Multiple comparisons analysis of serological data from an area of low Plasmodium falciparum transmission

Abstract

Background: As a nation reduces the burden of falciparum malaria, identifying areas of transmission becomes increasingly difficult. Over the past decade, the field of utilizing malaria serological assays to measure exposure has grown rapidly, and a variety of serological methods for data acquisition and analysis of human IgG against falciparum antigens are available. Here, different immunoassays and statistical methods are utilized to analyse samples from a low transmission setting and directly compare the estimates generated.

Methods: A subset of samples (n = 580) from a 2012 Haitian nationwide malaria survey was employed as sample population of low falciparum endemicity. In addition to the Haitian samples, samples from 247 US residents were used as a reference population of 'true seronegatives'. Data acquisition was performed through standard ELISA and bead-based multiplex assays assaying for IgG antibodies to the Plasmodium falciparum antigens MSP-1p19, MSP-1p42(D), MSP-1p42(F), and AMA-1. Appropriate parametric distributions and seropositivity cutoff values were determined by statistical measures.

Results: Data from both assays showed a strong positive skew, and the lognormal distribution was found to be an appropriate statistical fit to the Haitian and American populations. The American samples served as a good serological true negative population for the multiplex assay, but not for ELISA-based data. Mixture model approaches to determine seronegative and seropositive populations from the Haitian data showed a high degree of distribution overlap-likely due to the historical low falciparum transmission in this nation. Different fittings to the reversible catalytic model resulted depending upon the immunoassay utilized and seropositivity cutoff method employed. Data were also analysed through fitting to penalized B-splines, presenting another possible analytical tool for the analysis of malaria serological data.

Conclusions: Standardization of serological techniques and analyses may prove difficult as some tools can prove to be more useful depending on the area and parasite in question, making clear interpretation a vital pursuit. The presented analysis in the low-endemic nation of Haiti found malaria-naive US residents to be an appropriate seronegative reference population for the multiplex assay, and this assay providing consistent estimates between MSP-1 and AMA-1 antigens of percent seropositives for this low-endemic population.

Fig. 1

Histograms of samples from malaria-naive persons (n = 247) as analysed by ELISA and Luminex assays. Charts are overlaid with normal (solid line) and lognormal (dashed line) fitted curves. The x-axes are truncated to the upper limit of the observed data: OD units, 0.25; MFI units, 240

Fig. 2

Histograms of samples (n = 580) from a region of low-endemic P. falciparum transmission. The x-axes display the entire dynamic range of each assay. OD units, 0–3.5; MFI units, 0–30,000

Fig. 3

Overlay of histogram from malaria-naive population (grey bars), on sample population (open bars). The x-axis for both immunoassays is truncated as in Fig. 1 to show comparative overlay

Fig. 4

Seroprevalence curves for MSP-1 and AMA-1 antigens based on different immunoassays, fitted distributions, and seropositive cutoff determinations. The MSP-1p19 antigen was chosen to represent MSP-1 response as a whole. Curves represent ELISA (black lines) and multiplex assays (grey lines) with normal (solid lines) and lognormal (dashed lines) distributions. Seropositivity was based on mean + 3SD cutoff value for all comparisons. a Curves generated when using malaria-naive persons as reference. Note ELISA normal and lognormal seroprevalence curves for the MSP-1 antigen overlap. b Curves generated by finite mixture model approaches

Fig. 5

Penalized B-splines fitted to continuous data for each antigen MSP-1p42(D) no markers; MSP-1p42(F) square markers; MSP-1p19 triangle markers; AMA-1 circle markers. Inset box displays area under the curve (AUC) calculations for each antigen by each assay