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.

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.

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.