Background: Fasciolosis is a shared disease between humans and livestock caused by hepatic trematodes; Fasciola hepatica and F. gigantica. Differentiate between the two species of this genus is essential. High-Resolution Melting (HRM) Analysis represents a new approach to this issue. This method can be performed right after termination of Real-Time PCR. This technique has not been used for identification of adult F. hepatica and F. gigantica genotypes. The aim of this study was to determine Fasciola species by using HRM in isolates taken from Iran, respectively. Methods: Ninety-three Fasciola spp. samples were collected from infected slaughtered animals in different regions of Iran, including North West (Ardebil Province) and South East (Zahedan Province) during 2016. Genomic DNA from the samples was extracted using a DNA extraction kit and then after Real-Time PCR amplification, HRM was done. Results: Overall, 59 and 34 isolates were identified as F. hepatica and F. gigantica, respectively. The percentages of each species from animals were as follows: sheep (F. hepatica, 80.39% and F. gigantica, 19.61%), cattle (F. hepatica, 42.85% and F. gigantica, 57.15%). Conclusion: HRM technique developed in the present study is a powerful, rapid and sensitive technique for epidemiological survey and molecular identification between F. hepatica and F. gigantica.
Background: We aimed at genotyping and evaluating the predominance of G. duodenalis assemblages isolated from patients referred to medical laboratories in Khorramabad, Iran from Nov 2015 to Sep 2016. Hence, the development of a costeffective HRM approach to determine genotypes of G. duodenalis based on the triosephosphate isomerase (tpi) gene was examined and the genotyping results with and without diarrhea was compared. Methods: Seventy G. duodenalis positive fecal samples were collected. A microscopic confirmation for the presence of Giardia spp. was performed, cysts of 70 Giardia spp. positive specimens were concentrated using sucrose flotation technique and sucrose solution PCR amplification was performed on 69 of 70 (98.5%) samples, and High Resolution Melting (HRM) analysis was performed using a software. Results: The results showed two distinct genotypes (assemblages A and B) of G. duodenalis but infections with mixture of both assemblages were not detected. The genotypes of G. duodenalis showed that the sub assemblage AI, BIII and BIV were present in a proportion of 68.1%, 20.3% and 11.6% respectively in samples. Assemblage AI was significantly (P<0.05) more frequently found in patients with diarrhea. Conclusion: The sub-assemblage AI, BIII, and BIV are more zoonotic potential. According to the comparison of the results of this study with the results of previous studies in this area and around of it, as well as the way people live and keep pets. This pattern established in Khorramabad city. HRM can be an ideal technique to detect and genotyping of G. duodenalis in clinical samples.
Background: It is difficult to make an exact morphological distinction between Fasciola hepatica and Fasciola gigantica. We used High Resolution Melting analysis (HRM) method to differentiate the F. hepatica species from F. gigantica in order to differentiate them. Methods: Overall, 80 adult liver flukes were collected from infected slaughtered animals including cattle, sheep and goats from Lorestan Province, western Iran from Sep 2015 to Aug 2017. Genomic DNA was extracted using commercial DNA extraction kit. The multilocus sequences of mDNA including COX1, COX3 and ND6 were amplified employing real-time PCR & HRM analysis. Specific and universal primer pairs were designed for differentiation Fasciola spp. Results: Universal primers cannot be used to distinguish between these two species, but in the contrary, specific primer pairs of each species could differentiate them properly. Molecular identification using specific primer pairs were consistent. Conclusion: HRM is a simple, fast and reliable method for detecting and differentiating F. hepatica from F. gigantica and can be used for diagnostic and epidemiological purposes.
Background: Toxoplasma gondii is an obligate intracellular protozoan with worldwide distribution. Diagnosis of toxoplasmosis is a very critical issue, especially in pregnant women and immunocompromised patients. The aim of this study was rapid detection of T. gondii DNA in peripheral blood samples (PBS) employing HRM technique and using RE gene. Methods: Totally, 242 samples from pregnant women and human immunodeficiency virus (HIV) patients were collected from different hospitals and medical centers of Tehran during Oct 2017 to Dec 2018. High resolution melting analysis (HRM) using partial sequences of repetitive element (RE) gene was done and compared with ELISA test. Results: Overall, 51 were positive for acute toxoplasmosis that among them, 12 and 20 reported as positive in pregnant women and HIV+ patients, respectively using HRM technique. Among 70 patients in chronic phase of disease, 10 and 3 samples were reported as positive for pregnant women and HIV+ patients respectively. From 121 negative control, 3 (4.62%) samples associated with HIV+ patients, showed positive real-time PCR and HRM analysis results. Conclusion: For the first time, HRM technique via employing RE gene was used for detection of T. gondii infection in PBS. This method is suitable, helpful and in parallel with serological methods for early diagnosis of acute as well as active form of toxoplasmosis in pregnant women and HIV+ patients. The use of techniques based on melt curve and through employing next-generation dyes for diagnosis of T. gondii would be accessible for patients in developing countries.
Background: Asymptomatic malaria, which usually exists in low parasitemia, acts as the Plasmodium species reservoirs contributing towards malaria transmission. This situation hinders malaria elimination programs in endemic areas, thus necessitating an active case detection with a high sensitive method and treatment of cases. This is why we used a High Resolution Melting (HRM) assay to monitor the trend of asymptomatic malaria in a malaria endemic area of Iran which is under elimination program. Methods: The peripheral blood was sampled from 271 clinically approved non-febrile individuals from a malaria endemic zone of southeastern Iran for asymptomatic malaria prevalence detection by microscopy, Rapid Diagnostic Tests (RDTs) and HRM methods. The HRM assay was done based on the amplification of 18S SSU rRNA gene. Results: The HRM assay revealed infections from three individuals out of 271 (1.1% asymptomatic malaria prevalence) from the participants, two Iranian natives with Plasmodium vivax infection and one Pakistani immigrant with P. falciparum infection. Neither microscopy nor RDTs detected Plasmodium spp infections from the 271 non-febrile individuals. The nucleotide sequencing analysis of the positive controls used in this study showed a close homology with the reference gene bank sequences of P. falciparum 3D7 (CPO16995.1) and P. vivax Sal-1(UO3079.1). Conclusion: This study revealed a low frequency of asymptomatic malaria trend within malaria endemic areas of southeastern Iran which are under intense elimination program and also the ability of HRM assay in detecting low Plasmodium spp parasitemia beyond the limits of microscopy and RDTs.
Background: The liver flukes of the Fasciola species and Dicrocoelium spp. are recognised as parasites of domestic and wild herbivores. Both species of F. hepatica and F. gigantica as well as D. dendriticum are distributed in Iran. The present study aimed to identify Fasciola spp. and Dicrocoelium spp. using mitochondrial Cox1 (cytochrome c oxidase I) gene by HRM method. Methods: Totally, thirty infected liver specimens were collected from the sheep (n:23) and cattle (n:7) at the abattoirs of Qazvin Province, northwest Iran in 2022. DNA extraction and PCR amplification of Cox1 gene were conducted by HRM technique. DnaSP v.5.0 was used for compression of diversity indices of ribosomal 28S rDNA and mitochondrial Cox1 markers of Dicrocoelium spp. The taxonomic status of Dicrocoelium spp. was performed by sequencing and phylogenetic analysis. Results: Overall, 26 and 4 isolates were identified as F. hepatica and F. gigantica, respectively. D. dendriticum was the sole infecting species of Dicrocoelium revealed by HRM analysis. Genomic analysis showed a moderate (28S rDNA genes: 0.600±0.215) to high (Cox1: 0.733±0.155) haplotype diversity for D. dendriticum. Conclusion: The parasite-dependent mitochondrial gene (Cox1) could identify a higher genetic diversity of D. dendriticum compared to nuclear 28S rDNA gene. HRM technique in the present study found to be a reliable technique for identification and genetic diversity of liver flukes but more comprehensive and in-depth studies in different parts of the country are needed.
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