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

The study was conducted in the northwestern Province of Tak in Thailand, on the bank of Moei River, bordering with Myanmar. The study sites are located 51 km apart: community samples were collected in the hamlet Mae Salid Noi (17° 28' 4.7202", 98° 1' 48.5106"), and malaria clinic samples were collected in the town of Mae Tan (17° 13' 49.0146", 98° 13' 55.6212"). The climate in this region is tropical. Average temperature ranges from 20.2°C in December to 29.3°C in April [11]. Rainy season is from May to early October with annual rainfall of 2,300 mm. Malaria transmission is low, unstable, and peaks in May-August, coincident with the rainfall [8]. In May 2012, expert microscopy analysis of blood smears collected during a comprehensive mass blood survey of the population of Mae Salid Noi (n = 558) detected one (0.17%) positive infection with P. falciparum. The predominant malaria vectors are Anopheles dirus, Anopheles maculatus and Anopheles minimus. Five malaria species that cause human infection are found in Thailand, but P. falciparum and P. vivax are vastly predominant [1,2,11,21,22].

Firstly, active case detection (ACD) surveys were conducted in Mae Salid Noi, and resident’s health status history was recorded weekly during a five-month period, from March to July 2012. For the molecular evaluation of parasite prevalence study, whole blood samples were collected during a community mass blood survey (MBS) in May 2012 from individuals aged >10 years old (range 11 to 95), resulting in 219 samples. This represented 39.2% of the population of the hamlet, from a total of 558 residents. This number of samples enabled us to detect 6.5% margin of error, using alpha 0.05. Secondly, 61 whole blood samples were collected from individuals >15 years old presenting at the malaria clinic of Mae Tan in May 2012, where passive case detection (PCD) is routinely conducted. All sample donors gave written informed consent. The study was approved by the Institutional Review Boards of the Pennsylvania State University protocol (number 34319); University of California Irvine (number 2012–9123); Thai Ministry of Health (number 0435.3/857), and EC of the Department of Disease Control Ministry of Public Health, Thailand protocol number 7/54-479.

From each study participant, approximately 300 μl of whole blood was collected from a finger prick into a Microvette CB300 capillary blood collector with Lithium-Heparin (Sarstedt, Newton, NC), centrifuged to separate cellular and plasma fractions, then immediately frozen at −80°C for shipment to University of California Irvine for analysis. Upon thawing, plasma was removed, aliquoted and stored at −80°C until use. Total genomic DNA was isolated from the pelleted cellular fraction using DNeasy Blood and Tissue kit (Qiagen, Valencia, CA), and further purified with Genomic DNA Clean and Concentrator (Zymo Research, Irvine, CA), according to manufacturer’s instructions. Purified genomic DNA samples were kept at −20°C until use.

Field microscopy is performed by local trained staff who provides the first result, and positive cases were treated per national malaria treatment guidelines. Expert microscopy was performed later at Mahidol University by an expert microscopist, with over three decades of experience [23], who provided the final microscopy result. Thin and thick smears were prepared from each blood sample, stained with Giemsa solution, and examined for >200 leukocytes for a thick film and >200 microscopic fields with a 100× objective. Molecular detection of P. falciparum (Pf) and P. vivax (Pv) parasites in blood samples was performed by qPCR in the 219 MBS and 61 PCD samples using a SYBR Green detection method as described in Rougemont et al. [24]. Species-specific primers were designed to detect P. falciparum and P. vivax 18S rRNA gene: for P. falciparum, the forward primer sequence was 5′-AGTCATCTTTCGAGGTGACTTTTAGATTGCT-3′ and the reverse was 5′-GCCGCAAGCTCCACGCCTGGTGGTGC-3′; for P. vivax, the forward primer sequence was 5′-GAATTTTCTCTTCGGAGTTTATTCTTAGATTGC-3′ and the reverse was 5′GCCGCAAGCTCCACGCCTGGTGGTGC-3′. Amplification was performed in 20 μl reactions containing 2 μl of genomic DNA, 10 μl 2XSYBR Green qPCR Master Mix (Thermo Scientific, Waltham, MA), and 0.5 μM of each primer, in a CFX96 Touch Real-Time PCR Detection System (BIORAD, Hercules, CA). After initial denaturation at 95°C for 3 min, 45 cycles of 94°C for 30 sec, 55°C for 30 sec, and 68°C for 1 min were followed by a final step of 95°C for 10 sec. This was then followed by a melting curve from 65°C to 95°C with 0.5°C increments for 5 sec. Samples were tested in triplicates and a given sample was considered positive if the lower 95% confidence interval for adjusted Ct value was greater than 0. The detection limit of this method was 50 parasites/mL of blood.

A protein microarray displaying 500 P. falciparum and 515 P. vivax polypeptides printed as in vitro transcription translation (IVTT) reactions was manufactured as described previously [25]. Gene accession numbers follow annotation published on PlasmoDB [26,27]. The protein targets on this array, named Pf/Pv500, were down-selected from larger microarray studies published [28-30] and unpublished data collected at UCI, based on seroreactivity and antigenicity to humans. Quality control of array slides revealed over 99% protein expression efficiency of in vitro reactions spotted, as determined by detection of N- and C- terminal polyhistidine and influenza haemagglutinin epitope tags, respectively. Protein amount was consistent between multiple subarrays, with signal intensity of anti-6xHis tag probing showing a minimal R² = 0.78 and a maximal R² = 0.93 between subarrays and slides. For large proteins printed on the microarray as overlapping polypeptides or individual exons, the exon position relative to the full molecule and the segment of the ORF are indicated when applicable [30]. Information on the microarray platform is publicly available on NCBI’s Gene Expression Omnibus and is accessible through GEO Platform accession number GPL18316. For P. falciparum polypeptides spotted on the microarray, the distribution of parasite life stage of maximum expression, based on information from PlasmoDB according to Le Roch et al. [31], is as follows: merozoite 24%, early ring 22%, late schizogony and early trophozoite 14%, early schizogony 11%, late trophozoite 8%, late ring 5% and unknown 2%. Life stage expression information is not available for P. vivax proteins at this time.

From the 219 whole blood samples from the community MBS screened by qPCR for Plasmodium infection, a subset of samples were selected for Pf/Pv500 microarray probing, using the following criteria: i) being 15 years or older in age, and ii) had recorded lack of malaria symptoms (as defined above) during household visits two months prior and two months after blood collection. This yielded 93 samples. Sixty plasma samples from patients from the malaria clinic were also probed (one plasma of the original 61 clinic samples was compromised and not tested on the array). Twelve plasma samples from unexposed donors from the United States, with no travel history to malaria endemic regions, were used as controls for serology comparisons. Probing of plasma samples on the microarray was previously described in Baum et al. [32]. The raw and normalized data of antibody binding to proteins on the Pf/Pv500 microarray is publicly available through NCBI's Gene Expression Omnibus Series accession number GSE55265.

For analysis of antibody binding to Pf or Pv polypeptides on the microarray the following steps were taken: (i) the mean background signal of antibody binding to 24 control spots of IVTT reaction without DNA template (no DNA control spots) were subtracted from each plasma’s raw values of antibody binding measured as the mean signal intensity of spots of printed polypeptides; negative or zero values after background subtraction were assigned a net value of 1; (ii) net values were log₂-transformed for data normalization. Normalized data was used for statistical analyses and for figure representations of the data; (iii) to determine which polypeptides were considered seroreactive by plasma from the Thai study cohort, Significance Analysis for Microarrays (SAM) [33] was performed comparing the intensity of antibody binding to the proteins on the array between the exposed plasma from Thailand (n = 153) and unexposed controls from the USA (n = 12). The test was performed using MeV 4.8.1, with the following parameters: median and 90th percentile of False Discovery Rate, 0.15% and 0.92%, respectively; median and 90th percentile of Number of False Significant Genes, 0.66 and 4.19, respectively. This resulted in 458 polypeptides being considered significantly seroreactive in exposed Thai plasma and all further analyses considered only this set. (iv) Individual plasma samples were considered seropositive for a polypeptide if the sample’s signal intensity value was above the upper 99% confidence interval value of the unexposed control group. For analysis of intensity of response, (v) ANOVA testing with post hoc Tukey-Kramer (Tukey’s Honestly Significant Difference, HSD) was used for pairwise comparison of the mean signal intensity amongst the plasma groups using JMP9.0; significance tests were 2-sided and set at the 0.05 level for type I error. (vi) Z-scores of signal intensity were calculated as the number of standard deviations above or below the mean signal intensity of the unexposed group. (vii) ROC analysis was performed using ROCR package for R to obtain AUC (area under the curve) values and Mann–Whitney U test values (Benjamini-Hochberg-corrected) for each seroreactive protein, in comparisons of intensity of antibody binding between samples from asymptomatic malaria (MBS) (n = 13) and symptomatic malaria cases (PCD) (n = 26) to identify serological markers significantly associated with asymptomatic infections.