Loop-mediated isothermal amplification for rapid and reliable diagnosis of tuberculous meningitis

Diagnosis of tuberculous meningitis (TBM) is often difficult. A reliable, simple, and rapid diagnostic test that can be performed in any standard laboratory could be helpful in TBM diagnosis. In this study, a loop-mediated isothermal amplification assay (LAMP) was evaluated to rapidly detect and diagnose TBM infection and was compared to the performance of nested PCR. Six specific primers were used to recognize the IS6110 genomic sequence from Mycobacterium tuberculosis, which included one forward outer primer, one reverse outer primer, two respective inner primers, and two loop primers. The optimum reaction temperature and time were 63°C and 60 min, respectively. Nested PCR was performed targeting the IS6110 region from M. tuberculosis using a commercial kit. The LAMP method yielded a sensitivity of 88.23% and a specificity of 80%, compared to the nested-PCR assay, which yielded a sensitivity of 52.9% and a specificity of 90% for TBM diagnosis. Comparative experiments showed that the LAMP assay is a rapid, sensitive, and specific method to detect TBM infection and that it is superior to the nested-PCR assay. LAMP is very simple, and it can be performed in any laboratory and in rural settings.     

Development of an in vitro model to study clot lysis activity of thrombolytic drugs

Background

Thrombolytic drugs are widely used for the management of cerebral venous sinus thrombosis patients. Several in vitro models have been developed to study clot lytic activity of thrombolytic drugs, but all of these have certain limitations. There is need of an appropriate model to check the clot lytic efficacy of thrombolytic drugs. In the present study, an attempt has been made to design and develop a new model system to study clot lysis in a simplified and easy way using a thrombolytic drug, streptokinase.

Methods

Whole blood from healthy individuals (n = 20) was allowed to form clots in a pre-weighed sterile microcentrifuge tubes; serum was removed and clot was weighed. After lysis by streptokinase fluid was removed and remnants of clot were again weighed along with the tube. Percentage of Clot lysis was calculated on the basis of the weight difference of microcentrifuge tubes obtained before and after clot lysis.

Results

There was a significant percentage of clot lysis observed when streptokinase was used. On the other hand with water (negative control), minimal (2.5%) clot lysis was observed. There was a significant difference between clot lysis done by streptokinase and water.

Conclusion

Our study could be a rapid and effective methodology to study clot-lytic effect of newly developed drugs as well as known drugs.