Netrapal Singh

Diagnosis of extrapulmonary tuberculosis by PCR

During the last two decades, the resurgence of tuberculosis (TB) has been documented in both developed and developing nations, and much of this increase in TB burden coincided with human immunodeficiency virus (HIV) epidemics. Since then, the disease pattern has changed with a higher incidence of extrapulmonary tuberculosis (EPTB) as well as disseminated TB. EPTB cases include TB lymphadenitis, pleural TB, TB meningitis, osteoarticular TB, genitourinary TB, abdominal TB, cutaneous TB, ocular TB, TB pericarditis and breast TB, although any organ can be involved. Diagnosis of EPTB can be baffling, compelling a high index of suspicion owing to paucibacillary load in the biological specimens. A negative smear for acid-fast bacilli, lack of granulomas on histopathology and failure to culture Mycobacterium tuberculosis do not exclude the diagnosis of EPTB. Novel diagnostic modalities such as nucleic acid amplification (NAA) can be useful in varied forms of EPTB. This review is primarily focused on the diagnosis of several clinical forms of EPTB by polymerase chain reaction (PCR) using different gene targets.

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 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.

Gene Xpert MTB/RIF Assay: A New Hope for Extrapulmonary Tuberculosis

The diagnostic efforts for extrapulmonary tuberculosis (TB) have been severely hampered by the lack of diagnostic tests that are accurate, simple to use and can be applied at the point of clinical care. This has been further enhanced by the widespread inability to test for drug resistance. Gene Xpert® MTB/RIF test is a novel test for the diagnosis of extrapulmonary TB and rifampicin resistance. This unique test can be used almost everywhere with minimal technical expertise, enabling diagnosis of extrapulmonary TB and simultaneous assessment of rifampicin resistance to be completed within 2 h. In low-income countries, however, its cost, environmental limitations and difficulties involved in supply and maintenance are major obstacles. While it may be suitable for major reference hospitals, operational research is needed to evaluate the test and its additional yield above high-quality smear microscopy. In high TB burden countries like India, WHO in 2010 endorsed some guidelines for implantation of Gene Xpert® MTB/RIF test for diagnosis of TB and drug resistance.

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.

Genetic analysis in intra- and inter-specific crosses of tree willows

Twenty one full-sib families derived from intra- and interspecific controlled crosses were analysed to study different genetic parameters. Estimates of the general combining ability (GCA) variance were found to be less than the specific combining ability (SCA) variance for all the parameters. The dominance variance was more than the additive variance for all the parameters studied. The degree of dominance ranged between 1.73 (per cent successful crosses) and 5.35 (% germination). Moderately high (0.42 for % successful crosses) to low (0.15 for seed per capsule) narrow sense heritibilities and high heritability in broad sense (0.74 to 0.99) for per cent successful crosses and seeds per catkin was recorded. GCA effects were positive and significant for S. tetrasperma (TFB), S. tetrasperma (TWE) and S. tetrasperma (LNM) male parents for all the parameters while S. alba (SE- 63–007) exhibited significantly positive effects for number of seeds/catkin. Among females, S. tetrasperma (LP), S. tetrasperma (LNF) and S. tetrasperma (LN) depicted positive and significant GCA effects for number of seeds/catkin and per cent germination, respectively whereas crosses, S. matsudana (PN-227) x S. alba (Kashmiri) and S. tetrasperma (LNF) x S. tetrasperma (LNM) exhibited positive and significant SCA effects for per cent successful crosses. Cross S. matsudana (PN-227) x S. alba (Kashmiri) showed positive significant SCA effects for number of seeds/capsule.

Studies on heterotic parameters in Populus deltoides

Exploitation of genetic variability of Populus deltoides Bartr. through control crossing involving Line x Tester mating design was attempted with the objective of selection of suitable clones as well as parents for hybridization in future and identification of best families and best individuals with in family for releasing first stage clones. Lines ST-72, D-121, G-48, exhibited the highest positive GCA effects for plant height, collar diameter, plant height at first branch and root length. In general G-48 also proved to be the best combiner for survival percentage, inter nodal length and leaf area. Among the testers ST-63 and ST-70 expressed highest GCA effects and excelled for a number of desirable characters. Full sib families (F1s) of D-121 x ST-70 and G-48 x ST-63 showed desirable SCA for plant height and collar diameter and therefore F1 hybrids of these crosses are recommended at first stage of selections. The percent contribution of line x tester interactions was higher than that of lines and testers except number of leaves per plant. 13 traits expressed higher SCA variance than GCA variance and dominant gene effect (σ2D) were found higher than additive gene effect (σ2A) except in root length. For recurrent selection based on GCA effects, parents G-48, ST-70, D-121 and ST-63 appeared more appropriate in crossing programme directed towards clonal improvement of Poplar in India.

Genetic analysis of poplar (Populus deltoides Bartr.) clones for early generation selection

The study aimed to select the best clones from a field trial of 69 newly developed clones and two check clones of Populus deltoides after 3 years of field performances. Growth traits were assessed for three growing seasons. Significant differences were detected among clones for plant height, stem diameter, estimated trunk volume and sylleptic branch number. Plant height, stem diameter and trunk volume had low to moderate (0.21 to 0.41) broad sense heritability (h2) values. Sylleptic branch number showed high genetic advance (77.53%) despite moderate h2. In nursery trial, the h2 estimates were moderate to high h2 (0.61 to 0.69) for growth traits. It is possible to apply selection on the basis of principal component analysis (PCA) accounting for a large part of the total variance in the observed growth relying on trunk volume followed by stem diameter, plant height and sylleptic branch number. The promising clones viz. FRI-AM-53, -70, -51, -6 and -45 outperformed the control clone G-48. Stem diameter showed high (0.97) genetic correlation with trunk volume and proved to be a reliable criterion for selecting for trunk volume.The set of 25 most promising clones based on PCA1 ranking will be effective for selection of clones at the age of three years.