Genetic Diversity and Phylogenetic Study of Leishmania Species in Iran by Multilocus Sequence Typing.

Background: Leishmaniasis is an important public health parasitic infection, which is endemic in many parts of the world, including Iran. We aimed to investigate genetic diversity and phylogenetic relationship among different Leishmania isolates using multi-locus sequence typing (MLST). Methods: Totally, 41 isolates collected either from patients referred to Leishmaniasis Diagnostics and Treatment Center at Tehran University of Medical Sciences, Tehran, Iran or from animals during 2019-2021, were subjected to the study. They included L. major and L. tropica from human, L. infantum from canine, and L. turanica from rodents from different endemic foci of Iran analyzed using MLST including gp63, g6pdh, lack, nagt, and hsp70 genes. Results: A total of 5010 bps was analyzed from each isolate. The three targets, nagt, lack, and g6pdh, generated better topology comparing to the other genes. In the 44 isolates, 22 haplotypes (STs) were identified. Leishmania tropica contained the highest number of haplotypes (n=12) comparing to L. major (n=8), L. infantum (n=1) and L. turanica (n=1). All five genomic loci caused separation of Iranian Leishmania species at the species level, indicating conservation of these genes in the Leishmania parasite. Conclusion: The highest number of haplotypes belonged to L. tropica, indicating that the genetic diversity of this species is higher than that of L. major. It was further confirmed that the MLST is a suitable method to examine genetic variation of Leishmania parasites with respect to evolutionary and epidemiological studies.

Optimization of the heterologous expression and purification of Plasmodium falciparum generative cell specific 1 in Escherichia coli.

Generative cell specific 1 (GCS1) or Hapless2 (Hap2) is a main transmission-blocking vaccine (TBV) candidate against malaria. Experience has shown that this protein is difficult to express in heterologous hosts. In a study, Plasmodium falciparum GCS1 (PfGCS1) could be expressed in fusion with Glutathione S Transferase (GST). Since the large fusions could influence the immunogenicity of the recombinant antigens, in the current study, we tried to express PfGCS1 protein without large fusion tags with an appropriate yield and purity in E. coli. To this end, pfgcs1 gene was codon-optimized and cloned in pET23a plasmid. The expression was evaluated in different E. coli hosts [E. coli BL21(DE3), E. coli BL21(DE3) pLysS, E. coli Rosetta(DE3), and E. coli Rosettagami(DE3)] and media cultures. In addition, the effect of post-induction times, inducer concentration, temperature, and supplementation of glucose and ethanol to culture media were evaluated. The obtained results revealed that rPfGCS1 protein was expressed in all examined E. coli hosts and media cultures with different yields, with the best yield in E. coli BL21(DE3), and E. coli Rosetta(DE3) hosts in TB medium, 16 h post-induction. The expression of rPfGCS1 was confirmed by western blotting using anti-His antibodies. Expression in low temperature at 20 °C and addition of glucose and ethanol to TB media could improve the expression of rPfGCS1. We could express and purify rPfGCS1 without a large fusion protein with an appropriate yield and purity in E. coli Rosetta(DE3). We will evaluate this antigen as TBV candidate against P. falciparum transmission in the future.

Sequence analysis, intra-genotyping variation, and phylogenetic study of nad1 gene in Echinococcus granulosus sensu lato genotypes from intermediate hosts in southwestern Iran.

Cystic echinococcosis (CE) is caused by the larval stage of Echinococcus granulosus sensu lato (s. l.). The disease is cosmopolitan, and Iran is a highly endemic area for CE. This parasite exhibits high genetic diversity, which can be related to its life cycle, transmission, and pathogenesis. This study was aimed at determining the phylogenetic relationship and intra-genotyping variation of E. granulosus s.l. in a vast area in the southwest of Iran (SWI). Eighty hydatid cyst isolates of intermediate hosts (i.e., cattle, sheep, goat, buffalo, camel, and human) were collected. The sequence analysis of the nad1 gene exhibited the three genotypes of G1 (n = 70, 87.5%), G3 (n = 8, 10%), and G6/G7 (n = 2, 2.5%). Also, 16, 2, and 1 unique haplotypes were identified for the G1, G3, and G6/G7 genotypes, respectively. According to the phylogenetic tree topology, the nad1 gene similarities were found for some G1 isolates in some vast areas, and the G1 genotype showed a heterogeneous population worldwide. The only SWI G6/G7 haplotype was at a distant position in E. canadensis clade, indicating the notable difference of this haplotype from other isolates from Iran and other countries. The presence of the G6/G7 genotype in the SWI may be due to the transmission of the genotype from other regions or the role of camel/wild boar or other possible hosts in the expansion of this genotype in SWI. The results of the present study can be used in CE control programs, molecular epidemiology, and phylogenetic studies in Iran and other countries for future goals.

Taxonomy, Population Structure and Genetic Diversity of Iranian Leishmania Strains of Cutaneous and Visceral Leishmaniasis.

BACKGROUND: Despite the broad distribution of leishmaniasis in Iran, there is a little genetic information about the causative agents and epidemiological status of the disease. Genetic diversity of the parasite is suggested to be one of the factors, which influences the clinical manifestations of the disease. In this study, we investigated the genetic variations, population structure, and evolutionary history of Leishmania species from endemic foci of Iran. METHODS: Fifty-two isolates from humans, canines, and rodents from different endemic foci of Iran were used to sequence the N-acetyl glucosamine-1-phosphate transferase (Nagt) gene. Phylogenetic and structure analyses were performed to investigate inter- and intra-species diversity of the Leishmania isolates. RESULTS: In total, 10 haplotypes including L. major (n = 6), L. tropica (n = 2), L. infantum (n = 1) and L. turanica (n = 1) were identified across 52 isolates. Haplotype diversity (Hd) ranged from zero for L. infantum and L. turanica to 0.78 ± 0.136 for L. major. This study identified population structure of Leishmania isolates from different geographical regions of Iran. The results of the phylogenetic tree showed 4 distinct clades for each species of Leishmania. In addition, the highest intraspecies diversity was observed among L. major isolates. No correlation was observed between species and geographic distribution of haplotypes. CONCLUSIONS: Leishmania isolates were identified at the species level using the Nagt gene, low variation within species indicates conservation of this gene in Leishmania. The results provide knowledge into the evolutionary history of Iranian Leishmania isolates.

Infection rate and genetic diversity of Giardia duodenalis assemblage C in Iranian stray dogs, targeting the glutamate dehydrogenase gene.

BACKGROUND AND AIM: Giardia duodenalis is one of the most common enteric protozoan parasites in vertebrates, such as humans, domestic and wild animals, causing giardiasis. To the best of our knowledge, little is known about the genetic diversity of G. duodenalis assemblages. This study aimed to identify genetic diversity of G. duodenalis assemblages in Iranian stray dogs. MATERIALS AND METHODS: A total of 450 fecal samples were collected from 2015 to 2016 from stray dogs of Northwest Iran. All specimens were observed microscopically following concentration and flotation techniques. Subsequently, DNA samples were extracted, amplified, and sequenced targeting the glutamate dehydrogenase gene. RESULTS: The overall prevalence of G. duodenalis in infected dogs was estimated at 1.6%, based on microscopic and molecular diagnoses. Sequencing and phylogenetic analyses indicated a high level of genetic diversity of assemblage C (haplotype diversity; 0.802). CONCLUSION: The pairwise sequence distances between the identified isolates of assemblage C showed an intradiversity of 0.3%-1.3% and identity of 98.7%-100%. Current findings indicate that a significant genetic diversity of G. duodenalis assemblage C haplotypes is unequivocally circulates among stray dogs in Northwest Iran.

Prevalence and molecular analysis of Sarcocystis infections in cattle in Northwest Iran and the first global report of S. gigantea in cattle.

Cattle are intermediate host for several species of Sarcocystis, including S. cruzi, S. hirsuta, and S. hominis with high prevalence worldwide. The present study aimed to determine the prevalence of Sarcocystis infection, species identification, and phylogenetic analysis of the parasite in cattle in Northwest Iran. The samples of diaphragm and esophagus from 290 cattle were collected from slaughterhouses in Northwest Iran and subjected to macroscopic, microscopic, and histopathology examinations, PCR-RFLP, sequencing and phylogenetic analyses. Tissue cysts of Sarcocystis spp. were detected in 92% of cattle by digestion and microscopic tests. Based on the PCR-RFLP and specific PCR, 87.9% and 1.03% of isolates were identified as S. cruzi, and S. hominis, respectively. Macrocyst was seen in a single sample that was identified as S. gigantea. The haplotype network exhibited the extension of the various haplotypes of S. cruzi between neighboring provinces in Northwest Iran. Heterogeneity analysis of S. cruzi 18S-rRNA sequences indicated genetic diversity among S. cruzi isolates (Haplotype diversity: 0.733-0.854) consisting 16 haplotypes; however, the nucleotide differences showed low diversity (0.01481 to 0.03351). Pair wise sequence distance matrix amongst S. cruzi sequences indicated an intra-species divergence of 0%-7.8% and identity of 92.6%-100%. Sarcocystis infection is highly prevalent in cattle in Northwest Iran, with the predominance of S. cruzi, and genetic variants of this species are unequivocally distributing in Northwest provinces. First global detection of S. gigantea in cattle reflects new insights of transmission dynamic and biology of this parasite in Iran.

Molecular survey of ITS1 spacer and Rickettsia infection in human flea, Pulex irritans.

The human flea is an important ectoparasite causing serious public health problems worldwide. Planning and monitoring the control programs against this vector require the knowledge of population structure and vector competence. This study was carried out to identify molecular structure of internal transcribed spacer 1 (ITS1) of ribosomal gene and its capability in the survey of Pulex irritans populations as well as to investigate Rickettsia infection in these populations. Flea samples were collected via human baits from animal farms in two districts of Zanjan Province, northwest of Iran. The ITS1 region and the partial Rickettsia gltA gene were amplified from the samples of human flea, and 30 amplicons were sequenced. The 1136 collected fleas consisted of 1079 (94.98%) P. irritans, 36 (3.17%) Ctenocephalides canis, and 21 (1.85%) Ctenocephalides felis. Molecular investigation of 182 human fleas detected the infection of Rickettsia sp. in 4.9%. The ITS1 region covered 957 bp and contained three tandem units of 98-99 bp, starting at positions 145, 245, and 331. Multiple alignments of ITS1 sequences showed single-nucleotide polymorphism at position 798, which caused the substitution of cytosine for adenine in the novel haplotype. High frequency of P. irritans and its Rickettsia infection requires the application of vector control measures, and full characterization of Rickettsia sp. and its potential to cause disease in humans. Regarding the consistency of ITS1 region and its ability to differentiate insect communities, further investigations are recommended to identify the role of selective factors in maintenance of this spacer.

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.

PCR-based Diagnosis of Toxoplasma Parasite in Ocular Infections Having Clinical Indications of Toxoplasmosis.

BACKGROUND: The diagnosis of ocular toxoplasmosis is mainly based on clinical features. However, ocular fluid testing by PCR may be very helpful for approval or rejection of this etiology. In this study, we utilized a nested-PCR technique, targeting the B1 partial sequence to analyze the aqueous and vitreous samples for evaluating the presence of the Toxoplasma DNA. METHODS: Fifty aqueous or vitreous humor samples were obtained from patients with clinical features of ocular toxoplasmosis admitted to ophthalmology hospitals and clinics in Iran, within 2014. The samples were subsequently subjected to DNA extraction and purification. For nested amplification of the Toxoplasma B1 gene, two primer pairs were used. The outer and inner primers are expected to produce a 193 bp and a 96 bp fragments, respectively. RESULTS: The first-round PCR resulted in the detection of T. gondii in 58% of samples by amplification of the expected 193bp DNA fragment. The nested-PCR using the inner primers, detected 15 additional samples from those with negative amplicons in the first round PCR (overall positivity of 88%). In addition, vitreous samples showed relatively more positive cases than aqueous humor in detection of the infection. CONCLUSION: The nested-PCR protocol using the B1 gene, with the high detection power, could be a useful complimentary method to clinical diagnose of ocular toxoplasmosis.

Molecular detection of Taenia spp. in dogs' feces in Zanjan Province, Northwest of Iran.

AIM: Echinococcus and Taenia spp. are important but neglected zoonotic helminths of dogs. Dogs as the most relevant definitive hosts harbor several species of Taenia and Echinococcus simultaneously in their gastrointestinal lumen which are morphologically indistinguishable. In this study, we used a multiplex polymerase chain reaction (PCR) method to identify Taeniid infections which seem to be highly distributed in the study region. MATERIALS AND METHODS: A total of 450 dog fecal samples were collected from eight different areas of Zanjan province, northwest of Iran, and examined using a flotation method followed by multiplex PCR for detection and identification of parasites' eggs. RESULTS: Gastrointestinal parasites were found in 86 out of 450 fecal samples (19.1%) by microscopic examination. Taeniid eggs were observed in 5.6% of samples, containing 0.45%, 3.8%, and 1.3% Echinococcus granulosus, Taenia spp., and mix infection of both E. granulosus and Taenia spp., respectively. Echinococcus multilocularis was absent in the samples. CONCLUSION: A relatively low rate of E. granulosus (1.8%) was observed in this study. However, risks of this parasite should not be overlooked, and control programs need to be extended for this species and other Taeniid spp. In particular, dogs are recommended to be dewormed more frequently.

Dogs' Gastrointestinal Parasites and their Association with Public Health in Iran.

INTRODUCTION: Dogs harbour zoonotic parasites that cause serious infections in humans, such as visceral larva migrans, ocular larva migrans, cystic echinococcosis, and alveolar echinococcosis. Studies on dogs' gastrointestinal parasites in different geographical locations are required to increase knowledge of the risk of canine zoonoses in human populations. MATERIAL AND METHODS: The presence of parasites was examined in 450 faecal samples collected from eight zones of Zanjan province, northwest Iran from June to November 2015. The samples were examined using the sedimentation concentration method and modified Ziehl-Neelsen staining. RESULTS: Gastrointestinal parasites were found in 86 (19.1%) faecal samples. Sarcocystis spp. (7.3%), Taenia/Echinococcus spp. (5.6%), Toxocara spp. (1.8%), and Cystoisospora spp. (1.6%) were the most common parasites observed. The other detected parasites consisted of Dicrocoelium dendriticum (0.7%), Eimeria spp. (0.7%), Cryptosporidium spp. (0.4%), Physaloptera spp. (0.4%), Giardia spp. (1.3%), and Spirocerca lupi (1.3%). The lowest parasite infection rates belonged to Trichuris vulpis and Acanthocephalans (0.2% each). CONCLUSION: This study provides current information on the infection rates in dog populations in Zanjan Province. Furthermore, the study shows a high prevalence of gastrointestinal parasitic infections, including zoonotic ones and particularly Taenia/Echinococcus spp., potentially transmissible to humans and thus relevant to public health.

Outbreak of ovine congenital toxoplasmosis in Iran, confirmed by different diagnostic methods.

Toxoplasma gondii is a zoonotic obligatory intracellular protozoan parasite with the capability to infect all warm-blooded animals. One of the great concerns is that it can lead to ovine abortion in sheep growing industry. Different diagnostic methods such as serology, pathology, immunohistochemistry, bioassay and molecular detection have been used in order to detect ovine abortion associated with T. gondii. In this case, an outbreak of congenital toxoplasmosis based on serological, macroscopic, pathological detection and isolation of T. gondii by bioassay is described and emphasized on the importance of this route of transmission that caused lamb losses and an increase in possible sources of infection for human and environment.

Genetic characterization of livestock and human hydatid cyst isolates from northwest Iran, using the mitochondrial cox1 gene sequence.

Cystic echinococcosis (CE), caused by larval stages of the tapeworm Echinococcus granulosus, is one of the most important zoonoses distributed worldwide. Genotype analysis of the parasite isolates from various hosts is required to better understand the host specificity and transmission routes. The aim of this study was to identify the genotypes of E. granulosus isolated from humans and domestic animals from northwest of Iran (Zanjan Province) using the mitochondrial cox1 gene sequence. A total of 86 hydatid cysts including 49 sheep and 28 cattle isolates from the slaughterhouse and nine human isolates from surgical wards of local hospitals were collected. The isolates were subjected to DNA extraction, PCR amplification, and sequence. Eighty-two (95.35 %) isolates, including 47 sheep, 26 cattle, and all nine human isolates, were determined as G1 genotype, and the remaining four (4.65 %), including two sheep and two cattle isolates, were identified as G3 genotype. From the cox1 sequence data, 13 different haplotypes (10 G1s and three G3s) were detected and named as EGH1-EGH13 (GenBank accession numbers, KP859559-KP859571). EGH1 was the major variant among the haplotypes, and it was identified in 46 (53.49 %) isolates (31 sheep, 14 cattle, and one human). Alignment of the partial cox1 sequences showed 12 point mutations including seven (58.3 %) synonymous and five (41.7 %) non-synonymous substitutions. Based on the results, G1 was the major genotype of E. granulosus in northwest of Iran affecting sheep, cattle, and humans. In addition, a minor group of G3 genotype was found to be circulating in this region.

Detection of mixed-species infections of Plasmodium falciparum and Plasmodium vivax by nested PCR and rapid diagnostic tests in southeastern Iran.

Coexistence of two species of Plasmodium in a single host has disrupted the diagnosis and treatment of malaria. This study was designed to evaluate the ability of rapid diagnostic test (RDT) kits for the diagnosis of mixed-species malaria infections in southeastern Iran. A total of 100 malaria patients were included in the study out of 164 randomly suspected symptomatic malaria patients from May to November 2012. Nested polymerase chain reaction (PCR) was also used to judge the ability of microscopy versus RDT kits for detecting mixed species. The sensitivity of light microscopy for the detection of mixed-species malaria infections was 16.6% (95% confidence interval [CI] = 3-49.1). Nested PCR revealed 12 patients with mixed-species infection. The CareStart Pv/Pf Combo kit detected 58% of the mixed-species infections, which were determined by nested PCR (sensitivity = 58.3%; 95% CI = 28.5-83.5). For identifying P. falciparum, P. vivax, and mixed-species infections, the concordance rates (kappa statistics) of microscopy and CareStart Pv/Pf Combo kit with nested PCR were 0.76 and 0.79, respectively (P = 0.001). This study underlines the effectiveness of RDT kits to improve the differentiation of mixed-species malaria infections in endemic areas where the prevalence of chloroquine resistance is high.

In vitro activity of Alkanna frigida extracts in comparison with glucantime against Leishmania major.

BACKGROUND AND OBJECTIVES: Current chemotherapies of cutaneous leihmaniasis have faced to some problems and limitations; Development of new leishmanicidal drugs from different sources like herbal plants, are crucially important. The objective of the present study was evaluation of in vitro activity of Alkanna frigida extracts in comparison with glucantime against Leishmania major. MATERIALS AND METHODS: L. major promastigotes were exposed to different concentrations of the A. frigida extracts, processed by ethyl acetate, ethanol, hexane and chloroform. The inhibitory effect, as the IC50, were calculated after 24, 48 and 72 hours by linear regression analysis values of the concentrations employed. RESULTS: The significant inhibition was observed after 24 and 48 hours with different concentrations of compounds (p < 0.05 in all tests). All extracts had potent activity against proliferation of the promastigotes, comparing to the untreated negative control. It could compete with the glucantime efficacy in some concentrations. Ethyl acetate and ethanol extractions showed potent IC50 value, 106 µg/ml and 86 µg/ml, respectively. Hexane and chloroform extractions had poor efficacy after 24 hours; however, the efficacy increased after 48 and 72 hours. CONCLUSION: The results indicated that the A. frigida has appropriate inhibitory effects on the growth of L. major promastigotes in vitro and can be of herbal targets for further investigation in vivo.

Evaluation of anti-toxoplasma IgG, IgM, and IgA in mothers with spontaneous abortion in Zanjan, Northwest Iran.

Toxoplasma gondii is one of the major agents of infectious abortions and due to its worldwide distribution can threat healthy pregnant women who had no previous exposure to this parasite. The present study was designed to investigate the contribution of T. gondii to spontaneous abortions in Zanjan, Northwest of Iran, using ELISA method. Blood Samples were collected from 264 mothers referred to the provincial hospitals of Zanjan due to spontaneous abortion. The sera were isolated and subjected to evaluate the anti-Toxoplasma IgG, IgM and IgA antibodies. The results showed IgG positive (IgG(+)) in 99 cases (37.5%). A total of 68 women (25.8%) showed seroconversion with IgM or IgA or both IgM and IgA. They included: IgM(+) in 21 (8.0%), IgA(+) in 23 (8.7%) and both IgM(+) and IgA(+) in 24 (9.1%) subjects. In 23 cases, positive titers of IgM and IgG were accompanied. In general, the analysis of anti-Toxoplasma antibody patterns, showed that about 17% of the spontaneous abortions were associated with serological patterns of acute infection. According to these findings, a considerable proportion of spontaneous abortions can be attributed to T. gondii in the study area.

Frequency of HLA-DRB1 alleles in rheumatoid arthritis patients in Zahedan, southeast Iran.

BACKGROUND AND OBJECTIVES: Analysis of the role of different alleles of human leukocyte antigen (HLA) in rheumatoid arthritis (RA) patients is necessary in many populations and geographical areas. The aim of the present study was to investigate the frequency of HLA-DRB1 alleles in RA patients, comparing with that in control group in southeast Iran. DESIGN AND SETTING: Case-control study of rheumatoid arthritis patients referred to rheumatology clinic at university hospital. PATIENTS AND METHODS: The frequency of HLA-DRB1 alleles was determined in 79 RA patients and 93 healthy subjects in Zahedan, southeast Iran. HLA-DRB1 allele types were identified by polymerase chain reaction with sequence-specific primer (PCR-SSP). RESULTS: The HLA-DRB1FNx0110 allele showed a significantly higher frequency in patients with RA (OR=2.698, 95% CI=1.087-6.699, P=.045), while the frequency of DRB1FNx0103 allele in these subjects was significantly lower than that in the control group (OR=0.447, 95% CI=0.2285-0.8729, P=.021). The frequencies of DRB1FNx0101, DRB1FNx0104, DRB1FNx0107, DRB1FNx0109, DRB1FNx0111, DRB1FNx0113, DRB1FNx0114, DRB1FNx0115, DRB1FNx0116 were not significantly different between RA subjects and the control group. CONCLUSION: The data suggest that the DRB1FNx0110 allele is a risk factor and DRB1FNx0103 is protective for RA in this population.