Fig 1.
Comparative standard operating protocol of LAMP assay (performed with column extracted DNA) vs. direct blood lysis (DBL)-LAMP assay (performed with direct blood lysed supernatant).
Fig 2.
Receiver operating curve (ROC) curve analysis for assessing the diagnostic accuracy of LAMP assay.
(A) ROC curve for LAMP assay for diagnosis of VL at validation sites. The AUCROC for LAMP assay was 0.975; p <0.0001 depicting it to be an excellent diagnostic test (B)-ROC curve analysis showing comparison of rK39 RDT and LAMP. The AUCROCs for rK39 RDT and LAMP were 1.00 and 0.975 respectively. No remarkable difference between two AUCROCs 0.0254 (SE, 0.0125, p -0.1053).
Table 1.
Sensitivity and specificity of LAMP assay for diagnosis of VL at the two validation sites.
Fig 3.
Schematic representation of procedure and results obtained for validation study of LAMP assay at RMRIMS, Patna, India and IMS, BHU, Varanasi, India.
Sensitivity and specificity of DBL-LAMP assay for diagnosis of VL.
Fig 4.
Simplified Direct Blood Lysis (DBL)-LAMP assay for diagnosis of VL.
(A)- Visual appearance of amplified product on addition of diluted SYBR Green I for LAMP and DBL-LAMP assay at the end of reaction. The DBL-LAMP assay was performed using direct blood lysed supernatant instead of column extracted DNA; as described in the methods section. Negative samples remained orange whereas positives turned instantly green on giving brief spin allowing mixing of SYBR Green I (1:10 diluted) at the end of the reaction. (B)- Analytical sensitivity and specificity of DBL-LAMP assay for diagnosis of VL. (C)-ROC curve for DBL-LAMP assay for diagnosis of VL. The AUCROC for DBL-LAMP assay was 0.965; p<0.0001 indicative of an excellent diagnostic test. (D)- Comparative ROC curve analysis for DBL-LAMP assay vs rK39 RDT. The AUCROCs for rK39 RDT and DBL-LAMP were 1.00 and 0.965 respectively. No remarkable difference between the AUCROCs 0.0347(SE 0.0176, p-1.971).