A Sarcoptes scabiei specific isothermal amplification assay for detection of this important ectoparasite of wombats and other animals

Background: The globally distributed epidermal ectoparasite, Sarcoptes scabiei, is a serious health and welfare burden to at-risk human and animal populations. Rapid and sensitive detection of S. scabiei infestation is critical for intervention strategies. While direct microscopy of skin scrapings is a widely utilised diagnostic method, it has low sensitivity. PCR, alternatively, has been shown to readily detect mite DNA even in microscopy-negative skin scrapings. However, a limitation to the latter method is the requirements for specialised equipment and reagents. Such resources may not be readily available in regional or remote clinical settings and are an important consideration in diagnosis of this parasitic disease. Methodology: A Loop Mediated Isothermal Amplification(LAMP) assay targeting the ITS-2 gene for S. scabiei was developed and evaluated on clinical samples from various hosts, previously screened with conventional S. scabies-specific PCR. Species specificity of the newly developed LAMP assay was tested against a range of DNA samples from other arthropods. The LAMP assays were performed on a real-time fluorometeras well as thermal cyclerto evaluate an end-point of detection. Using skin scrapings, a rapid sample processing method was assessed to eliminate extensive processing times involved with DNA extractionsprior to diagnostic assays, including LAMP. Results: The S. scabiei LAMP assay was demonstrated to be species-specific and able to detect DNA extractedfrom a single mite within a skin scraping in under 30 minutes. Application of this assay to DNA extractsfrom skin scrapings taken from a range of hosts revealed 92.3% congruence (with 92.50% specificity and 100% sensitivity) to the conventional PCR detection of S. scabiei. Preliminary results have indicated that diagnostic outcome from rapidly processed dry skinscrapings using our newly developed LAMP is possible in approximately 40 minutes. Discussion: We have developed a novel, rapid and robust molecular assay for detecting S. scabiei infesting humans and animals. Based on these findings, we anticipate that this assay will serve an important role as an ancillary diagnostic tool at the point-of-care, complementing existing diagnostic protocols for S. scabiei.;