The laboratory diagnosis of Strongyloides stercoralis (S. stercoralis) at the Department of Clinical Parasitology (DCP) by the routine methods of microscopy and Strongyloides culture is not sensitive due to the, usually, low parasite load and intermittent larval excretion of the parasite. Serology (enzyme-linked immunosorbent assay) suffers from a lack of specificity because Strongyloides antibodies are known to cross- react with schistosomal, filarial and other helminthic antibodies in serological tests. Moreover, antibody levels are slow to decline after successful treatment therefore serology cannot be used to monitor point of cure. A missed diagnosis of strongyloidiasis in immunocompromised patients or those about to undergo iatrogenic immune suppression may have severe, even fatal, consequences. The disease is poorly studied because of the lack of sensitive, specific and cost-effective tests. Therefore, the decision was made to evaluate and validate nucleic acid amplification techniques (NAATs) for the diagnosis of S. stercoralis for use in a well- resourced specialist referral parasitology laboratory. A novel loop mediated isothermal amplification (LAMP) assay was also developed for use in resource- limited regions. The study was conducted over two years (2014-2016) and examined 284 residual diagnostic samples. The cohort was drawn from patients attending a central London western travel medicine (WTM) clinic. The NAATs chosen for this study were a published real- time PCR (qPCR) assay (ten Hove et al., 2009) and a novel LAMP assay. The NAATs were compared to the combined reference standard of microscopy, culture and serology for the diagnosis of S. stercoralis in stool samples. The development of the novel LAMP assay for use in resource- limited areas included the investigation of methods for rapid, simple and cost- effective DNA extraction. The qPCR and LAMP assays detect target DNA within areas on either side of the S. stercoralis 18S rRNA genome hypervariable region (Hasegawa et al., 2009). In this study the LAMP and qPCR assays demonstrated a limit of detection of 10-3 and 10-4, respectively for S. stercoralis DNA detection in clinical samples. Specificity was determined for the LAMP and qPCR assays to be 100% and 94.83%, respectively and the cost per test was calculated as £4.80 and £8.21, respectively. In this study, persistence of S. stercoralis DNA in clinical samples was improved when the samples were stored at -20oC. While the LAMP assay has a shorter turnaround time and is less costly than qPCR, the superior efficiency of qPCR detection of S. stercoralis DNA in clinical samples established that the qPCR assay was a more suitable addition to the diagnostic repertoire at a high- throughput WTM clinic. The LAMP assay showed promise for deployment in resource- limited areas and as a point- of- care test but further work is required to optimise the LAMP assay for these purposes.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:759746 |
| Date | January 2017 |
| Creators | Bowers, Katherine |
| Publisher | University of Westminster |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://westminsterresearch.westminster.ac.uk/item/q56v6/development-and-clinical-performance-of-nucleic-acid-amplification-techniques-for-the-diagnosis-of-strongyloides-stercoralis |
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