Comments on "Genetic characterization and phylogenetic analysis of Fasciola species based on ITS2 gene sequence, with first molecular evidence of intermediate Fasciola from water buffaloes in Aswan, Egypt".

Dear Editor-in-Chief, In Annals of Parasitology 2021, 67(1), 55-65, a paper entitled "Genetic characterization and phylogenetic analysis of Fasciola species based on ITS2 gene sequence, with first molecular evidence of intermediate Fasciola from water buffaloes in Aswan, Egypt" was published with great interest [1]. After reading the article carefully and critically, we think some points should be noted. Fasciola species are meiotically functional diploid, can produce sperm and temporarily and store in the seminal vesicles. This type is named spermic fluke [2]. On the other hand, intermediate Fasciola with morphological characteristics intermediates between F. hepatica and F. gigantica with no sperm or aspermic and no sperm in seminal vesicles. However, this is also seen in older flukes [3-5]. It seems that morphological studies based on spermatogenesis ability were necessary for this study. Also, this parasite's anthelmintic resistance is due to aspects of biology, and population structure depends on genetic diversity [6]. We question whether there are any documents about and sequences of mitochondrial markers as COX (Cytochrome Oxidase) and NAD (Nicotinamide Adenine Dinucleotide) to analyze intraspecific phylogenetic relationship in addition to nuclear gene? In Table 3, the pairwise distances between three groups of Fasciola spp. from different livestock animals were low, ranging from 0.004 to 0.01 with an overall mean of 0.008. Genetic diversity is described as a tendency of genetic characteristics to vary and serves as a way for the population to adapt to changing hosts and environments [7]. The nature of the nuclear gene (ITS) is instability. It is better to use mitochondrial sequence data to compare diversity. Also, genetic discrimination grade from infra population to meta population is annotated by Fst value ranging; 0 to 1. Fst values between 0-0.05 indicated a low genetic differentiation population [8]. It seems that by calculating Fst and showing the gene migration based on mitochondrial sequences data of specimens, this study's species population will be obtained. Also, Tajima's D and Fu's F in all loci populations based on GenBank data may show the Fasciola haplotypes' population proximity. Here we recommend, that Omar et al. [1] studies that molecular phylogeny with mitochondrial DNA efectively used for appropriate diferentiation of haplotypes and spermatogenic ability by carmen allium staining helps them find the physiological aspects. Of course, more prominent populations are needed to find intermediate types. [1] Omar M.A, Elmajdoub L.O., Ali A.O., Ibrahim D.A., Sorour S.S., Al-Wabel M.A., Suresh M., Metwally A.M. 2021. Genetic characterization and phylogenetic analysis of Fasciola species based on ITS2 gene sequence, with first molecular evidence of intermediate Fasciola from water buffaloes in Aswan, Egypt. Annals of Parasitology 67: 55-65. doi:10.17420/ap6701.312 [2] Sanderson A. 1953. Maturation and probable gynogenesis in the liver fluke, Fasciola hepatica L. Nature 172: 110-112. doi:10.1038/172110a0 [3] Hayashi K., Ichikawa-Seki M., Mohanta U.K., Singh T.S., Shoriki T., Sugiyama H., Itagaki T. 2015. Molecular phylogenetic analysis of Fasciola flukes from eastern India. Parasitology International 64: 334-338. https://doi.org/10.1016/j.parint.2015.04.004 [4] Ichikawa-Seki M., Tokashiki M., Opara M.N., Iroh G., Hayashi K., Kumar U.M., Itagaki T. 2017. Molecular characterization and phylogenetic analysis of Fasciola gigantica from Nigeria. Parasitology International 66: 893-897. doi:10.1016/j.parint.2016.10.010 [5] Rouhani S., Raeghi S., Mirahmadi H., Fasihi Harandi M., Haghighi A., Spotin A. 2017. Identification of Fasciola spp. in the east of Iran, based on the spermatogenesis and nuclear ribosomal DNA (ITS1) and mitochondrial (ND1) genes. Archives of Clinical Infectious Diseases 12:e57283. doi:10.5812/archcid.57283 [6] Hodgkinson J., Cwiklinski K., Beesley N., Paterson S., Williams D., Devaney E. 2013. Identification of putative markers of triclabendazole resistance by a genome-wide analysis of genetically recombinant Fasciola hepatica. Parasitology 140: 1523. doi:10.1017/S0031182013000528 [7] Bozorgomid A., Rouhani S., Harandi M.F., Ichikawa- Seki M., Raeghi S. 2020. Genetic diversity and distribution of Fasciola hepatica haplotypes in Iran: molecular and phylogenetic studies. Veterinary Parasitology: Regional Studies and Reports 19: 00359. [8] Rouhani S., Raeghi S., Spotin A. 2017. Spermatogenic and phylo-molecular characterizations of isolated Fasciola spp. from cattle, North West Iran. Pakistan Journal of Biological Sciences 20: 204-209.

Toxoplasma gondii and human immunodeficiency virus co-infection in western Iran: A cross sectional study

Objective:To determine Toxoplasma gondii (T. gondii) as a cause of morbidity and mortality in human immunodeficiency virus (HIV) infected patients by ELISA method and to investigate toxoplasmosis-associated risk factors.Methods:This cross sectional study was conducted with 385 patients with HIV/AIDS referred to the behavior disease consultation center in Kermanshah, Iran between May 2012 and June 2013. Anti-Toxoplasma IgG and IgM antibodies were measured by using the ELISA method. Also, the relationship between the infection and age, gender, education, occupation, antiretroviral status, CD4+ cell counts and some other factors of the patients were assessed. P<0.05 was considered statistically significant.Results:Among the HIV-positive patients, 40.8% (157/358) and 2.6% (10/358) patients were positive for anti-T. gondii IgG and IgM antibodies, respectively. The mean CD4+ count in the patients with HIV was 327 cells/μL. A significant correlation was observed between the toxoplasmosis infections with the age group (P<0.05). Nevertheless, no statistically significant difference was found between gender, living area, educational level, occupational, marital status, antiretroviral status, prophylaxis, CD4+ cell count and toxoplasmosis.Conclusions:This study revealed that the patients with HIV infection were at the high risk of developing toxoplasmosis disease especially those patients who do not receive antiretroviral therapy and prophylaxis. The development and use of the effectiveness-based guidelines for controlling and the prevention reactivation of the latent T. gondii infection is required.

Toxoplasma gondii and human immunodeficiency virus co-infection in western Iran: A cross sectional study

Objective: To determine Toxoplasma gondii (T. gondii) as a cause of morbidity and mortality in human immunodeficiency virus (HIV) infected patients by ELISA method and to investigate toxoplasmosis-associated risk factors. Methods: This cross sectional study was conducted with 385 patients with HIV/AIDS referred to the behavior disease consultation center in Kermanshah, Iran between May 2012 and June 2013. Anti-Toxoplasma IgG and IgM antibodies were measured by using the ELISA method. Also, the relationship between the infection and age, gender, education, occupation, antiretroviral status, CD4+ cell counts and some other factors of the patients were assessed. P<0.05 was considered statistically significant. Results: Among the HIV-positive patients, 40.8% (157/358) and 2.6% (10/358) patients were positive for anti-T. gondii IgG and IgM antibodies, respectively. The mean CD4+ count in the patients with HIV was 327 cells/µL. A significant correlation was observed between the toxoplasmosis infections with the age group (P<0.05). Nevertheless, no statistically significant difference was found between gender, living area, educational level, occupational, marital status, antiretroviral status, prophylaxis, CD4+ cell count and toxoplasmosis. Conclusions: This study revealed that the patients with HIV infection were at the high risk of developing toxoplasmosis disease especially those patients who do not receive antiretroviral therapy and prophylaxis. The development and use of the effectiveness-based guidelines for controlling and the prevention reactivation of the latent T. gondii infection is required.

Genetic Diversity and Phylogenetic Analysis of the Iranian Leishmania Parasites Based on HSP70 Gene PCR-RFLP and Sequence Analysis

Despite the broad distribution of leishmaniasis among Iranians and animals across the country, little is known about the genetic characteristics of the causative agents. Applying both HSP70 PCR-RFLP and sequence analyses, this study aimed to evaluate the genetic diversity and phylogenetic relationships among Leishmania spp. isolated from Iranian endemic foci and available reference strains. A total of 36 Leishmania isolates from almost all districts across the country were genetically analyzed for the HSP70 gene using both PCR-RFLP and sequence analysis. The original HSP70 gene sequences were aligned along with homologous Leishmania sequences retrieved from NCBI, and subjected to the phylogenetic analysis. Basic parameters of genetic diversity were also estimated. The HSP70 PCR-RFLP presented 3 different electrophoretic patterns, with no further intraspecific variation, corresponding to 3 Leishmania species available in the country, L. tropica, L. major, and L. infantum. Phylogenetic analyses presented 5 major clades, corresponding to 5 species complexes. Iranian lineages, including L. major, L. tropica, and L. infantum, were distributed among 3 complexes L. major, L. tropica, and L. donovani. However, within the L. major and L. donovani species complexes, the HSP70 phylogeny was not able to distinguish clearly between the L. major and L. turanica isolates, and between the L. infantum, L. donovani, and L. chagasi isolates, respectively. Our results indicated that both HSP70 PCR-RFLP and sequence analyses are medically applicable tools for identification of Leishmania species in Iranian patients. However, the reduced genetic diversity of the target gene makes it inevitable that its phylogeny only resolves the major groups, namely, the species complexes.