Genus Rhabditis have been reported to infect human digestive system [12-15], urinary system [16, 17, 19], and even outer ear canal ; and excreting larvae in stool and urine. As the parasitological diagnosis of S. stercoralis is the detection of parasite larvae in stool exams and other biological materials such as such as sputum, duodenal aspirates, gastric biopsies, cervical smear or CSF liquid , therefore, this study pointing out to the challenge for discrimination between S. stercoralis and Rhabditis species. Due to the differences in the managements of infected patients with S. stercoralis , the exact differentiations of these two parasites is necessary.
The essential of early diagnosis of strongyloidiasis, especially in immunocompromised individuals, urged the efforts for development of molecular biology technique, mostly based on PCR, during recent years. Although, such techniques are highly sensitive and specific, they are recommended as confirmatory tests . Besides, such molecular techniques have not been developed for detection of Rhabditis spp. recovered from human stool samples, and on the other hand, identification of such rare parasites using morphological criteria requires expert microscopists that may be lacking among new generation of laboratory staff . Therefore, in order to attempt for prevention of misdiagnosis between these parasites, the present study was designed to characterize human retrieved isolates of S. stercoralis and Rhabditis spp. based on the amplification of cox1 gene. For this purpose, ten S. stercoralis and three Rhabditis spp. isolates were obtained from infected human stool samples and molecular approach was carried out by utilizing cox1 gene.
Although studies comparing quantitative polymerase chain reaction (qPCR) with microscopy methods for soil-transmitted helminthes are imperfect, they do show a significant increase in sensitivity and specificity of qPCR compared with labor-intensive traditional microscopic techniques . The cox1 gene of mitochondrial DNA, which has been reported to mutate more rapidly than 18S rDNA, is useful sources of sequence data for study on different populations of Strongyloides species  and a useful target for molecular diagnosis of strongyloidiasis in human stool samples . Presently, utilization of molecular-based methods in identification of Rhabditis species in human is very rare in clinical settings . The sequence alignments of this study illustrated a considerable difference between nucleotide sequences of S. stercoralis and Rhabditis species. The inter-species genetic variation between S. stercoralis and Rhabditis spp. was 13.5 to 14.5%. According to pairwise distance of the current study isolates, intra-species genetic variation within S. stercoralis nucleotide sequences was 0 to 3.5%. Pairwise difference in cox1 gene among interbreeding strains of a nematode species was reported to be usually less than 6% and that between distinct species in a genus was more than 10% . Pairwise difference in nucleotides sequence of cox1 gene among isolates of S. stercoralis from humans, apes and dogs was less than 4%, regardless of the host and locality of the isolates . The average pairwise distances of cox1 nucleotide sequences were 3.6% for S. stercoralis isolates from human and dog collected mainly from Myanmar .
According to the phylogenetic analysis of current study, S. stercoralis isolates were located in a distinct clade from Rhabditis species. Rhabditis spp. clade was included three isolates, all merely from our study, having 100% similarity with each other. There was not any available cox1 gene sequence of Rhabditis species with human origin or other pathogenic isolates to be included in the constructed tree for comparison. The phylogenetic tree indicated that the ten sequences of S. stercoralis consisted of five haplotypes. The haplotype one, which included four isolates, showed 100% homology with GenBank registered sequences of human isolates from Thailand (KY081240), Myanmar (LC179147), and dog isolate from Cambodia (KX226374). The haplotypes two, three and four, each including one isolate, have not been already recorded from other regions in the world. Moreover, haplotype five, locating solely in separate cluster and including three completely similar isolates, was distinctive from any other haplotypes in the tree. Despite the small number of S. stercoralis isolates studied presently, five different haplotypes were recovered which four of them were considered as new reports. Thus far, it seems S. stercoralis represents diverse haplotypes and needs to be speculated further using more isolates from different regions of the world in order to investigate its genetic variations.
Among our isolates of S. stercoralis, haplotype one indicated 100% homology with a dog isolate from Cambodia (KX226374), belonging to the population indistinguishable from the population of S. stercoralis isolated from humans in Cambodia . In mentioned study, two genetically different populations of Strongyloides spp. were found in dogs, one of which that the majority of the worms belonged, appeared to be restricted to dogs; the other population was shared with humans . In another study, using nuclear and mitochondrial markers, phylogenetic relationships among S. stercoralis isolates from several human and dog populations in multiple countries of East Asia were examined . Accordingly, two distinct lineage of S. stercoralis were present, referring to as type A parasites isolated both from humans and dogs, and type B parasites founding exclusively in dogs and not adapted to infect humans. All these findings suggest the possibility of zoonotic potential of S. stercoralis and the possibility that human S. stercoralis originated from dogs. Thus, dogs might be considered as a reservoir for human S. stercoralis. Further studies with larger samples of human and dog isolates from different geographical areas is recommended for assessing this assumption particularly in region where people have close contact with dogs.