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 β-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. duodenalispurified 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.

Intestinal Schistosomiasis and Giardiasis Co-Infection in Sub-Saharan Africa: Can a One Health Approach Improve Control of Each Waterborne Parasite Simultaneously?

“…Identification of cysts in concentrated faecal smear via formalin/ether concentration techniques, flotation techniques or immunofluorescent antibody microscopy [68] Detection of species-specific antigens in faecal samples using ELISA or lateral-flow test strips [11,[78][79][80] Detection and amplification of species-specific DNA in faecal samples using PCR/qPCR [78], (LAMP and RPA assays have also been developed [81][82][83]).…”

“…While POC-RDTs have many advantages over light-microscopy, microscopy remains less financially expensive to carry out and so it is the favoured method of diagnosis during routine monitoring and control programmes with only limited financial resources available [68,75,78]. In addition, and though promising, assays such as LAMP and RPA to detect species-specific parasite DNA currently require further assessment and validation before their upscaled and routine use in such control programmes [81,85]. Nevertheless, continued development, assessment and validation of POC-RDTs is widely advocated as affordable and sensitive point-of-care diagnostics, capable of detecting low levels of infection within individuals able to maintain disease transmission, are sorely needed [86].…”

“…Away from the lake at Biiso and Busingiro, the prevalence of giardiasis and intestinal schistosomiasis declined, suggesting that the risk of infection is perhaps higher on the lake shoreline. This study also attempted to evaluate a new POC-RPA RDT onsite, as well as a pilot assessment of giardiasis in local livestock and companion animals [81]. Ultimately, the RPA assay did not perform as well as expected, being in need for further optimisation of stool DNA extraction protocols.…”

Rapid Detection of Pathogens in Wound Exudate via Nucleic Acid Lateral Flow Immunoassay

“…Among the isothermal amplification methods that can be interacted with LFA-based analysis, especially the recombinase polymerase amplification (RPA) has gained in importance as a cost-effective and reliable technology for POC diagnostics [ 37 ]. In the last years several articles were published that successfully combined RPA and LFA [ 36 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 ]. However, only a minority of them showed direct amplification of target nucleic acids from crude lysate [ 45 , 46 , 47 , 48 , 49 , 50 ].…”

“…However, only a minority of them showed direct amplification of target nucleic acids from crude lysate [ 45 , 46 , 47 , 48 , 49 , 50 ]. Most of these publications used purified nucleic acids [ 38 , 39 , 40 , 41 , 42 , 43 , 44 ] or commercial nucleic acid isolation kits [ 36 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 ] that require at least 6–13 hands-on steps and/or substantial sample preparation time (10–60 min), which adds to the total process time. Thus, these workflows do not meet the requirements of a rapid and simple POC test.…”