Dried Blood Spots (DBS): A suitable alternative to using whole blood samples for diagnostic testing of visceral leishmaniasis in the post-elimination era

BackgroundSerum or whole blood collection, processing, transport and storage still present significant challenges in low resource settings where mass surveillance is required to sustain disease elimination. Therefore, in this study, we explored the diagnostic efficacy of dried blood spots (DBS) as a minimally invasive and potentially cost-effective alternative sampling technique to whole blood sampling procedures for subsequent detection of Leishmania donovani antibodies or DNA.Methodology and principal findingsArchived serum, DNA samples from whole blood of visceral leishmaniasis (VL) cases and healthy controls, and DBS from corresponding cases and controls, were used. Both molecular and serological assays were optimized to detect L. donovani antibodies or DNA in DBS elute and results were compared against those obtained with whole blood. Serological assays (both rK28 ELISA and rK39 ELISA) of DBS samples showed sensitivity and specificity of 100% and had excellent agreement with results from whole blood samples (kappa value ranged from 0.98-1). Bland-Altman analysis of OD values from rK28-ELISA with DBS elute and patients' serum showed an excellent agreement (ICC = 0.9) whereas a good agreement (ICC = 0.8) was observed in the case of rK39-ELISA. However, qPCR and RPA of DBS samples had a diminished sensitivity of 76% and 68%, respectively, and poor agreement was observed with the whole blood samples.ConclusionOur results demonstrate that DBS offer excellent diagnostic efficiency for serological assays and represent a viable alternative to whole blood sampling procedures. Since the inception of the National Kala-azar Elimination Program (NKEP) in 2005, visceral leishmaniasis (VL) cases have declined significantly in Bangladesh. Bangladesh has achieved the elimination targets set by the WHO and continuous strides have been enforced by the government and non-government organizations in the present maintenance phase. To sustain the outcomes of the NKEP, move towards a zero-transmission goal and subvert the endgame challenges, an effective surveillance strategy is a primary focus. As part of this strategy, we examined whether using dried blood spots (DBS), rather than whole blood samples as currently recommended, could serve as a minimally invasive sampling technique prior to subsequent diagnostic tests. We determined the accuracy of molecular and serological assays using DBS samples compared with whole blood samples. While using DBS, excellent diagnostic accuracy was observed for serological assays (rK28 ELISA and rK39 ELISA), whereas sensitivity and specificity using molecular assays for DNA detection (qPCR and RPA) declined in comparison to whole blood. Given the low cost and field feasibility of DBS, we believe this simple sample collection technique coupled with serology-based detection of L. donovani meets the requirements for mass surveillance towards detecting residual VL transmission.

Automated summary

The study demonstrates the excellent diagnostic efficiency of dried blood spots (DBS) for serological assays and suggests it as a viable alternative to whole blood sampling procedures. However, the sensitivity and specificity of molecular assays using DBS for DNA detection are lower compared to whole blood. The low cost and field feasibility of DBS, coupled with serology-based detection, make it suitable for mass surveillance in detecting residual visceral leishmaniasis transmission.