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Application of a recombinase polymerase amplification (RPA) assay and pilot field testing for Giardia duodenalis at Lake Albert, Uganda.

ABSTRACT: BACKGROUND:Giardia duodenalis is a gastrointestinal protozoan causing 184 million cases of giardiasis worldwide annually. Detection is by microscopy or coproantigen assays, although sensitivity is often compromised by intermittent shedding of cysts or trophozoites, or operator expertise. Therefore, for enhanced surveillance field-applicable, point-of-care (POC), molecular assays are needed. Our aims were to: (i) optimise the recombinase polymerase amplification (RPA) assay for the isothermal amplification of the G. duodenalis ?-giardin gene from trophozoites and cysts, using published primer and probes; and (ii) perform a pilot field validation of RPA at a field station in a resource-poor setting, on DNA extracted from stool samples from schoolchildren in villages around Lake Albert, Uganda. Results were compared to an established laboratory small subunit ribosomal RNA (SSU rDNA) qPCR assay with additional testing using a qPCR targeting the triose phosphate isomerase (tpi) DNA regions that can distinguish G. duodenalis of two different assemblages (A and B), which are human-specific. RESULTS:Initial optimisation resulted in the successful amplification of predicted RPA products from G. duodenalis-purified gDNA, producing a double-labelled amplicon detected using lateral flow strips. In the field setting, of 129 stool samples, 49 (37.9%) were positive using the Giardia/Cryptosporidium QuikChek coproantigen test; however, the RPA assay when conducted in the field was positive for a single stool sample. Subsequent molecular screening in the laboratory on a subset (n?=?73) of the samples demonstrated better results with 21 (28.8%) RPA positive. The SSU rDNA qPCR assay resulted in 30/129 (23.3%) positive samples; 18 out of 73 (24.7%) were assemblage typed (9 assemblage A; 5 assemblage B; and 4 mixed A+B). Compared with the SSU rDNA qPCR, QuikChek was more sensitive than RPA (85.7 vs 61.9%), but with similar specificities (80.8 vs 84.6%). In comparison to QuikChek, RPA had 46.4% sensitivity and 82.2% specificity. CONCLUSIONS:To the best of our knowledge, this is the first in-field and comparative laboratory validation of RPA for giardiasis in low resource settings. Further refinement and technology transfer, specifically in relation to stool sample preparation, will be needed to implement this assay in the field, which could assist better detection of asymptomatic Giardia infections.

SUBMITTER: Molina-Gonzalez SJ

PROVIDER: S-EPMC7275508 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Application of a recombinase polymerase amplification (RPA) assay and pilot field testing for Giardia duodenalis at Lake Albert, Uganda.

Molina-Gonzalez Sandra J SJ Bhattacharyya Tapan T AlShehri Hajri R HR Poulton Kate K Allen Stephen S Miles Michael A MA Arianitwe Moses M Tukahebwa Edridah M EM Webster Bonnie B Russell Stothard J J Bustinduy Amaya L AL

Parasites & vectors 20200606 1

<h4>Background</h4>Giardia duodenalis is a gastrointestinal protozoan causing 184 million cases of giardiasis worldwide annually. Detection is by microscopy or coproantigen assays, although sensitivity is often compromised by intermittent shedding of cysts or trophozoites, or operator expertise. Therefore, for enhanced surveillance field-applicable, point-of-care (POC), molecular assays are needed. Our aims were to: (i) optimise the recombinase polymerase amplification (RPA) assay for the isothe ...[more]

PMID: 32505215

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Project description:Equine piroplasmosis (EP) is a severe disease of horses caused by the tick-borne protozoa Theileria equi (T. equi) and Babesia caballi (B. caballi). Infectious carriers are not always symptomatic, meaning there is a risk to non-enzootic areas. Regulatory tests for EP include sero-epidemiological methods for equine babesiosis, but these lack specificity due to cross-reactivity with other Babesia species. In this study, we present a real-time quantitative recombinase polymerase amplification (qRPA) method for fast simultaneous detection of both T. equi and B. caballi. In this method, primers and probes targeting the 18S rRNA gene of both T. equi and B. caballi, the ema-1 gene of T. equi and the bc48 gene of B. caballi were designed and evaluated. The sensitivity of qRPA was evaluated using the pUC57 plasmid DNA containing the target gene. For the pUC57-bc48 gene DNA, the R2 value was 0.983 for the concentration range 0.2 ng (4.1 × 107 DNA copies) to 2.0 fg (4.1 × 101 DNA copies). For the pUC57-ema gene DNA, the R2 value was 0.993 for the concentration range 0.2 ng (5.26 × 107 DNA copies) to 2.0 fg (5.26 × 102 DNA copies). For the pUC57-Bc18S gene DNA the R2 value was 0.976 for the concentration range 2.0 ng (4.21 × 108 DNA copies) to 2.0 fg (4.21 × 102 DNA copies). For the pUC57-Te18S gene DNA, the R2 value was 0.952 (Fig. S3b) for the concentration range 2.0 ng (4.16 × 108 DNA copies) to 2.0 fg (4.16 × 102 DNA copies). Furthermore, a duplex qRPA analysis was developed and optimized and the results showed that primers and probes targeting for the bc48 gene of B. caballi and the 18S rRNA gene of T. equi is the best combination for a duplex qRPA analysis in one reaction. The developed duplex qRPA assay has good specificity, and had negative amplification for several similar parasite. For DNA extracted from real horse blood specimens, this qRPA method has comparable sensitivity to traditional qPCR, but a simpler and more rapid operating process to obtain positive amplification. The qRPA, including the duplex strategy described here, could allow fast identification of the EP-causing T. equi and B. caballi, showing great potential for on-site EP screening of horses.

Dataset Information

Application of a recombinase polymerase amplification (RPA) assay and pilot field testing for Giardia duodenalis at Lake Albert, Uganda.

Publications

Application of a recombinase polymerase amplification (RPA) assay and pilot field testing for Giardia duodenalis at Lake Albert, Uganda.

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