ABSTRACT
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.
Subject(s)
Molecular Diagnostic Techniques/methods , Multiplex Polymerase Chain Reaction , Mycobacterium tuberculosis/isolation & purification , Pleural Effusion/microbiology , Tuberculosis, Pulmonary/diagnosis , Adolescent , Adult , Bacterial Proteins/genetics , Diagnostic Tests, Routine/methods , Female , Humans , Male , Middle Aged , Mycobacterium tuberculosis/genetics , Sensitivity and Specificity , Tuberculosis, Pulmonary/microbiology , Young AdultABSTRACT
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.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Bacterial Proteins/genetics , Mycobacterium tuberculosis/isolation & purification , Real-Time Polymerase Chain Reaction/methods , Tuberculosis/diagnosis , Adolescent , Adult , Bacterial Typing Techniques , Case-Control Studies , Colony Count, Microbial , DNA, Bacterial/isolation & purification , Evaluation Studies as Topic , Female , Humans , Male , Middle Aged , Sensitivity and Specificity , Tuberculosis/microbiology , Young AdultABSTRACT
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.
Subject(s)
Multiplex Polymerase Chain Reaction/methods , Mycobacterium tuberculosis/isolation & purification , Polymerase Chain Reaction/methods , Tuberculosis/diagnosis , Bacteriological Techniques/methods , DNA Transposable Elements/genetics , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Early Diagnosis , Female , Gene Amplification , Humans , Male , Mycobacterium tuberculosis/genetics , Polymerase Chain Reaction/standards , Sensitivity and SpecificityABSTRACT
Immuno-PCR (PCR-amplified immunoassay; I-PCR) is a novel ultrasensitive method combining the versatility of ELISA with the sensitivity of nucleic acid amplification of PCR. The enormous exponential amplification power of PCR in an I-PCR assay leads to at least a 10(2)-10(4)-fold increase in sensitivity compared with an analogous ELISA. I-PCR has been used to detect many biological molecules such as proto-oncogenes, toxins, cytokines, hormones, and biomarkers for autoimmune and Alzheimer's diseases, as well as microbial antigens and antibodies, and it can be adapted as a novel diagnostic tool for various infectious and non-infectious diseases. Quantitative real-time I-PCR has the potential to become the most analytically sensitive method for the detection of proteins. The sensitivity and specificity of a real-time I-PCR assay can be enhanced further with the use of magnetic beads and nanoparticles. This review is primarily focused on the detection of potential viral, bacterial and parasitic antigens by I-PCR assay, thus enabling their application for immunological research and for early diagnosis of infectious diseases.
Subject(s)
Antigens/analysis , Clinical Laboratory Techniques/methods , Communicable Diseases/diagnosis , Diagnostic Tests, Routine/methods , Real-Time Polymerase Chain Reaction/methods , Antigens/genetics , Antigens/immunology , Biomedical Research/methods , Enzyme-Linked Immunosorbent Assay/methods , Humans , Sensitivity and SpecificityABSTRACT
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.