Loop-mediated isothermal amplification as a reliable assay for Toxocara canis infection in pet dogs.

Keeping of infected dogs as pet results in the potential transmission risk factors for shedding helminthic infections such as toxocariasis. Lack of accurate identification of Toxocara canis eggs in non-dewormed infected pet dogs remains a diagnostic concern among researchers. In this study, dog owners were asked to fill up a questionnaire regarding their pets and their attitude towards the deworming regimen. One hundred faecal samples were collected from pet dogs (Northwest Iran) and were subsequently identified by the ZnSo4 flotation technique, PCR and loop-mediated isothermal amplification (LAMP) assays. The DNA of the recovered T. canis eggs was then extracted and amplified by LAMP and PCR. Furthermore, ITS2 amplicons were sequenced for appraisal of the phylogenetic analysis. Nine, 5 and 11% of T. canis infections were identified by microscopy, PCR and LAMP, respectively. It was detected that LAMP was 10 times (10-10to 10-13 g/µl) more sensitive than PCR (10-10to 10-12 g/µl). The kappa value between LAMP and PCR indicated a faint concurrence (0.463). The kappa coefficient between LAMP and flotation technique indicated a strong agreement (0.667). The highest infection rate (n = 11) was detected in non-dewormed pet dogs, particularly those less than 3 months old (P < 0.05). None of the infected dogs had a history of walking and kennelled behaviours in public places. The LAMP assay can address as a simple, rapid and highly sensitive technique for detecting low burden of T. canis eggs in infected pet dogs. It was proposed that the dog holder's awareness is insufficient to implement regular deworming schedules. Additionally, regional policymakers should broadly revise anthelmintic treatment guidelines.

Assessment of the global pattern of genetic diversity in Echinococcus multilocularis inferred by mitochondrial DNA sequences.

The aim of this review was to assess our current knowledge on phylogeography and global genetic structure of Echinococcus multilocularis populations originating from rodents, wild canid hosts, and human. Six bibliographic databases were searched from 1990 to 2017, identifying a total of 110 publications. The cytochrome c oxidase subunit 1 (cox1) and cytochrome b (cytb) sequences of E. multilocularis from Asia, Europe, and North Americas were analyzed to estimate the diversity and neutrality indices, and genetic differentiation. A total of 69 (cox1, 36.7%) and 16 haplotypes (cytb, 19.2%) were grouped into various geographical clades. A parsimonious haplotype network demonstrated a star-like feature with haplo-groups Em2 (Asia: 36%), Em105 (Eastern Tibetan plateau: 4.8%), Em46 (Europe: 9.1%), Em73, (Europe: 2.7%) and Em92 (North Americas: 4.3%) as the most common haplotypes. A relatively high level of genetic diversity was detected in rodent-derived E. multilocularis isolates (Haplotype diversity: 0.944), wild canids (Hd: 0.912), and human origin (Hd: 0.704). The highest number of haplotypes (n = 59) and the highest haplotype diversity (0.969) were identified in the Asian and European populations, respectively. Cladistic phylogenetic tree indicated the European clade has a sister relationship with the Asian clade. However, some North American haplotypes were assigned to the European clade together with haplotypes from Poland. The statistically significant Fst values indicated that E. multilocularis populations of Asian-European, Asian-North American, and European-North American origins were genetically differentiated (Fst: 0.22624 to 0.43059). An occurrence of distinct parasite populations suggests that E. multilocularis derived from glacial refugia have been plausibly sustained by indigenous hosts during the Pleistocene Epoch.

Genetic variability and discrimination of low doses of Toxocara spp. from public areas soil inferred by loop-mediated isothermal amplification assay as a field-friendly molecular tool.

ABSTRACT: Aim: One of the main diagnostic problems of conventional polymerase chain reaction (PCR) is indiscrimination of low parasitic loads in soil samples. The aim of this study is to determine the genetic diversity and identification of Toxocara spp. from public areas soil inferred by loop-mediated isothermal amplification (LAMP) assay. MATERIALS AND METHODS: A total of 180 soil samples were collected from various streets and public parks of northwest Iran. The DNA of recovered Toxocara eggs were extracted and amplified by PCR and LAMP following ZnSO4 flotation technique. The amplicons of internal transcribed spacer-2 gene were sequenced to reveal the heterogeneity traits of Toxocara spp. In addition, Toxocara canis sequences of southwest Iran were directly retrieved to compare gene flow between two distinct populations. RESULTS: Toxocara spp. eggs were found in 57, 14 and 77 of soil samples using the microscopy, PCR and LAMP (detection limit 1-3 eggs/200 g soil), respectively. 7.7% of isolates were identified as T. canis by PCR method, while LAMP was able to detect 27.2%, 15.5% and 12.2% as Toxocara cati, T. canis and mixed infections, respectively. The kappa coefficient between LAMP and microscopy indicated a strong agreement (0.765) but indicated a faint agreement among LAMP-PCR (0.203) and PCR-microscopy (0.308) methods. A pairwise fixation index (Fst) as a degree of gene flow was generally low (0.02156) among Toxocara populations of northwest and southwest Iran. CONCLUSIONS: The statistically significant Fst value indicates that the T. canis populations are not genetically well differentiated between northwest and southwest Iran. This shows that here is possibly an epidemiological drift due to the transfer of alleles. The LAMP assay because of its shorter reaction time, more sensitivity, and simultaneous detection of environmental contamination to be appears as valuable field diagnosis compared to PCR. Therefore, the detection of low Toxocara spp. loads from public area soils will help to expand epidemiological understanding of toxocariasis and establishing preventive strategies in resource-limited endemic of Iran.