Common asymptomatic and submicroscopic malaria infections in Western Thailand revealed in longitudinal molecular and serological studies: a challenge to malaria elimination

Abstract

Background: Despite largely successful control efforts, malaria remains a significant public health problem in Thailand. Based on microscopy, the northwestern province of Tak, once Thailand's highest burden area, is now considered a low-transmission region. However, microscopy is insensitive to detect low-level parasitaemia, causing gross underestimation of parasite prevalence in areas where most infections are subpatent. The objective of this study was to assess the current epidemiology of malaria prevalence using molecular and serological detection methods, and to profile the antibody responses against Plasmodium as it relates to age, seasonal changes and clinical manifestations during infection. Three comprehensive cross-sectional surveys were performed in a sentinel village and from febrile hospital patients, and whole blood samples were collected from infants to elderly adults. Genomic DNA isolated from cellular fraction was screened by quantitative-PCR for the presence of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. Plasma samples were probed on protein microarray to obtain antibody response profiles from the same individuals.

Results: Within the studied community, 90.2 % of Plasmodium infections were submicroscopic and asymptomatic, including a large number of mixed-species infections. Amongst febrile patients, mixed-species infections comprised 68 % of positive cases, all of which went misdiagnosed and undertreated. All samples tested showed serological reactivity to Plasmodium antigens. There were significant differences in the rates of antibody acquisition against P. falciparum and P. vivax, and age-related differences in species-specific immunodominance of response. Antibodies against Plasmodium increased along the ten-month study period. Febrile patients had stronger antibody responses than asymptomatic carriers.

Conclusions: Despite a great decline in malaria prevalence, transmission is still ongoing at levels undetectable by traditional methods. As current surveillance methods focus on case management, malaria transmission in Thailand will not be interrupted if asymptomatic submicroscopic infections are not detected and treated.

Keywords: Asymptomatic; Low transmission; Molecular screening; Plasmodium falciparum; Plasmodium vivax; Protein microarray; Serology; Submicroscopic; Surveillance; qPCR.

Fig. 1

Heatmap of Z-scores of intensity of antibody binding to P. falciparum and P. vivax proteins on the microarray, segregated by decade of age. Samples were collected from the community in Mae Salid Noi (n = 298) and from febrile patients of the hospital/malaria clinic in Mae Tan (n = 83). Healthy community and non-malaria illness samples were qPCR-negative for Plasmodium sp., community malaria and febrile malaria samples were qPCR-positive for Plasmodium sp.. The decade of age of sample donors is shown at the top of heatmap signals, and correspond to years of age as: 1 (up to 10 years old), 2 (11–20), 3 (21–30), 4 (31–40), 5 (41–50) and 6 (51–60+). The youngest age available from community samples was 3 years old, and from PCD, 5 years old. The colored gradient indicates the number of standard deviations above the mean signal intensity of unexposed adult controls from the USA (Z-score). Individual samples appear as columns, seroreactive proteins appear as rows

Fig. 2

Age-dependent increase in intensity (a) and breadth (b) of antibody binding to Plasmodium antigens by 298 plasma samples collected during community MBS in Mae Salid Noi. The average of responses to 184 P. falciparum antigens are shown in blue, responses to 142 P. vivax antigens are shown in red. Top of bars represent the mean value, error bars represent 95 % confidence interval of the mean. Wilcoxon p values for comparison of responses to the two Plasmodium species are shown as asterisks: *<0.05, **≤0.01, ***<0.001; p values greater than 0.05 are not shown. Linear regression lines show 95 % CI as shaded area outside line, the equation for each regression is shown

Fig. 3

Antibody responses to antigens with significant seasonal changes. The intensity of antibody responses of children and adults with confirmed malaria in March 2013 (cases, panels a, c), and matched uninfected controls (b, d) are shown for antigens with at least one significant change (p < 0.05) in antibody levels between two time points. Antigens are identified in the figures by their microarray index number, and their corresponding gene ID and protein description are found in Additional file 4. The box indicates the first and third quartiles, the line inside the box the median pixel intensity value. The whiskers represent the minimum and maximum value

Fig. 4

Comparisons of intensity (a) and breadth (b) of antibody responses to Plasmodium antigens by plasma samples from asymptomatic (purple) and febrile (green) malaria cases, segregated by age group. Healthy community samples (qPCR-negative for Plasmodium) are shown in grey. The number of samples (n) in each age group appears in parenthesis. Top of bars represent the mean pixel intensity value of antibody binding to 326 antigens, error bars represent 95 % confidence interval of the mean. For box whisker plots, the box indicates the first and third quartiles, the line inside the box the median. The whiskers represent the minimum and maximum value. Wilcoxon test p values comparing the means between the asymptomatic and febrile cases within each age group are shown as asterisks: **<0.01, ***<0.001; p values greater than 0.05 are not shown