Lena Chng1, Deborah C Holt2,3, Matt Field4,5, Joshua R Francis2,6, Dev Tilakaratne6,7, Milou H Dekkers8, Greg Robinson1, Kate Mounsey9, Rebecca Pavlos10, Asha C Bowen2,10,11, Katja Fischer1, Anthony T Papenfuss12, Robin B Gasser13, Pasi K Korhonen13, Bart J Currie2,6, James S McCarthy1, Cielo Pasay1. 1. QIMR Berghofer Medical Research Institute, Brisbane, Australia. 2. Menzies School of Health Research, Charles Darwin University, Darwin, Australia. 3. College of Health and Human Sciences, Charles Darwin University, Darwin, Australia. 4. Centre for Tropical Bioinformatics and Molecular Biology and Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, Australia. 5. Genome Informatics, John Curtin School of Medical Research, Australian National University, Canberra, Australia. 6. Royal Darwin Hospital, Tiwi, Australia. 7. Darwin Dermatology, Tiwi, Australia. 8. Queensland Animal Science Precinct, University of Queensland, Gatton, Australia. 9. University of Sunshine Coast, Sippy Downs, Australia. 10. Wesfarmers Centre for Vaccines and Infectious Diseases, Telethon Kids Institute, University of Western Australia, Perth, Australia. 11. Department of Infectious Diseases, Perth Children's Hospital, Perth, Australia. 12. The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia. 13. Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary Sciences, The University of Melbourne, Parkville, Australia.
Abstract
BACKGROUND: The suboptimal sensitivity and specificity of available diagnostic methods for scabies hampers clinical management, trials of new therapies and epidemiologic studies. Additionally, parasitologic diagnosis by microscopic examination of skin scrapings requires sample collection with a sharp scalpel blade, causing discomfort to patients and difficulty in children. Polymerase chain reaction (PCR)-based diagnostic assays, combined with non-invasive sampling methods, represent an attractive approach. In this study, we aimed to develop a real-time probe-based PCR test for scabies, test a non-invasive sampling method and evaluate its diagnostic performance in two clinical settings. METHODOLOGY/PRINCIPAL FINDINGS: High copy-number repetitive DNA elements were identified in draft Sarcoptes scabiei genome sequences and used as assay targets for diagnostic PCR. Two suitable repetitive DNA sequences, a 375 base pair microsatellite (SSR5) and a 606 base pair long tandem repeat (SSR6), were identified. Diagnostic sensitivity and specificity were tested using relevant positive and negative control materials and compared to a published assay targeting the mitochondrial cox1 gene. Both assays were positive at a 1:100 dilution of DNA from a single mite; no amplification was observed in DNA from samples from 19 patients with other skin conditions nor from house dust, sheep or dog mites, head and body lice or from six common skin bacterial and fungal species. Moderate sensitivity of the assays was achieved in a pilot study, detecting 5/7 (71.4% [95% CI: 29.0% - 96.3%]) of clinically diagnosed untreated scabies patients). Greater sensitivity was observed in samples collected by FLOQ swabs compared to skin scrapings. CONCLUSIONS/SIGNIFICANCE: This newly developed qPCR assay, combined with the use of an alternative non-invasive swab sampling technique offers the possibility of enhanced diagnosis of scabies. Further studies will be required to better define the diagnostic performance of these tests.
BACKGROUND: The suboptimal sensitivity and specificity of available diagnostic methods for scabies hampers clinical management, trials of new therapies and epidemiologic studies. Additionally, parasitologic diagnosis by microscopic examination of skin scrapings requires sample collection with a sharp scalpel blade, causing discomfort to patients and difficulty in children. Polymerase chain reaction (PCR)-based diagnostic assays, combined with non-invasive sampling methods, represent an attractive approach. In this study, we aimed to develop a real-time probe-based PCR test for scabies, test a non-invasive sampling method and evaluate its diagnostic performance in two clinical settings. METHODOLOGY/PRINCIPAL FINDINGS: High copy-number repetitive DNA elements were identified in draft Sarcoptes scabiei genome sequences and used as assay targets for diagnostic PCR. Two suitable repetitive DNA sequences, a 375 base pair microsatellite (SSR5) and a 606 base pair long tandem repeat (SSR6), were identified. Diagnostic sensitivity and specificity were tested using relevant positive and negative control materials and compared to a published assay targeting the mitochondrial cox1 gene. Both assays were positive at a 1:100 dilution of DNA from a single mite; no amplification was observed in DNA from samples from 19 patients with other skin conditions nor from house dust, sheep or dog mites, head and body lice or from six common skin bacterial and fungal species. Moderate sensitivity of the assays was achieved in a pilot study, detecting 5/7 (71.4% [95% CI: 29.0% - 96.3%]) of clinically diagnosed untreated scabiespatients). Greater sensitivity was observed in samples collected by FLOQ swabs compared to skin scrapings. CONCLUSIONS/SIGNIFICANCE: This newly developed qPCR assay, combined with the use of an alternative non-invasive swab sampling technique offers the possibility of enhanced diagnosis of scabies. Further studies will be required to better define the diagnostic performance of these tests.
Authors: Michael Marks; Jodie McVernon; James S McCarthy; Wendemagegn Enbiale; Christopher Hanna; Olivier Chosidow; Daniel Engelman; Kingsley Asiedu; Andrew Steer Journal: PLoS Negl Trop Dis Date: 2022-08-30