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. 2023 Oct 20;17(10):e0011680.
doi: 10.1371/journal.pntd.0011680. eCollection 2023 Oct.

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

Prakash Ghosh  1 , Rajashree Chowdhury  1 , Md Abu Rahat  1 , Faria Hossain  1 , Nur E Arpha  2 , Mojca Kristan  3 , Matthew Higgins  3 , Ahmed Abd El Wahed  4 , Yasuyuki Goto  5 , M M Towhidul Islam  6 , Susana Campino  3 , Mary Cameron  3 , Malcom S Duthie  7 , Rashidul Haque  8 , Dinesh Mondal  1
Affiliations

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

Prakash Ghosh et al. PLoS Negl Trop Dis. .

Abstract

Background: Serum 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 findings: Archived 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.

Conclusion: Our results demonstrate that DBS offer excellent diagnostic efficiency for serological assays and represent a viable alternative to whole blood sampling procedures.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Multiple ROC Curve constructed from the OD (450nm) values obtained from conducting rK39 ELISA in serum and DBS samples.
Fig 2
Fig 2. Multiple ROC Curve generated from the OD (450nm) values obtained from rK28 ELISA in serum and DBS samples.
Fig 3
Fig 3. Measure of agreement presented as Cohens Kappa coefficients between molecular and serological methods employing different clinical specimens (WB–whole blood; DBS–dried blood spot; S–serum).
Fig 4
Fig 4. Differences in Ct values by qPCR assay between whole blood (WB) and dried blood spot (DBS) extracted DNA.
Fig 5
Fig 5. Differences in TT values by RPA assay between whole blood (WB) and dried blood spot (DBS) extracted DNA.
Fig 6
Fig 6. Bland-Altman analysis to determine the agreement between serological assays (A: rK28 ELISA; B: rK39 ELISA) using different clinical samples (DBS–dried blood spots).
Fig 7
Fig 7. Index-based surveillance to gauge residual Leishmania infection using a filter paper blood sampling method.
(A) Identification of index VL case and selection of its contacts for screening. (B) Collection of blood on filter paper and drying blood spots [39]. (C) Transport of filter papers with samples to the central facility. (D) Suitable buffer is applied to elute the analytes/biomarkers from filter paper. (E) Downstream analysis (ELISA, qPCR or RPA etc.) is performed to identify Leishmania infection. [The figure was generated in paid BioRender].
See this image and copyright information in PMC

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