MOLECULAR CHARACTERIZATION OF SCABIES MITE (Sarcoptes scabieivar.hominis) IN PAKISTAN

Abstract

Scabies is a human skin disease due to the burrowing ectoparasite Sarcoptes scabiei var. hominis resulting in intense itching and inflammation and manifesting as a skin allergy. Currently limited information is known about the genetic diversity of Sarcoptes scabiei mites in human and also little is known about the specific scabies molecules involved in the inflammatory and immunologic responses. Due to insufficient mite material and lack of in vitro propagation system for antigen preparation, scabies is a challenging disease to diagnose. To examine the extent of the genetic variation in the DNA sequences of Sarcoptes scabiei var. hominis, mites were collected from scabietic patients by visiting the dermatology clinics of private and government hospitals from different localities of Pakistan. Individual mite gDNA was first amplified using ITS-2 and 16S by PCR, however, later amplicons were sequenced directly. Sequence analysis of Pakistani isolates by mitochondrial 16S rRNA gene showed greater sequence variability with 0.451M average evolutionary divergence (AED) as compared to ITS-2 sequences having a level of 0.01M AED. Moreover, results derived from Neighbor-joining tree showed that ITS-2 sequences did not show any host segregation and geographical isolation, whereas 16S indicated presence of both host adapted and geographically segregated populations of S. scabiei. These results suggested that 16S rRNA appeared to be suitable for examining genetic diversity among human mite populations as compared to ITS-2. Moreover different varieties of Sarcoptes mites belonged to different host species and geographic regions recommended a common gene pool which represented existence of a single species. For allergen characterization, full-length S. scabiei tropomyosin (Sar s 10) was cloned xxiii and expressed in pET-15b and assessed for reactivity with IgE antibodies from human sera. IgE binding was observed to Sar s 10 with sera collected from crusted and ordinary scabies, House Dust Mite (HDM) positive and naive subjects and a diagnostic sensitivity of <30% was observed. S. scabiei paramyosin (Sar s 11) was cloned and expressed in pET-28a in three overlapping fragments designated as Sspara1, Sspara2 and Sspara3. IgE and IgG binding was observed to Sspara2 and Sspara3 antigens with sera collected from crusted and ordinary scabies, and HDM positive subjects but no binding was observed with sera collected from naive subjects. Sspara 2 displayed excellent diagnostic potential with 98% sensitivity and >90% specificity observed for IgE binding and 70% sensitivity for IgG. In contrast the diagnostic efficiency of Sspara 3, was 84% for IgE binding and 40% for IgG binding. In combination Sspara 2 and Sspara3 provided an IgE sensitivity of 98%. These results demonstrate the genus-specific scabies mite epitopesare able to detect IgE reactivity with high sensitivity. The developed ELISA represents a marked improvement for the clinical diagnosis of scabies and helps direct future development of a specific diagnostic tool for scabies. Keywords: Sarcoptes scabiei var. hominis, rDNA ITS-2, MtDNA-16S, Tropomyosin, Paramyosin, Recombinant, IgE, Diagnosis