Date Published: May 19, 2018
Publisher: The American Society of Tropical Medicine and Hygiene
Author(s): Christine F. Markwalter, Lauren E. Gibson, Lwiindi Mudenda, Danielle W. Kimmel, Saidon Mbambara, Philip E. Thuma, David W. Wright.
A rapid, on-bead enzyme-linked immunosorbent assay for Plasmodium lactate dehydrogenase (pLDH) and Plasmodium falciparum histidine-rich protein 2 (HRP2) was adapted for use with dried blood spot (DBS) samples. This assay detected both biomarkers from a single DBS sample with only 45 minutes of total incubation time and detection limits of 600 ± 500 pM (pLDH) and 69 ± 30 pM (HRP2), corresponding to 150 and 24 parasites/μL, respectively. This sensitive and reproducible on-bead detection method was used to quantify pLDH and HRP2 in patient DBS samples from rural Zambia collected at multiple time points after treatment. Biomarker clearance patterns relative to parasite clearance were determined; pLDH clearance followed closely with parasite clearance, whereas most patients maintained detectable levels of HRP2 for 35–52 days after treatment. Furthermore, weak-to-moderate correlations between biomarker concentration and parasite densities were found for both biomarkers. This work demonstrates the utility of the developed assay for epidemiological study and surveillance of malaria.
Quantitative laboratory measurement of malarial protein biomarkers helps define disease prevalence, distribution, and infection intensities. In the context of malaria elimination, sensitive detection methods are useful for determining response to interventions on a population level, ensuring that low-density infections are identified, and informing the development of improved point-of-care diagnostics. However, the logistics and biohazard risk of venous whole blood sample collection, preservation, and transportation from the field to the laboratory often pose challenges to large studies.
In this work, the on-bead SCSD ELISA for pLDH and HRP2 was adapted for use with DBS samples. For mock DBS samples, the assay was highly reproducible and could detect pLDH as low as 600 ± 500 pM and HRP2 as low as 69 ± 30 pM, corresponding to 150 and 24 parasites/μL in our in-house culture, respectively. Using the DBS SCSD ELISA, we demonstrated the need for controlled DBS storage; the detectability of both pLDH and HRP2 from DBS decreased nearly 70% after 8 days of storage at room temperature. Next, we applied the DBS SCSD ELISA to patient DBS samples from rural Zambia to measure pLDH and HRP2. In these samples, weak-to-moderate correlations between biomarker concentration and parasite density were found for both biomarkers, and the overall concentrations of HRP2 were several orders of magnitude higher than those of pLDH. Finally, biomarker clearance patterns relative to parasite clearance were studied. It was found that pLDH clearance followed closely with parasite clearance, whereas 87% of patients had detectable levels of HRP2 for 35–52 days after treatment. This work demonstrated the utility of the SCSD ELISA for quantifying pLDH and HRP2 from DBS samples and its potential for future application in epidemiological studies.