Diagnosis of tuberculosis by nanoparticle-based immuno-PCR assay based on mycobacterial MPT64 and CFP-10 detection.

Aim: To improve the diagnostic accuracy of immuno-PCR (I-PCR) in tuberculosis (TB) patients by using functionalized gold nanoparticles (AuNPs) coupled with detection antibodies and oligonucleotides, and magnetic beads (MBs) conjugated with capture antibodies in the liquid phase. Materials & methods: MB-coupled AuNP-based I-PCR (MB-AuNP-I-PCR) assay was designed to detect a cocktail of Mycobacterium tuberculosis MPT64 and CFP-10 proteins in bodily fluids of TB patients. Results: The sensitivities of 89.3 (n = 94) and 78.1% (n = 73) were observed in pulmonary TB and extrapulmonary TB patients, respectively, with specificities of 97.9-98.3%. Notably, the sensitivities attained by MB-AuNP-I-PCR in smear-negative pulmonary TB and extrapulmonary TB patients were significantly higher (p < 0.05-0.001) than Magneto-ELISA and GeneXpert assay. Conclusion: The improved technology, as well as enhanced diagnostic accuracy of MB-AuNP-I-PCR, may lead to development of an attractive diagnostic kit.

Detection of mycobacterial CFP-10 (Rv3874) protein in tuberculosis patients by gold nanoparticle-based real-time immuno-PCR.

Aim: Timely and reliable diagnostic test for tuberculosis (TB) is immediately required. Attempts were made to improve the technology and diagnostic potential of real-time immuno-PCR (RT-I-PCR). Methods: We designed gold nanoparticle (GNP)-based RT-I-PCR (GNP-RT-I-PCR) assay for the detection of Mycobacterium tuberculosis CFP-10 (Rv3874) protein in clinical samples of TB patients. Results: A wide quantitative detection range of CFP-10 was found to be 0.5-5 × 104 pg/ml in bodily fluids of TB patients, which can evaluate the progression of disease. Moreover, sensitivities of 83.7 and 76.2% were observed in pulmonary (n = 49) and extrapulmonary TB (n = 42) patients, respectively, with specificities of 93.5-93.8% (n = 63). Conclusion: Conjugation of detection antibodies and oligonucleotides to functionalized GNPs of GNP-RT-I-PCR is relatively easier, compared with streptavidin-biotin/succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate system employed in RT-I-PCR. Our assay also showed better diagnostic performance than RT-I-PCR, which may provide a viable platform for the development of an efficient TB diagnostic test.

Detection of Mycobacterium tuberculosis lipoarabinomannan and CFP-10 (Rv3874) from urinary extracellular vesicles of tuberculosis patients by immuno-PCR.

Extracellular vesicles (EVs), the small circulating vesicles released from urine samples of tuberculosis (TB) patients, contain a pool of biomarkers. We recently detected Mycobacterium tuberculosis lipoarabinomannan (LAM) and CFP-10 (Rv3874) biomarkers from the urinary EVs of pulmonary TB (PTB) and extrapulmonary TB (EPTB) patients by immuno-polymerase chain reaction (I-PCR) assay and the results were compared with the analogous enzyme-linked immunosorbent assay (ELISA). The detection limits of both purified LAM and CFP-10 were determined to be 1 fg/mL with I-PCR, which was 106 times lower than ELISA. Detection of LAM and CFP-10 biomarkers in urinary EVs of TB patients by I-PCR showed superiority over ELISA. Notably, LAM I-PCR revealed sensitivities of 74.3 and 67.9% in PTB (n = 74) and EPTB (n = 53) patients, respectively, with specificities of 91.5-92.8% (n = 116). Moreover, the sensitivities attained with LAM I-PCR were significantly higher (P < 0.01) than with CFP-10 I-PCR. After further improving the sensitivity and specificity of the assay, our I-PCR based on LAM detection in urinary EVs may be used as an adjunct test for rapid diagnosis of TB.

Quantitative detection of a cocktail of mycobacterial MPT64 and PstS1 in tuberculosis patients by real-time immuno-PCR.

AIM: There is an urgent need to design a reliable diagnostic test for tuberculosis (TB). METHODS: Real-time immuno-PCR (RT-I-PCR) assay was devised for the quantitative detection of a cocktail of mycobacterial MPT64 (Rv1980c) and PstS1 (Rv0934) in TB patients. RESULTS: A broad dynamic range of 0.95 pg/ml-95 ng/ml of MPT64+PstS1 was detected in TB patients. In smear-positive (n = 59) and smear-negative (n = 42) pulmonary TB cases, sensitivities of 93.2 and 83.3% were observed, respectively with 92.8% specificity, whereas a sensitivity of 77.9% and a specificity of 91.3% were observed in extrapulmonary TB cases (n = 86). Furthermore, significantly reduced MPT64+PstS1 concentrations (p < 0.001) were noticed in patients on therapy by RT-I-PCR as compared with untreated patients. CONCLUSION: Our RT-I-PCR assay revealed high sensitivity especially for the rapid diagnosis of smear-negative pulmonary TB and paucibacillary extrapulmonary TB samples, which could also monitor the dynamics of disease in patients on therapy.

Bronchoscopic lung cryobiopsy: An Indian association for bronchology position statement.

BACKGROUND: Bronchoscopic lung cryobiopsy (BLC) is a novel technique for obtaining lung tissue for the diagnosis of diffuse parenchymal lung diseases. The procedure is performed using several different variations of technique, resulting in an inconsistent diagnostic yield and a variable risk of complications. There is an unmet need for standardization of the technical aspects of BLC. METHODOLOGY: This is a position statement framed by a group comprising experts from the fields of pulmonary medicine, thoracic surgery, pathology, and radiology under the aegis of the Indian Association for Bronchology. Sixteen questions on various technical aspects of BLC were framed. A literature search was conducted using PubMed and EMBASE databases. The expert group discussed the available evidence relevant to each question through e-mail and a face-to-face meeting, and arrived at a consensus. RESULTS: The experts agreed that patients should be carefully selected for BLC after weighing the risks and benefits of the procedure. Where appropriate, consideration should be given to perform alternate procedures such as conventional transbronchial biopsy or subject the patient directly to a surgical lung biopsy. The procedure is best performed after placement of an artificial airway under sedation/general anesthesia. Fluoroscopic guidance and occlusion balloon should be utilized for positioning the cryoprobe to reduce the risk of pneumothorax and bleeding, respectively. At least four tissue specimens (with at least two of adequate size, i.e., ≥5 mm) should be obtained during the procedure from different lobes or different segments of a lobe. The histopathological findings of BLC should be interpreted by an experienced pulmonary pathologist. The final diagnosis should be made after a multidisciplinary discussion. Finally, there is a need for structured training for performing BLC. CONCLUSION: This position statement is an attempt to provide practical recommendations for the performance of BLC in DPLDs.

Comparative evaluation of GeneXpert MTB/RIF and multiplex PCR targeting mpb64 and IS6110 for the diagnosis of pleural TB.

AIM: Diagnosis of pleural TB poses serious challenges due to paucibacillary nature of specimens and there is an urgent need to devise a reliable diagnostic test. METHODS: We compared GeneXpert Mycobacterium tuberculosis/rifampin assay and the multiplex PCR (M-PCR) targeting mpb64 (Rv1980c) and IS6110 in pleural fluids (n = 78) of pleural TB patients and non-TB controls. RESULTS: The sensitivities of 89.6 and 33.3%, and specificities of 96.7 and 100%, were observed with M-PCR and Xpert assay, respectively. CONCLUSION: M-PCR showed superiority over Xpert assay and may facilitate an efficient diagnosis of pleural TB.

Development of real-time immuno-PCR for the quantitative detection of mycobacterial PstS1 in tuberculosis patients.

A novel indirect real-time immuno-polymerase chain reaction (RT-I-PCR) assay, an evolution of I-PCR, was developed for the quantitative detection of Mycobacterium tuberculosis PstS1 (Rv0934) with a wide dynamic range of 10ng/mL to 1pg/mL in body fluids of tuberculosis (TB) patients, which may monitor the dynamics of disease.

Immuno-PCR, a new technique for the serodiagnosis of tuberculosis.

Rapid and accurate diagnosis of tuberculosis (TB) is essential to control the disease. The conventional microbiological tests have limitations and there is an urgent need to devise a simple, rapid and reliable point-of-care (POC) test. The failure of TB diagnostic tests based on antibody detection due to inconsistent and imprecise results has stimulated renewed interest in the development of rapid antigen detection methods. However, the World Health Organization (WHO) has emphasized to continue research for designing new antibody-based detection tests with improved accuracy. Immuno-polymerase chain reaction (I-PCR) combines the simplicity and versatility of enzyme-linked immunosorbent assay (ELISA) with the exponential amplification capacity and sensitivity of PCR thus leading to several-fold increase in sensitivity in comparison to analogous ELISA. In this review, we have described the serodiagnostic potential of I-PCR assays for an early diagnosis of TB based on the detection of potential mycobacterial antigens and circulating antibodies in body fluids of TB patients.

Serodiagnostic potential of immuno-PCR using a cocktail of mycobacterial antigen 85B, ESAT-6 and cord factor in tuberculosis patients.

A novel indirect immuno-polymerase chain reaction (I-PCR) assay was developed for the detection of circulating anti-Ag85B (antigen 85B, Rv1886c), anti-ESAT-6 (early secretory antigenic target-6, Rv3875) and anti-cord factor (trehalose 6,6'-dimycolate) antibodies from the sera samples of pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) patients and the results were compared with an analogous enzyme-linked immunosorbent assay (ELISA). We covalently attached the amino-modified reporter DNA to the dithiothreitol (DTT)-reduced anti-human IgG antibody through a chemical linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC). The detection of cocktail of anti-Ag85B, anti-ESAT-6 and anti-cord factor antibodies was found to be superior to the detection of individual antibodies. The sensitivities of 89.5% and 77.5% with I-PCR and 70.8% and 65% with ELISA were observed in smear-positive and smear-negative PTB cases, respectively with high specificity (90.9%). On the other hand, a sensitivity of 77.5% with I-PCR and 65% with ELISA was observed in EBTB cases. The detection of cocktail of antibodies by I-PCR is likely to improve the utility of existing algorithms for TB diagnosis.

Comparative Evaluation of Several Gene Targets for Designing a Multiplex-PCR for an Early Diagnosis of Extrapulmonary Tuberculosis.

PURPOSE: Diagnosis of extrapulmonary tuberculosis (EPTB) poses serious challenges. A careful selection of appropriate gene targets is essential for designing a multiplex-polymerase chain reaction (M-PCR) assay. MATERIALS AND METHODS: We compared several gene targets of Mycobacterium tuberculosis, including IS6110, devR, and genes encoding MPB-64 (mpb64), 38kDa (pstS1), 65kDa (hsp65), 30kDa (fbpB), ESAT-6 (esat6), and CFP-10 (cfp10) proteins, using PCR assays on 105 EPTB specimens. From these data, we chose the two best gene targets to design an M-PCR. RESULTS: Among all gene targets tested, mpb64 showed the highest sensitivity (84% in confirmed cases and 77.5% in clinically suspected cases), followed by IS6110, hsp65, 38kDa, 30kDa, esat6, cfp10, and devR. We used mpb64+IS6110 for designing an M-PCR assay. Our M-PCR assay demonstrated a high sensitivity of 96% in confirmed EPTB cases and 88.75% in clinically suspected EPTB cases with a high specificity of 100%, taking clinical diagnosis as the gold standard. CONCLUSION: These M-PCR results along with the clinical findings may facilitate an early diagnosis of EPTB patients and clinical management of disease.

Diagnosis of tuberculosis based on the detection of a cocktail of mycobacterial antigen 85B, ESAT-6 and cord factor by immuno-PCR.

Attempts were made to enhance the sensitivity of immuno-PCR assay based on the detection of cocktail of mycobacterial antigen 85B (Rv1886c), ESAT-6 (Rv3875) and cord factor (trehalose 6,6'-dimycolate) in pulmonary and extrapulmonary TB patients. Detection of Ag85B was found to be superior to the detection of cocktail in TB patients.

Diagnosis of pulmonary and extrapulmonary tuberculosis based on detection of mycobacterial antigen 85B by immuno-PCR.

We developed a novel indirect sandwich immuno-polymerase chain reaction (I-PCR) assay for the detection of mycobacterial antigen 85B (Ag85B, 30kDa, Rv1886c) in pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) patients. The amino-modified reporter DNA was covalently attached with the antidetection antibody through a heterobifunctional cross-linking agent succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. The detection limit of Ag85B by I-PCR was found to be 1 femtogram (fg)/mL, which was 10(6)-fold lower than an analogous enzyme-linked immunosorbent assay (ELISA). The sensitivities of 85% and 77% with I-PCR and 77.6% and 62.5% with ELISA were observed in smear-positive and smear-negative PTB patients, respectively, with high specificity. On the other hand, sensitivities of 84% and 63.7% with I-PCR and 68% and 47.5% with ELISA were observed in confirmed and clinically suspected EPTB cases, respectively, with high specificity.

Tracheal compression due to anomalous innominate artery in an adult.

A 47-year-old-female presented with dyspnea and unproductive cough for 4 months. General examination revealed pulsatile swelling in the midline below the thyroid cartilage present since childhood. Computed tomography-angiography of the neck showed right innominate artery dilated, elongated and coursing above downward, anterior to the trachea below the thyroid, compressing the trachea and origin of the right subclavian artery higher up. A case of anomalous innominate artery causing symptomatic compression of the trachea in adults is a rare entity.