Fig 1.
Flow chart of sample processing for multiplex PCR detection of intestinal parasites.
Table 1.
Quantitative multiplex PCR set up overview.
Table 2.
Semi-quantitative multiplex PCR set up overview.
Fig 2.
Multiplex to singleplex PCR Ct comparison.
Assay optimization to determine effects of multiplex PCR set up on sensitivity and efficiency of PCRs compared to singleplex PCR using plasmid standard curve controls containing all PCR products.
Fig 3.
Overall parasite prevalence comparison between multiplex PCR and microscopy.
Data presented combined for all 680 study participants, as well as individually for Timor-Leste (467 participants) and Cambodia (213 participants), showing higher recorded percentage prevalence across all target organisms by Multiplex PCR.
Table 3.
Multiplex PCR and microscopy parasite prevalence agreement statistics.
Fig 4.
Diagrams depict polyparisitism observed in the 680 combined Timor-Leste and Cambodia samples in both (A) Microscopy and (B) Multiplex PCR. Pie graph depicts total number of parasites per sample and the venn diagram details the specific division of STH coinfections for Microscopy (259 Ascaris and/or hookworm positive samples) and multiplex PCR (504 Ascaris and/or hookworm positive samples). *Microscopy unable to differentiate Hookworm species N. americanus and Ancylostoma spp. -considered only as ‘hookworms’ for polyparasitism comparison.
Fig 5.
Intensity of infection for Ascaris spp. and hookworm positive samples as determined by sodium nitrate flotation.
Timor-Leste microscopy produced 219 Ascaris positive samples (200 EPG average), 97 hookworm positive samples (40 EPG average). Cambodia microscopy produced 54 hookworm positive samples (60 EPG average).
Fig 6.
Multiplex PCR Ct-value frequency distribution for hookworm and Ascaris spp. positive samples.
Graphs show the infection intensity distribution, with lower Ct-values indicating higher infection intensities, presented for Timor-Leste hookworm (353) and Ascaris (259), as well as Cambodia Hookworm (80) positive samples. As Ct-values are expressed in a continuous format, values were rounded up to the nearest integer to produce categorical data for frequency analysis.
Fig 7.
Relationship between EPG and intensity converted PCR Ct-values.
Graph shows strong linear relationship (P<0.001) between sodium nitrate flotation determined EPG and Multiplex PCR Intensity upon universal log10 transformation. (95% Confidence Intervals: Ascaris- slope 1.028 to 1.089; Y-intercept 0.6405 to -0.4333; X-intercept 0.4205 to 0.5895. Necator—slope 0.3151 to .7344; Y- intercept -0.6150 to 0.7130; X-intercept -2.253 to 0.8413). * PCR Intensity = 10–0.298*Ct +9.81.