Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity Of Intestinal Parasite Infections in a Controlled Clinical Trial

Abstract

Background: Accurate quantitative assessment of infection with soil transmitted helminths and protozoa is key to the interpretation of epidemiologic studies of these parasites, as well as for monitoring large scale treatment efficacy and effectiveness studies. As morbidity and transmission of helminth infections are directly related to both the prevalence and intensity of infection, there is particular need for improved techniques for assessment of infection intensity for both purposes. The current study aimed to evaluate two multiplex PCR assays to determine prevalence and intensity of intestinal parasite infections, and compare them to standard microscopy.

Methodology/principal findings: Faecal samples were collected from a total of 680 people, originating from rural communities in Timor-Leste (467 samples) and Cambodia (213 samples). DNA was extracted from stool samples and subject to two multiplex real-time PCR reactions the first targeting: Necator americanus, Ancylostoma spp., Ascaris spp., and Trichuris trichiura; and the second Entamoeba histolytica, Cryptosporidium spp., Giardia. duodenalis, and Strongyloides stercoralis. Samples were also subject to sodium nitrate flotation for identification and quantification of STH eggs, and zinc sulphate centrifugal flotation for detection of protozoan parasites. Higher parasite prevalence was detected by multiplex PCR (hookworms 2.9 times higher, Ascaris 1.2, Giardia 1.6, along with superior polyparasitism detection with this effect magnified as the number of parasites present increased (one: 40.2% vs. 38.1%, two: 30.9% vs. 12.9%, three: 7.6% vs. 0.4%, four: 0.4% vs. 0%). Although, all STH positive samples were low intensity infections by microscopy as defined by WHO guidelines the DNA-load detected by multiplex PCR suggested higher intensity infections.

Conclusions/significance: Multiplex PCR, in addition to superior sensitivity, enabled more accurate determination of infection intensity for Ascaris, hookworms and Giardia compared to microscopy, especially in samples exhibiting polyparasitism. The superior performance of multiplex PCR to detect polyparasitism and more accurately determine infection intensity suggests that it is a more appropriate technique for use in epidemiologic studies and for monitoring large-scale intervention trials.

Fig 1. Flow chart of sample processing for multiplex PCR detection of intestinal parasites.

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.

Fig 4. Polyparasitism.

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.}