Abstract
BackgroundGiardia 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.ResultsInitial 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.ConclusionsTo 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.
Highlights
Giardia duodenalis is a gastrointestinal protozoan causing 184 million cases of giardiasis worldwide annually
recombinase polymerase ampli‐ fication (RPA) optimisation at London School of Hygiene and Tropical Medicine (LSHTM) As shown in Fig. 1b, the RPA with DNA extracted from the G. duodenalis cultures generated amplicons of the predicted sizes from the β-giardin gene
The 153-bp FAM-biotin double-labelled product could be reliably identified by the PCRD lateral flow cassette; cyst DNA diluted to 104 generated RPA products detectable by lateral flow and confirmed by gel electrophoresis (Fig. 1c)
Summary
Giardia duodenalis is a gastrointestinal protozoan causing 184 million cases of giardiasis worldwide annually. Our aims were to: (i) optimise the recombinase polymerase ampli‐ fication (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. Giardia duodenalis (syns G. lamblia and G. intestinalis) is a flagellated protozoan parasite of the human gastrointestinal tract as well as a range of other mammals worldwide. It is acquired by ingestion of viable cysts present in faecal-contaminated water, food or on fomites [1]. On the basis of molecular characterisation, G. duodenalis is divided into eight genetic assemblages (A–H), of which A and B are considered human-specific [3]; it is important to identify which assemblages are present during validation of any new molecular diagnostic based on species-specific DNA loci
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