Genetic diversity and seroprevalence of Toxoplasma gondii in COVID­19 patients; a first case-control study in Iran.

BACKGROUND: Toxoplasmosis is a serious or life-threatening disease in immunosuppressed patients and pregnant women. This study examined the likely association between Toxoplasma gondii infection and COVID-19 patients with moderate illness. METHODS: Seventy blood samples were collected from patients at the Health Reference Laboratory of Tabriz, Northwest Iran from April 2021 to September 2021. In addition, 70 healthy subjects of the same age (37 ± 15 years) and sex distribution were ethnically matched. Sera samples were examined for the detection of anti-Toxoplasma antibodies using ELISA. Nested-PCR targets were amplified based on the B1 and GRA6 genes. GRA6 amplicons were subjected to sequencing and phylogenetic analysis. RESULTS: The seroprevalence of toxoplasmosis based on IgG titer was 35.7% in the COVID­19 patients and 27.1% in the control group, representing not to be associated with the Toxoplasma seropositivity in COVID­19 patients (P = 0.18) compared to healthy subjects. Anti-T. gondii IgM was not found in any of the patients and healthy individuals. According to PCR amplification of the B1 and GRA6 genes, the frequency of T. gondii in COVID-19 patients was 14.2% (10/70). However, no T. gondii infection was detected in the healthy group. The CD4+T cell count was relatively lower in toxoplasmosis-infected patients (430-450 cells/mm3) than in control group (500-1500 cells/mm3). High genetic diversity (Hd: 0.710) of the type I strain of T. gondii was characterized in the patients. Present results showed that consumption of raw vegetables and close contact with stray cats can increase the transmission of T. gondii to COVID-19 patients (P < 0.01). CONCLUSIONS: The current study revealed that T. gondii type I infection is unequivocally circulating among the COVID-19 patients in Tabriz; However, no significant association was observed between the occurrence of Toxoplasma and the severity of COVID-19. To make more accurate health decisions, multicenter investigations with a larger sample size of different ethnic groups of the Iranian population are needed.

Sero-molecular detection, multi-locus genotyping, and clinical manifestations of ocular toxoplasmosis in patients in northwest Iran.

BACKGROUND: Our goal was to use molecular techniques to verify and characterise clinical diagnoses of ocular toxoplasmosis. Clinical cases were evaluated against IgM and IgG Toxoplasma antibodies, and IgG avidity was tested. B1 gene was assessed for molecular detection, and multi-locus genotyping were conducted to type Toxoplasma infections. METHODS: A cross-sectional study was conducted in 33 patients with suspected active ocular toxoplasmosis. Patients were examined by an ophthalmologist and clinical manifestations were recorded. Toxoplasma gondii IgG and IgM from serum samples were analysed by chemiluminescence immunoassay and ELISA. Acute vs chronic infection was evaluated by IgG avidity testing. Molecular diagnosis of T. gondii infection targeted the B1 gene, and the T. gondii genotype was determined by amplification of the GRA6, SAG2, SAG3, BTUB and APICO loci. The correlation of age, gender, occupation, education, contact with cats or soil, and the consumption of undercooked meat with the incidence of ocular toxoplasmosis was evaluated. RESULTS: Twenty-eight patients (84.8%) were seropositive, two (6%) were both IgG and IgM positive, while one (3%) showed IgG avidity <40%. Molecular testing confirmed toxoplasmosis in 27 patients (81.8%). Chorioretinal scarring (p=0.014) and posterior uveitis (p=0.004) was significantly associated with ocular toxoplasmosis patients. Multi-locus genotyping showed genotype I. Ocular toxoplasmosis showed no significant correlation with gender, age, behaviours, occupation or education. CONCLUSION: Clinical manifestations, serological and molecular detection of Toxoplasma were highly correlated in the diagnosis of ocular toxoplasmosis. Genotype I was predominant in ocular toxoplasmosis in northwest Iran. A larger comparative study should be conducted to provide a broader view of the molecular epidemiology of T. gondii genotypes and its role in toxoplasmosis.

Wound Myiasis in a Flamingo (Phoenicopterus ruber) Caused by Calliphora spp. Larvae from Northwest of Iran: A Case Report.

Myiasis is the infestation of animals or man tissues by parasitic dipterous fly larvae. Wound myiasis is the result of fly egg deposition on decaying flesh or pus discharging wounds. This case report describes a type of wound myiasis caused by Calliphora spp. in a Flamingo (Phoenicopterus ruber) from East Azerbaijan Province, Iran. A 3-yr-old female Flamingo was suffering in its left wing leading to an extensive discharging wound, which was heavily infested by maggots (fly larvae). The examination of external morphological characters of the second and third-instar larvae, posterior spiracles and internal cephalopharyngeal skeleton, led to the identification of the Calliphora spp. fly genus. Treatment consisted of removal of the larvae and surgical debridement, then spray of antibiotic and toxic drug. Following removal of larvae and treatment, the symptoms completely resolved within the last hour and remained asymptomatic several weeks later. This is the first report of wound myiasis in a Flamingo (Phoenicopterus ruber) by the facultative myiasis agent Calliphora spp. in Iran and the world.

Gene migration for re-emerging amebiasis in Iran's northwest-Iraq borders: a microevolutionary scale for reflecting epidemiological drift of Entamoeba histolytica metapopulations.

In the microevolutionary scales of Entamoeba isolates, the gene migration shows how Entamoeba spp. has epidemiologically drifted among border countries. Five hundred fecal samples were taken from patients suffering gastrointestinal disorders, abdominal pain, and diarrhea at Saggez, northwest Iran located within the border Iraq country. Following parasitological techniques, DNA samples were extracted and amplified by polymerase chain reaction (PCR) of 18S rRNA region to identify Entamoeba infections. To distinguish the Entamoeba spp., a multiplex PCR was conducted. Amplicons were sequenced to reconfirm their heterogeneity traits and phylogenetic analysis. Additionally, Entamoeba histolytica sequences of Iraq were retrieved from GenBank database. The suspected isolates were diagnosed as E. histolytica (2.2 %), Entamoeba moshkovskii (1 %), and Entamoeba dispar (0.4 %). Mixed Entamoeba infections did not detect among isolates. A parsimonious network of the sequence haplotypes displayed star-like features in the overall isolates containing E.h1, E.d2, and E.m3 as the most common haplotypes. According to analysis of molecular variance (AMOVA) test, high partial value of haplotype diversity (0.700 to 0.800) of E. histolytica was shown the total genetic variability within populations while nucleotide diversity was low among Iranian and Iraqi metapopulations. Neutrality indices of the 18S rRNA were shown negative values in E. histolytica populations which indicating significant deviations from neutrality. A pairwise fixation index (F-statistics [Fst]) as a degree of gene flow had a low value for all populations (0.001) while the number of migrants was 2.48. The statistically Fst value indicates that E. histolytica isolates are not genetically differentiated among shared isolates of Iran and Iraq. Occurrence of E.h1 between two regional populations indicates that there is dawn of Entamoeba flow due to transfer of alleles from one population to another population through host mobility and ecological alterations. To evaluate the hypothetical evolutionary scenario, further study is required to analyze Entamoeba spp. in the neighboring Middle East countries.

Gene flow for Echinococcus granulosus metapopulations determined by mitochondrial sequences: A reliable approach for reflecting epidemiological drift of parasite among neighboring countries.

In genetic diversity and population structure of Echinococcus granulosus, the gene flow can illustrate how the Echinococcus isolates have epidemiologically drifted among endemic neighboring countries. 51 isolates of hydatid cysts were collected from human, dog, cattle and sheep in northwest Iran, where placed co-border with Turkey. DNA samples were extracted, amplified and subjected to sequence analysis of NADH dehydrogenase subunit 1 (nad1) and cytochrome oxidase subunit 1 (cox1) genes. As well, sequences of Echinococcus at east to the southeast regions of Turkey were retrieved from GenBank database for the cox1 gene. The confirmed isolates were grouped as G1 (n = 74) and G3 (n = 6) genotypes. 31 unique haplotypes were identified inferred by the analyzed sequences of cox1 among two distinct populations. A parsimonious network of the sequence haplotypes displayed star-like features in the overall population containing TUR1, IR15 and IR22 as the most common haplotypes. According to AMOVA test, the high value of haplotype diversity (0.94758-0.98901) of E. granulosus was reflected the total genetic variability within populations while nucleotide diversity was low (0.00727-0.01046) in Iranian and Turkish metapopulations. Neutrality indices of the cox1 were shown negative values (-15.078 to -10.057) in Echinococcus populations which indicating a significant divergence from neutrality. A pairwise fixation index (Fst) as a degree of gene flow was partially high value for all populations (0.151). The statistically Fst value indicates that E. granulosus sensu stricto (G1-G3) are genetically moderate differentiated among Iranian and Turkish isolates. The occurrence of TUR1 and IR15 elucidate that there is possibly the dawn of domestication due to transfer of alleles between populations through the diffusion of stock raising or anthropogenic movements. To evaluate the hypothetical evolutionary scenario, further exploration is necessitated to analyze isolates from various host species in rest Middle East countries.

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.