Diagnosis of abdominal tuberculosis: Detection of mycobacterial CFP-10 and HspX proteins by gold nanoparticle-PCR amplified immunoassay.

Attempts were made to improve the efficacy of PCR amplified immunoassay (I-PCR) for diagnosing abdominal TB cases by utilizing the gold nanoparticle (AuNP)-based I-PCR, where AuNPs were functionalized with detection antibodies/oligonucleotides that exhibited 84.3% sensitivity and 95.1% specificity. This assay would improve the ongoing algorithms used in abdominal TB diagnosis.

Does gut brain axis has an impact on Parkinson's disease (PD)?

Parkinson's Disease (PD) is becoming a growing global concern by being the second most prevalent disease next to Alzheimer's Disease (AD). Henceforth new exploration is needed in search of new aspects towards the disease mechanism and origin. Evidence from recent studies has clearly stated the role of Gut Microbiota (GM) in the maintenance of the brain and as a root cause of various diseases and disorders including other neurological conditions. In the case of PD, with an unknown etiology, the GM is said to have a larger impact on the disease pathophysiology. Although GM and its metabolites are crucial for maintaining the normal physiology of the host, it is an undeniable fact that there is an influence of GM in the pathophysiology of PD. As such the Enteroendocrine Cells (EECs) in the epithelium of the intestine are one of the significant regulators of the gut-brain axis and act as a communication mediator between the gut and the brain. The communication is established via the molecules of neuroendocrine which are said to have a crucial part in neurological diseases such as AD, PD, and other psychiatry-related disorders. This review is focused on understanding the proper role of GM and EECs in PD. Here, we also focus on some of the metabolites and compounds that can interact with the PD genes causing various dysfunctions in the cell and facilitating the disease conditions using bioinformatical tools. Various mechanisms concerning EECs and PD, their identification, the latest studies, and available current therapies have also been discussed.

Diagnosis of pleural tuberculosis by multi-targeted loop-mediated isothermal amplification assay based on SYBR Green I reaction: comparison with GeneXpert® MTB/RIF assay.

BACKGROUND: Diagnosis of pleural tuberculosis (TB) is tedious owing to its close resemblance with malignant pleural effusion and sparse bacterial load in clinical specimens. There is an immediate need to design a rapid and dependable diagnostic test to prevent unnecessary morbidity/mortality. RESEARCH DESIGN AND METHODS: A multi-targeted loop-mediated isothermal amplification (MT-LAMP) was deliberated using mpt64 and IS6110 to diagnose pleural TB within pleural fluids/biopsies. MT-LAMP products were analyzed by gel-based and visual detection methods, viz. SYBR Green I, SYBR Green I+deoxyuridine triphosphate uracil-N-glycosylase (dUTP-UNG), and dry methyl green reactions. RESULTS: In a pilot study, while assessing pleural TB/non-TB control subjects (n = 40), both SYBR Green I+dUTP-UNG/gel-based MT-LAMP assays exhibited better sensitivity/specificity than SYBR Green I and dry methyl green MT-LAMP. Since it is facile to work with SYBR Green I+dUTP-UNG than gel-based MT-LAMP, we validated the performance of SYBR Green I+dUTP-UNG in a higher number of specimens (n = 97), which revealed somewhat higher sensitivity (85.2 vs. 81.5%) and specificity (97.7 vs. 90.7%) than SYBR Green I MT-LAMP. Furthermore, the sensitivity attained by SYBR Green I+dUTP-UNG MT-LAMP was significantly higher (p < 0.001) than GeneXpert. CONCLUSIONS: Our SYBR Green I+dUTP-UNG MT-LAMP is a simple and reliable method to diagnose pleural TB, which may translate into a point-of-care test.

Quantification of mycobacterial proteins in extrapulmonary tuberculosis cases by nano-based real-time immuno-PCR.

Aim: Diagnosis of extrapulmonary tuberculosis (EPTB) is difficult, and a rapid and dependable diagnostic test is urgently needed. Methods: A nano-based assay, SYBR Green magnetic bead-coupled gold nanoparticle-based real-time immuno-polymerase chain reaction (MB-AuNP-RT-I-PCR) was studied for the quantitative detection of Mycobacterium tuberculosis MPT-64+CFP-10 proteins in clinically suspected EPTB patients. Results: A wide range (270 fg/ml-9.9 ng/ml) of MPT-64+CFP-10 was quantified by MB-AuNP-RT-I-PCR in EPTB cases, whereas magneto-ELISA demonstrated a narrow range (1.8-10 ng/ml). Furthermore, high sensitivity (88.2%) and specificity (100%) were attained by MB-AuNP-RT-I-PCR in EPTB (n = 51) and non-TB control (n = 49) subjects, respectively. Both MB-AuNP-I-PCR/magneto-ELISA exhibited significantly lower (p < 0.05-0.01) sensitivities than MB-AuNP-RT-I-PCR. Conclusion: The MB-AuNP-RT-I-PCR described herein shows good diagnostic accuracy, which may translate into a credible diagnostic kit.

Extrapulmonary tuberculosis (EPTB) is a type of tuberculosis disease caused by the bacteria Mycobacterium tuberculosis (Mtb) that affect other regions of the body, rather than the lungs. Detecting EPTB is difficult, and a fast and reliable test is needed. This study developed a test based on a small particle, known as a nanoparticle, to identify Mtb in people with EPTB. The test shows good accuracy and could be used for routine testing.

Diagnosis of genitourinary tuberculosis: detection of mycobacterial lipoarabinomannan and MPT-64 biomarkers within urine extracellular vesicles by nano-based immuno-PCR assay.

We detected a cocktail of Mycobacterium tuberculosis lipoarabinomannan (LAM) and MPT-64 biomarkers within urine extracellular vesicles (EVs) of genitourinary TB (GUTB) patients by nano-based immuno-PCR (I-PCR) assay, i.e., magnetic bead-coupled gold nanoparticle-based I-PCR (MB-AuNP-I-PCR) and compared the results with I-PCR and Magneto-ELISA. The size (s) of urine EVs ranged between 52.6 and 220.4 nm as analyzed by transmission electron microscopy (TEM) and nanoparticle tracking analysis. Functionalized AuNPs (coupled with detection antibodies/oligonucleotides) were characterized by UV-vis spectroscopy, TEM, ELISA, PCR, Atomic Force Microscopy and Fourier Transform Infrared spectroscopy, while conjugation of capture antibodies with MBs was validated by UV-vis spectroscopy and Magneto-ELISA. Our MB-AuNP-I-PCR exhibited sensitivities of 85% and 87.2% in clinically suspected (n = 40) and total (n = 47) GUTB cases, respectively, with 97.1% specificity in non-TB controls (n = 35). These results were further authenticated by the quantitative SYBR Green MB-AuNP-real-time I-PCR (MB-AuNP-RT-I-PCR). Concurrently, I-PCR and Magneto-ELISA showed sensitivities of 68.1% and 61.7%, respectively in total GUTB cases, which were significantly lower (p < 0.05-0.01) than MB-AuNP-I-PCR. Markedly, a wide range (400 fg/mL-11 ng/mL) of LAM+MPT-64 was quantified within urine EVs of GUTB cases by SYBR Green MB-AuNP-RT-I-PCR, which can assess the disease dynamics. This study will certainly improve the current algorithms used in GUTB diagnostics.

Diagnosis of extrapulmonary tuberculosis by GeneXpert MTB/RIF Ultra assay.

INTRODUCTION: Diagnosis of extrapulmonary tuberculosis (EPTB) is an arduous task owing to different anatomical locations, unusual clinical presentations, and sparse bacillary load in clinical specimens. Although GeneXpert® MTB/RIF is a windfall in TB diagnostics including EPTB, it yields low sensitivities but high specificities in many EPTB specimens. To further improve the sensitivity of GeneXpert®, GeneXpert® Ultra, a fully nested real-time PCR targeting IS6110, IS1081 and rpoB (Rv0664) has been endorsed by the WHO (2017), wherein melt curve analysis is utilized to detect rifampicin-resistance (RIF-R). AREA COVERED: We described the assay chemistry/work design of Xpert Ultra and evaluated its performance in several EPTB types, that is, TB lymphadenitis, TB pleuritis, TB meningitis, and so on, against the microbiological reference standard or composite reference standard. Notably, Xpert Ultra exhibited better sensitivities than Xpert, but mostly at the compensation of specificity values. Moreover, Xpert Ultra exhibited low false-negative and false-positive RIF-R results, compared with Xpert. We also detailed other molecular tests, that is, Truenat MTBTM/TruPlus, commercial real-time PCR, line probe assay, and so on, for EPTB diagnosis. EXPERT OPINION: A combination of clinical features, imaging, histopathological findings, and Xpert Ultra are adequate for definite EPTB diagnosis so as to initiate an early anti-tubercular therapy.

We discussed the assay chemistry and evaluated performance of GeneXpert®MTB/RIF Ultra to identify the TB germs and resistance to one of the potent bactericidal drugs, that is, rifampicin in different types of extrapulmonary TB (EPTB, TB in organs other than lungs) and compared its performance with its ancestor, that is, GeneXpert. We briefly outlined the other molecular tests, such as Truenat MTB^TM/Truenat MTB^TM Plus, commercial real-time PCR, line probe assay, and so on for EPTB diagnosis. While evaluating Xpert Ultra results, the 'trace call' has been introduced, whose interpretation is important. By and large, Xpert Ultra assay outperformed in TB lymphadenitis, TB pleuritis, and TB meningitis when compared with Xpert, though limited information is available on other EPTB forms. Overall, the sensitivity of Xpert Ultra has been increased in most of EPTB cases, but mostly at the cost of specificity values.

Diagnosis of abdominal tuberculosis by multi-targeted (mpt64 and IS6110) loop-mediated isothermal amplification assay.

BACKGROUND AND AIM: Diagnosis of abdominal TB is an exigent task due to variable anatomical sites and non-specific clinical manifestations that closely resemble other diseases. Most of the available diagnostic modalities yield low sensitivities and need expertise to handle the specialized equipment. Hence, there is an urgent need to develop a rapid and reliable diagnostic test, so as to reduce the unnecessary morbidity. Therefore, we designed a multi-targeted loop-mediated isothermal amplification (MT-LAMP) for diagnosing abdominal TB. METHODS: We evaluated an MT-LAMP (using mpt64 and IS6110) to diagnose abdominal TB within ascitic fluids and intestinal/peritoneal biopsies and compared these results with multiplex-PCR (M-PCR) using the same targets. MT-LAMP products were analyzed by gel electrophoresis and visual detection methods, that is, hydroxy naphthol blue and SYBR Green I reaction. RESULTS: Sensitivities of 80.9% and 84.6% were obtained in suspected (n = 42) and total abdominal TB (n = 52) cases, respectively by gel-based MT-LAMP, with 97.3% (n = 37) specificity in non-TB controls. Notably, sensitivities attained by gel-based/SYBR Green I MT-LAMP in both clinically suspected and total abdominal TB cases were significantly higher (P < 0.05) than M-PCR. Furthermore, sensitivity obtained with SYBR Green I was equivalent to that of gel-based MT-LAMP, while somewhat lesser specificity (94.6%) was attained with SYBR Green I, compared with gel-based MT-LAMP. CONCLUSION: Both gel-based and SYBR Green MT-LAMP exhibited equivalent sensitivities to diagnose abdominal TB. Because SYBR Green LAMP is easier to perform than a gel-based assay, we are currently focused on improving the specificity of this assay so as to develop a diagnostic kit.

Quantitative detection of mycobacterial mannophosphoinositides in tuberculosis patients by real-time immuno-PCR assay.

A real-time immuno-PCR assay was deliberated to detect mycobacterial mannophosphoinositides (PIMs). A dynamic range of PIMs (0.9 pg/mL-10 ng/mL) was detected in TB patients, wherein 88.2% and 81.1% sensitivities were obtained in pulmonary TB and extrapulmonary TB respectively, with 96-96.4% specificity. This assay may translate into a diagnostic kit.

Diagnosis of peritoneal tuberculosis by real-time immuno-PCR assay based on detection of a cocktail of Mycobacterium tuberculosis CFP-10 and HspX proteins.

BACKGROUND: Diagnosis of peritoneal TB is difficult owing to unusual clinical manifestations and low sensitivities obtained with most of the available diagnostic modalities. Hence, there is an urgent need to design a reliable diagnostic test so that an early therapy is initiated. RESEARCH DESIGN AND METHODS: We designed a quantitative real-time immuno-PCR (RT-I-PCR) assay to detect a cocktail of Mycobacterium tuberculosis CFP-10 (Rv3874) and HspX (Rv2031c) proteins in clinical samples (ascitic fluids and peritoneal biopsies) of peritoneal TB patients, and results were compared with I-PCR/ELISA. RESULTS: A wide range of CFP-10+ HspX (0.6 pg/mL to 9.9 ng/mL) was detected in clinical samples of peritoneal TB patients by RT-I-PCR, whereas ELISA exhibited a narrow range (3 ng/mL to 11.5 ng/mL). Sensitivities of 81.5% and 65.7% and specificities of 92.5% and 90% were obtained in a total of 78 cases (comprising 38 peritoneal TB and 40 non-TB controls) by RT-I-PCR and I-PCR, respectively. Markedly, sensitivity obtained by RT-I-PCR was significantly higher than I-PCR (p = 0.0143) and ELISA (p = 0.0005). CONCLUSIONS: Our RT-I-PCR revealed good accuracy for the rapid diagnosis of peritoneal TB cases. After further improving the specificity and reducing the cost, this assay may develop into a diagnostic kit.

Recent updates in diagnosis of abdominal tuberculosis with emphasis on nucleic acid amplification tests.

INTRODUCTION: Abdominal tuberculosis (TB) is a common epitome of extrapulmonary TB (EPTB), wherein peritoneal and intestinal TB are the most prevalent forms. Diagnosis of abdominal TB is a daunting challenge owing to variable anatomical locations, paucibacillary nature of specimens and atypical clinical presentations that mimic other abdominal diseases, such as Crohn's disease and malignancies. In this review, we made a comprehensive study on the diagnosis of abdominal TB. AREA COVERED: Various modalities employed for abdominal TB diagnosis include clinical features, imaging, bacteriological tests (smear/culture), histopathological/cytological observations, interferon-gamma release assays and nucleic acid amplification tests (NAATs). Among NAATs, loop-mediated isothermal amplification assay, PCR, multiplex-PCR, nested PCR, real-time PCR and GeneXpert® MTB/RIF were discussed. Identification of circulating Mycobacterium tuberculosis cell-free DNA by real-time PCR within ascitic fluids is another useful approach. EXPERT OPINION: Several novel molecular/immunological methods, such as GeneXpert Ultra, aptamer-linked immobilized sorbent assay, immuno-PCR (I-PCR) and nanoparticle-based I-PCR have recently been developed for detecting pulmonary TB and several EPTB types, which may also be explored for abdominal TB diagnosis. Precise and prompt diagnosis of abdominal TB may initiate an early therapy so as to reduce the complications, i.e. abdominal pain, ascites, abdominal distension, intestinal obstruction/perforation, etc., and avoid surgical involvement.Plain Language SummaryAbdominal tuberculosis (TB) is a manifestation of extrapulmonary TB (EPTB), where peritoneal and intestinal TB are two major forms. Diagnosis of abdominal TB is difficult owing to low bacterial load present in clinical samples and non-specific clinical presentations as it mimics other diseases such as inflammatory bowel diseases, abdominal malignancies, etc. Bacteriological tests (smear/culture) almost fail owing to poor sensitivities and it is not always possible to get representative tissue samples for histopathological and cytological observations. In recent years, molecular tests i.e. nucleic acid amplification tests (NAATs), such as PCR/multiplex-PCR (M-PCR), nested PCR and GeneXpert are widely employed. Markedly, PCR/M-PCR and nested PCR exhibited reasonable good sensitivities/specificities, while GeneXpert revealed low sensitivity in most of the studies but high specificity, thus it could assist in differential diagnosis of intestinal TB and Crohn's disease. Further, novel molecular/immunological tests employed for pulmonary TB and other EPTB types were described and those tests can also be utilized to diagnose abdominal TB. Reliable and rapid diagnosis of abdominal TB would initiate an early start of anti-tubercular therapy and reduce the severe complications.

Insight into diagnosis of female genital tuberculosis.

INTRODUCTION: Female genital tuberculosis (TB) is a common form of extrapulmonary TB (EPTB) with varied clinical presentations, i.e. infertility, pelvic pain, and menstrual irregularities. Diagnosis of female genital TB is challenging predominantly due to paucibacillary nature of specimens and inconclusive results obtained by most of the routine laboratory tests. AREAS COVERED: This review has briefly summarized the epidemiology, clinical features, and transmission of female genital TB. Commonly used laboratory tests include bacteriological examination (smear/culture), tuberculin skin testing, interferon-γ release assays, imaging, laparoscopy/hysteroscopy, and histopathological/cytological observations. Furthermore, utility of nucleic acid amplification tests (NAATs), like loop-mediated isothermal amplification, PCR, multiplex-PCR, nested PCR, real-time PCR, and GeneXpert® could significantly improve the detection of female genital TB. EXPERT OPINION: Currently, there is no single test available for the efficient diagnosis of female genital TB, rather a combination of tests is being employed, which yields moderate diagnostic accuracy. The latest modalities developed for diagnosing pulmonary TB and other clinical EPTB forms, i.e. aptamer-linked immobilized sorbent assay, immuno-PCR (I-PCR), analysis of circulating cell-free DNA by NAATs, and identification of Mycobacterium tuberculosis biomarkers within extracellular vesicles of bodily fluids by I-PCR/nanoparticle-based I-PCR, may also be exploited to further improve the diagnosis of female genital TB.

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.

Detection of potential biomarkers associated with outrageous diseases and environmental pollutants by nanoparticle-based immuno-PCR assays.

Immuno-polymerase chain reaction (I-PCR) assay with advantages of both enzyme-linked immunosorbent assay (ELISA) and PCR exhibits several-fold enhanced sensitivity in comparison to respective ELISA, which has wide applications for ultralow detection of several molecules, i.e. cytokines, protooncogenes and biomarkers associated with several diseases. Conjugation of reporter DNA to the detection antibodies is the most crucial step of I-PCR assay that usually employs streptavidin-protein A, streptavidin-biotin conjugate or succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) system by a covalent binding. However, coupling of antibodies and oligonucleotides to nanoparticles (NPs) is relatively easier in the NP-based I-PCR (NP-I-PCR) that also displays better accuracy. This article is mainly focused on the detection of important biomarkers associated with several outrageous infectious and non-infectious diseases by NP-I-PCR assays, which would expedite an early initiation of therapy thus human health would be improved. Similarly, ultralow detection of environmental pollutants by these assays and their elimination would certainly improve human health.

Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay.

PURPOSE: Immuno-PCR (I-PCR), an ultrasensitive method, combines the versatility of ELISA with the exponential amplification capacity of PCR. Coupling of detection antibodies with the reporter DNA is a critical step of I-PCR. Gold nanoparticles (GNPs) and magnetic beads (MBs) are relatively easy to attach with the antibodies and DNA. Therefore, we designed MB-coupled GNP-based I-PCR (MB-GNP-I-PCR) assay for the detection of Mycobacterium tuberculosis antigen. METHODS: GNPs were synthesized by chemical reduction and seed-mediated synthesis. Functionalized GNPs were prepared by coupling GNPs with the detection antibodies and reporter DNA and were characterized. Detection limit of M. tuberculosis-specific purified early secreted antigenic target-6 (ESAT-6) (Rv3875) was determined by MB-GNP-I-PCR. RESULTS: Transmission electron microscopy revealed spherical and slightly polydispersed GNPs of ~20 and ~60 nm size. Coupling of antibodies to GNPs was indicated by a shift in absorption maxima from 524 to 534 nm, which was confirmed by transmission electron microscopy. A color reaction with ELISA and the presence of 76 bp product by PCR further validated the coupling of detection antibodies and signal DNA to the functionalized GNPs. Also, attachment of capture antibodies with MBs was confirmed by magneto-ELISA. Detection limit of purified ESAT-6 by MB-GNP-I-PCR was determined to be 10 fg/mL, 105-fold lower than analogous ELISA. Notably, no sample matrix effect was observed in the saliva samples of healthy individuals spiked with the purified ESAT-6. CONCLUSION: Unlike conventional I-PCR (solid format), MB-GNP-I-PCR (liquid format) is relatively simple with the reduced background signals, which can be further exploited for the clinical diagnosis of tuberculosis.

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.

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.

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.