The samples used in this study were collected as part of a longitudinal study in which the purpose was to evaluate the dynamic of submicroscopic Plasmodium infections in Colombia.
Dried blood spots (DBS) in Whatman® 903 protein saver card (GE Healthcare, US) were collected by passive case detection in the transversal phase of the study, conducted between August 2017 to March 2018 in four villages (California, Tangareal, Robles, and Candelillas) in Tumaco city located in the south of Colombia (1 850’N, 78845’W) (Fig.1). The first village represents a typical suburban zone. The following two sites are characterized as rural areas, and the last one is classified as a peri-urban zone. During the study, P. falciparum was reported as the predominant species (96%) in Tumaco with an API of 13.5 cases/1000 inhabitants in 2017 and 10.4 cases/1000 inhabitants in 2018. No entomological data was collected during the time of this study .
To compare the vector exposure between infected and uninfected individuals, all positive P. falciparum samples were selected (n=63) from the 958 people that were enrolled in the main study. All of these infections were afebrile (axillary temperature <37.5°C), and 48 (76.2%) were submicroscopic (detected by Loop-mediated isothermal amplification -LAMP or nested polymerase chain reaction- nPCR but not by light microscopy- LM). Furthermore, 50 uninfected samples were randomly selected by age (±5 years) and gender from the total of non-infected individuals by using an Excel random list.
ELISA antigens and SGE preparations
Anopheles albimanus and An. darlingi were maintained under insectary conditions until salivary gland dissection. Based on recent studies suggesting that time of colonization has an influence on arthropod salivary gland content , and that two different An. albimanus lineages are circulating in two geographically distant regions of Colombia, the potential differences in antibody responses against salivary content of two different strains of An. albimanus were evaluated, one from a long-stablished colony strain STECLA (STE) versus a recently colonized strain Cartagena (CTG). Briefly, An. albimanus strains originated from El Salvador (STE) and Colombia (CTG), respectively, and were maintained in the insectary at the CDC (Atlanta, GA, USA). The An. darlingi laboratory strain originated from Iquitos, Peru , and was maintained in the NAMRU-6 insectary (Iquitos, Loreto, Peru). Salivary glands from 8 to 10 days old female mosquitoes were extracted by dissection and pooled into 1X PBS . Mosquitoes were blood feed at day 3 or 4 after emergence. A pool of 100 salivary gland pairs from each strain was then frozen and thawed three times to prepare the SGE. The concentration of the SGE was determined using a NanoDrop™ (Thermo Scientific, Wilmington, DE, USA) and 50µL aliquots were stored at -80oC until use. The An. gambiae gSG6-P1 peptide was synthesized by Genscript (Piscataway, NJ, USA) and the P. falciparum Pf-MSP (Plasmodium falciparum Merozoite Surface Protein) peptide (Fitzgerald, USA) was used to evaluate exposure to malaria parasites.
Indirect ELISA (Enzyme Linked Immunosorbent Assay)
ELISA conditions were standardized as described elsewhere [23, 24]. Also, DBS samples were prepared as by eluting half of a card circle into 300uL of elution buffer (PBS 1X, Tween 20 0.05%) and incubated overnight at 4°C. Testing of serial dilutions (1:50, 1:100 and 1:200) showed better performance of the ELISA using a 1:50 dilution. Briefly, Nunc-Maxisorp 96-well plates (Nalgene Nunc International, Rochester, NY) were coated with 50 µL/well of gSG6-P1 peptide (2μg/mL), An. darlingi and An. albimanus SGE (1μg/mL) or Pf-MSP (1μg/mL) diluted 1X PBS. Plates were incubated overnight at 4°C and blocked with 200 µL of 5% skim milk solution in PBS-tween 20 (0.05%) (Blocking buffer) for 1.5 hours at 37°C. The DBS elution was used to prepare a 1:50 sample dilution in blocking buffer, this optimal dilution had been determined by preliminary experiments and 50 µL of diluted samples were added to each well (individual samples were tested in duplicate). Plates were incubated at 37°C for 1.5 hours, washed three times, then incubated 1h at 37°C with 50 µL/well of a 1/1,000 dilution of goat monoclonal anti-human IgG conjugated with horseradish peroxidase (AbCam, Cambridge, MA). After three final washes, colorimetric development was carried out using tetra-methyl-benzidine (Abcam) as a substrate. In parallel, each assessed microplate contained in duplicate: a positive control, a negative control, and a blank; wells containing no sample. The positive control was a pool of DBS of people with positive malaria diagnosis. The negative control was a sample of people from US (n=36) with no exposure to malaria parasites. The blank was composed by wells containing no sample. The reaction was stopped with 0.25 N sulfuric acid, and the optical density (OD) was measured at 450 nm.
All data from questionnaires and forms were entered into a Microsoft Access database, and statistical analyses were conducted in STATA 14 (StataCorp. 2015. Stata Statistical Software: Release 14. College Station, TX: StataCorp LP) and GraphPad Software V5. OD normalization and plate to plate variation was performed as described elsewhere . Briefly, antibody levels were expressed as the ΔOD value: ΔOD = ODx − ODb, where ODx represents the mean of individual OD in both antigen wells and ODb the mean of the blank wells. For each tested peptide, positive controls of each plate were averaged and divided by the average of the ODx of the positive control for each plate to obtain a normalization factor for each plate as previously described. Each plate normalization factor was multiplied by plate sample ΔOD to obtain normalized ΔOD that were used in statistical analyses. Assay variation of samples (inter and intra assay) tested in the study was below 20% and it was only included in the analysis serum samples with a coefficient of variation ≤20% duplicates between duplicate . The mean ΔOD of negative US controls plus 3 standard deviations (SD) was used to determine cut-off value for responsiveness to antigens. The ΔOD cut off value to determine exposure to malaria antigens as 0.263. The median of antibody level for each antigen in uninfected people (negative PCR and negative LM) in submicroscopic (positive PCR and negative LM) and microscopic (positive PCR and positive LM) carriers was estimated. The medians are shown with their respective interquartile ranges (IQR).
Odd ratios (OR) were calculated to evaluate risk of malaria. For this, the median was used to classify IgG antibody levels as high (ΔOD higher than the median) and low (ΔOD equal or lower than the median) and the samples were classified as cases (Asymptomatic and submicroscopic infections) and controls (uninfected). In addition, Spearman correlation coefficients were calculated to measure the strength of association between each Anopheles antigen with Pf-MSP IgG levels. Finally, a Mann-Whitney U-test was used to estimate differences between medians of each Anopheles antigen by the status of infection in the whole sample and by sites and a Kruskal–Wallis test to estimate differences between groups of infection. A multiple linear mixed regression model was constructed to determine the correlation between anti-Anopheles IgG levels (anti-gSG6-P1, CTG, STE, and An. darlingi) with anti Pf-MSP IgG levels. A random intercept at the village level was introduced in the model to correct the inter-village variations. The model was adjusted by Plasmodium infection, age and time of residence in a malarial endemic area; these factors showed significant p values in simple models.