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

Abstract

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.

Keywords: HNB; SYBR Green I; abdominal TB; diagnosis; multi-targeted LAMP.