Submicroscopic and asymptomatic Plasmodium falciparum and Plasmodium vivax infections are common in western Thailand - molecular and serological evidence

Abstract

Background: Malaria is a public health problem in parts of Thailand, where Plasmodium falciparum and Plasmodium vivax are the main causes of infection. In the northwestern border province of Tak parasite prevalence is now estimated to be less than 1% by microscopy. Nonetheless, microscopy is insensitive at low-level parasitaemia. The objective of this study was to assess the current epidemiology of falciparum and vivax malaria in Tak using molecular methods to detect exposure to and infection with parasites; in particular, the prevalence of asymptomatic infections and infections with submicroscopic parasite levels.

Methods: Three-hundred microlitres of whole blood from finger-prick were collected into capillary tubes from residents of a sentinel village and from patients at a malaria clinic. Pelleted cellular fractions were screened by quantitative PCR to determine parasite prevalence, while plasma was probed on a protein microarray displaying hundreds of P. falciparum and P. vivax proteins to obtain antibody response profiles in those individuals.

Results: Of 219 samples from the village, qPCR detected 25 (11.4%) Plasmodium sp. infections, of which 92% were asymptomatic and 100% were submicroscopic. Of 61 samples from the clinic patients, 27 (44.3%) were positive by qPCR, of which 25.9% had submicroscopic parasite levels. Cryptic mixed infections, misdiagnosed as single-species infections by microscopy, were found in 7 (25.9%) malaria patients. All sample donors, parasitaemic and non-parasitaemic alike, had serological evidence of parasite exposure, with 100% seropositivity to at least 54 antigens. Antigens significantly associated with asymptomatic infections were P. falciparum MSP2, DnaJ protein, putative E1E2 ATPase, and three others.

Conclusion: These findings suggest that parasite prevalence is higher than currently estimated by local authorities based on the standard light microscopy. As transmission levels drop in Thailand, it may be necessary to employ higher throughput and sensitivity methods for parasite detection in the phase of malaria elimination.

Figure 1

Heatmap of signal intensity of antibody binding to seroreactive polypeptides on the microarray. A three-colour gradient display of intensity of antibody binding to 281 P. falciparum and 177 P. vivax seroreactive polypeptides is shown for samples collected during a community-wide mass blood survey and passive case detection at a malaria clinic in Tak Province, Thailand. Samples are segregated according to infectious status and health condition (presenting symptoms or not) at the time of sample collection into four major groups: healthy, asymptomatic malaria, non-malaria illness and symptomatic malaria. Results from qPCR screening are shown as negative or the species (Pf, Pv or Pf + Pv mixed-species) identified in the sample. The coloured gradient represents Z-score values of signal intensity in relation to malaria-unexposed controls, ranging from 0 to ≥5. Individual samples appear as columns, ranked from left to right in their totals of binding to the array’s proteins; seroreactive polypeptides appear as rows, ranked from top to bottom in their mean values of antibody binding for all plasma.

Figure 2

Analysis of intensity and breadth of antibody response to P. f alciparum and P. vivax . Plasma samples from the community MBS or malaria clinic PCD are segregated by qPCR result. (A) Average signal intensity of antibody binding to seroreactive polypeptides, shown as the mean log2-transformed signal intensity with 95% CI (error bars) for each plasma group. (B) Breadth of antibody response by each plasma group, shown as a box-whisker plot of the number of antigens recognized by plasma antibodies. Each box indicates the first and third quartiles, and the line inside the box is the median. The 1.5× interquartile range is indicated by the vertical line bisecting the box.