Quantitative detection of PfHRP2 in saliva of malaria patients in the Philippines

Abstract

Background: Malaria is a global health priority with a heavy burden of fatality and morbidity. Improvements in field diagnostics are needed to support the agenda for malaria elimination. Saliva has shown significant potential for use in non-invasive diagnostics, but the development of off-the-shelf saliva diagnostic kits requires best practices for sample preparation and quantitative insight on the availability of biomarkers and the dynamics of immunoassay in saliva. This pilot study measured the levels of the PfHRP2 in patient saliva to inform the development of salivary diagnostic tests for malaria.

Methods: Matched samples of blood and saliva were collected between January and May, 2011 from eight patients at Palawan Baptist Hospital in Roxas, Palawan, Philippines. Parasite density was determined from thick-film blood smears. Concentrations of PfHRP2 in saliva of malaria-positive patients were measured using a custom chemiluminescent ELISA in microtitre plates. Sixteen negative-control patients were enrolled at UCLA. A substantive difference between this protocol and previous related studies was that saliva samples were stabilized with protease inhibitors.

Results: Of the eight patients with microscopically confirmed P. falciparum malaria, seven tested positive for PfHRP2 in the blood using rapid diagnostic test kits, and all tested positive for PfHRP2 in saliva. All negative-control samples tested negative for salivary PfHRP2. On a binary-decision basis, the ELISA agreed with microscopy with 100 % sensitivity and 100 % specificity. Salivary levels of PfHRP2 ranged from 17 to 1,167 pg/mL in the malaria-positive group.

Conclusion: Saliva is a promising diagnostic fluid for malaria when protein degradation and matrix effects are mitigated. Systematic quantitation of other malaria biomarkers in saliva would identify those with the best clinical relevance and suitability for off-the-shelf diagnostic kits.

Figure 1

Enrollment of patients. Flow chart showing the process for enrollment of patients, collection and analysis of samples.

Figure 2

Configuration of the sandwich ELISA for malaria antigens. The IgM capture antibody is immobilized on the microtitre plate and binds Pf HRP2. The biotinylated IgG detector antibody binds a different epitope of Pf HRP2. The use of the biotin-streptavidin system to immobilize peroxidase enzyme provides a degree of amplification. Schematic diagram represents the possibility of multivalent recognition of epitopes by the antibodies.

Figure 3

Typical calibration curves for recombinantPf HRP2 in buffer and saliva. Relative to buffer, saliva matrix yielded a greater signal at higher concentrations. With the addition of protease inhibitors to saliva, the signal and the LOD were reduced.

Figure 4

Estimated concentrations ofPf HRP2 in patient saliva. Example of the estimated concentration of PfHRP2 in patient saliva from one microtitre plate. The standard curve was calculated by fitting calibration samples by non-linear regression. The concentration of Pf HRP2 in patient saliva was then estimated from the standard curve.

Figure 5

Salivary levels ofPf HRP2. Aligned dot plot showing the median and interquartile ELISA signals for Pf HRP2 levels in malaria-positive patients and negative controls.