberculosis but is absent in all M. bovis BCG sub-strains. A study from Kerala, a south-western coastal state of India aimed to find out differences in RD1 region showed for the first time the presence of moaA3 gene in majority of their clinical isolates, that was absent in type strain H37Rv. We attempted to find out such polymorphism between type strains and the clinical isolates within RD1, targeting moaA3 gene among the clinical isolates of Tamil Nadu & Pondicherry, south-eastern coastal states of India

One hundred and sixteen clinical isolates of M. tuberculosis were included in the study. PCR using RD1DLa and RD1DRa primers was carried out to amplify a 652 bp fragment, encoding for cfp10 and esat 6 proteins of RD1. A second PCR using primers designed from the surrounding regions of moaA3 gene was done to confirm the presence of the full Open Reading Frame (ORF) in clinical isolates.

In M. tuberculosis H37Rv the expected 652 bp band was present. In BCG it was absent as expected, but a 386 bp fragment was amplified. Around 12/116 (10.3%) of our clinical isolates showed both 652 and 386 bp fragments. The additional 386 bp amplicon is a part of the moaA3 gene which codes for molybdopterin cofactor protein A in M. bovis. The second PCR amplified the flanking sequence of moaA3 and yielded the expected amplicon of 1254 bp in all those 10.3% of clinical isolates which had the 386 bp fragment. However the earlier study carried out in Kerala, reported the presence of moaA3 gene in majority (97%) of their clinical isolates.

This finding showed that there was regional variation presenting polymorphism in moA3 gene, among the strains of M. tuberculosis and further strengthens the speculation of genetic differences among the strains of Kerala and Tamil Nadu & Pondicherry, the South Indian states