Application and evaluation of native antigen B from Echinococcus granulosus sensu stricto and E. canadensis alone or mixture for serodiagnosis of human G1-G3 and G6/G7 genotypes cystic echinococcosis sera, using ELISA and Western blotting.

Cystic echinococcosis (CE) is one of the most important helminthic diseases in the world with different genotypes distribution. The application of specific genotype antigens together with sera from patients with specific cyst genotypes have not been reported, so far. The present study aimed to apply and evaluate native AgB from Echinococcus granulosus sensu stricto (Eg) and Echinococcus canadensis (Ec) alone or mixture for serodiagnosis of human G1-G3 and G6/G7 genotypes cystic echinococcosis sera, using ELISA and Western blotting. A total of 47 human sera along with 47 human CE cysts were collected. CE genotypes were determined. Native AgB were prepared from E. granulosus s.s and E. canadensis genotypes. ELISA and Western blot were performed on human specific G1-G3 and G6/G7 genotypes sera. Species specific native AgB were used alone or mixed. The sensitivity of ELISA using alone and mixed 1Eg-1Ec, 1Eg-2Ec, and 2Eg-1Ec of native AgB from E. granulosus s.s and E. canadensis genotypes for human G1-G3 sera were 92.10, 89.47, 97.37, 100, and 100%, respectively; while using AgBs, alone and mixed for human G6/G7 sera were 100%. The sensitivity of Western blotting using native AgB of E. granulosus s.s and E. canadensis genotypes alone and mixed 2Eg-1Ec were 78.95% and 100% for human G1-G3 and G6/G7 genotypes sera, respectively. The mixture of AgB from Echinoccus granulosus sensu stricto and Echinococcus canadensis genotypes increased ELISA sensitivity for the diagnosis of human CE. Preparation and application of native AgB from specific and prevalent genotypes of CE in endemic regions is recommended.

Evaluation of the knockdown resistance locus (kdr) in Anopheles stephensi (Diptera: Culicidae) in southeastern Iran.

Background & objectives: The resistance to insecticide among Anopheles stephensi population due to insecticide selection pressure has been previously reported from Iran. The current study was performed to evaluate the susceptibility of different insecticide reagents against An. stephensi by bioassay and molecular methods in Saravan County, a malaria-endemic area in southeastern Iran. Methods: An. stephensi mosquitoes were collected from different larval habitats in Saravan City, southeastern Iran in 2022. At first, the susceptibility of collected samples for DDT, permethrin, and deltamethrin were evaluated by bioassay test. The collected mosquitoes were then evaluated for the presence of different kdr mutations. Results: Insecticide susceptibility tests were conducted on the field population of An. stephensi from Saravan, revealing its potential resistance to pyrethroids and DDT. Of the 150 An. stephensi samples, 4 % carried the kdr L1014F mutation as heterozygous and the rest of them were homozygous L1014 wild type. Interpretation & conclusion: The current study revealed the presence of L1014F mutation for the first time in Iran. So, further monitoring of kdr mutations in the VGSC gene and resistance phenotypes should be performed.

Serological and molecular survey of zoonotic visceral leishmaniasis in stray dogs (Canis familiaris) from an endemic focus in Meshkin-Shahr district in Ardabil province, Iran.

BACKGROUND & OBJECTIVES: Visceral leishmaniasis (VL),a protozoan disease caused by Leishmania infantum is a major public health problem and cause of death among infants aged under 1 year and the elderly in endemic foci of Iran. The aim of this study is to determine the status of L.infantum infection in stray dogs from Meshkin-Shahr, a typical endemic area of VL in Iran. METHODS: Sixty-eight randomly trapped stray dogs in Meshkin-Shahr area were tested for L. infantum infection using the direct agglutination test (DAT) from June to October 2016. The confirmation of seropositive samples was performed by Microscopic slides of spleen, culture and then PCR. The molecular methods performed by ITS1-PCR, RFLP-PCR and kDNA-PCR. The allof kDNA -PCR products were sequenced. RESULTS: Out of 68 examined stray dogs, 17 (25.0%) were positive for L. infantum by DAT (1:320 titers or higher). Parasite test showed that all of seropositive samples have amastigote forms in their spleens but only 3 out of them could be cultured. The kDNA-PCR confirmed all of seropositive samples but ITS1-PCR and RFLP-PCR only confirmed 3 out of 17 (17.6%) seropositive samples. The sequenced products showed 94% homology with L. infantum. INTERPRETATION & CONCLUSION: The results showed a high prevalence of L. infantum infection in dogs in an endemic area of CVL and it provided key information for designing control programs against canine and human leishmaniasis.

Prevalence of Dirofilaria immitis in mosquitoes (Diptera) - systematic review and meta-analysis.

Knowledge of the vectors of dirofilariasis in the world beside the treatment of infected dog is crucial to establish mosquito vector-based control programs. The current systematic review and meta-analysis were conducted on published studies, documenting the prevalence of Dirofilaria immitis infected/infective mosquitoes from field surveys and laboratory experiments under controlled conditions. Articles up through 2019 from Scopus, PubMed, Embase, Web of Science, Google Scholar were screened systematically. The overall prevalence of D. immitis infected/infective mosquitoes was estimated using a random effect model. Meta-regression was used to identify factors related to high dirofilariasis prevalence in the vectors. In these studies, the detection method was not identified as a heterogeneity and the overall prevalence in both subgroups had overlap (7.9-34.9 and 1.5-48.5). The overall prevalence of infective stage was 2.6 (95% CI: 0.97-4.77 per 1,000) and 84.7 per 1000 (95% CI: 20.5-183.8 per 1,000) for the field survey/laboratory experiment, respectively. The higher overall prevalence of D. immitis infected/infective mosquitoes were reported across studies in which take place in Eastern Mediterranean Region office (EMRO), longitude: 80 to 110, latitude: 20 to 40, annual rainfall: 250 to 1000, sea level: 26 to 100 and <1,000, humidity: 66 to 70, during 2000 to 2005 by dissection methods. Our review determined that mosquito species within the genus Anopheles and to a less extent Culex were the main vectors of dirofilariasis.

Amino acid mutation in Plasmodium vivax dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes in Hormozgan Province, southern Iran.

Molecular analysis of antifolate resistance-associated genes-dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) of Plasmodium vivax is important in predicting the emergence of drug resistance to sulphadoxine-pyrimethamine (SP). The present study aimed to determine the polymorphism of dhfr and dhps genes in P. vivax field isolates. Samples from 80 microscopically diagnosed vivax malaria cases were collected from endemic areas of malaria in Hormozgan Province of Iran, from June 2010 to November 2015. The two sets of codons at position 33, 57, 58, 117, 173 of dhfr and 382, 383, and 553 of dhps genes were analysed by direct sequencing of PCR products. The majority of the isolates (70%) harboured a wild-type allele for P. vivax dhfr (Pvdhfr) and P. vivax dhps (Pvdhps). Mutations were detected in three codons of Pvdhfr (P33L, S58R and S117N) and single codon in Pvdhps (A383G). Novel mutations that have not been identified previously at codon 459 (D459A) of Pvdhps were also observed. The high prevalence of point mutation as well as the rising triple mutation of Pvdhfr and Pvdhps genotypes necessitate change in programmes and guidelines to eliminate P. vivax in future.

Demodex folliculorum (Trombidiformes: Demodecidae) infestation in medical students with facial dermatoses and healthy groups.

Demodex folliculorum (Simon, 1842) has been associated with various dermatological conditions. This study aimed to assess the prevalence of Demodex infestation in medical students with facial dermatoses compared with healthy medical students serving as controls. A total of 250 participants were enrolled, including 150 individuals with facial dermatoses and 100 healthy controls. Sampling was performed based on the standardized skin surface biopsy method. Demographic characteristics, specifically gender and age, were not statistically different between the patient and control groups. Among the facial dermatosis patients, 25 out of 150 (16.6%) were found to have Demodex infestation, while only three out of the 100 healthy controls (3%) exhibited infestations. The only identified species was D. folliculorum. The rates of Demodex infestation were significantly higher in the patients compared to the control groups. These findings indicate a higher prevalence of Demodex infestation among medical students with facial dermatosis, particularly in those diagnosed with folliculitis, acne vulgaris, and inflammatory papule, when compared to healthy controls. A better understanding of the relationship between D. folliculorum infestation and these dermatological conditions may lead to improved diagnostic and treatment strategies in the future.

Comparison of Isoenzyme Pattern of Echinococcus granulosus sensu stricto (G1-G3) and E. canadensis (G6/G7) Protoscoleces

Background: Different genotypes of Echinococcus granulosus sensu lato (s.l.) infect humans and ungulate animals, causing cystic echinococcosis. Simultaneous isoenzyme, as well as molecular characterizations of this parasite, has not yet been investigated in Iran. The present study aimed to evaluate the isoenzyme pattern of the E. granulosus sensu stricto (s.s.) and E. canadensis genotypes in Iran. Methods: A total of 32 (8 humans and 24 animals) cystic echinococcosis cysts were isolated from Shiraz, Tehran, Ilam, and Birjand from May 2018 to December 2020. The DNAs were extracted and their genotypes were determined by molecular methods. Enzymes were extracted from the cysts and subjected to polyacrylamide gel electrophoresis. The activities of glucose-6-phosphate sehydrogenase (G6PD), malate dehydrogenase (MDH), malic enzyme (ME), nucleoside hydrolyse 1 (NH1), and isocitrate dehydrogenase (ICD) were examined in the cyst samples using isoenzyme method and compared it with the genotyping findings. Results: DNA sequence analysis of the samples showed that the specimens contained 75% E. granulosus s.s. (G1) and 25% E. canadensis (G6) genotypes. The isoenzyme pattern of ICD in both genotypes produced a six-band pattern with different relative factors. The G6PD also produced two bands with different relative migrations in both genotypes. The MDH and NH1 systems revealed a two-band pattern, while only one band was generated in the ME enzyme in the E. granulosus s.s. genotype. In the E. canadensis, the MDH and NH1 enzymes showed one band, and the ME enzyme represented a two-band pattern. Conclusion: Our findings suggest that E. granulosus s.s. and E. canadensis genotypes have entirely different isoenzyme patterns for NH1, G6PD, MDH, and ME.

Thermotolerant Acanthamoeba spp. isolated from therapeutic hot springs in Northwestern Iran.

This study was conducted to address the distribution of Acanthamoeba genotypes in therapeutic hot springs in Iran. Sixty water and sediment samples were collected from bicarbonate, sulphur, and sodium chloride thermal springs in the northwest. All hot springs examined are used mainly for health purposes in Iran. Acanthamoeba were identified by both morphology and PCR (polymerase chain reaction). Genotype identification was based on the sequencing of a highly variable and informative region of Diagnostic Fragment 3 (stem 29-1 of 18S rRNA gene) within Acanthamoeba-specific amplimer (ASA.S1). Twenty percent of hot springs were contaminated with thermotolerant Acanthamoeba belonging to the potentially pathogenic T4 and T3 genotypes. A high number (91.7%) of strains showed growth at 37 °C, and eight isolates showed growth at 42 °C. A single isolate (HSNW2) was detected in waters at 70 °C. The presence of thermotolerant Acanthamoeba highlights a risk factor for susceptible individuals, as Acanthamoeba-related keratitis continues to rise in Iran. Periodic surveillance of thermal waters as well as improved filtration and disinfection is recommended to prevent disease related to pathogenic Acanthamoeba. This is the first comprehensive molecular study of Acanthamoeba genotypes in hot springs in Iran and the first to report the occurrence of the T3 genotype (corresponding to Acanthamoeba griffini) in thermal water sources in this country.

Cellulitis Caused by Hirudo orientalis Bites That Lead to an Allergic reaction.

The allergic reaction due to leech bites is frequently reported due to complications of leech therapy and also unwanted leech infestation. Regularly, the urticarial papules are common, and itching lasts less than 24 h. In the case of leech biting, dermal infection could be caused by leech gut bacterial flora such as Aeromonas spp and histamine from leech saliva. In this case report, a 30-year-old diabetic woman, who works in the field of leech breeding, was bitten by Hirudo orientalis during breeding. Her clinical signs were inflammation, swelling, pain, and redness in the back of her left hand. A microbiological examination revealed that the isolated leech was infected with Aeromona hydrophila. The risk of death due to anaphylactic shock and sepsis is high in some cases of underlying diabetes and immunocompromised individuals. The study pointed out the hazards of leech bites and proposed preventative measures such as using gloves and boots for farm workers.

Use of Recombinant CP2 and CP23 Antigens of Cryptosporidium parvum for Serodiagnosis of Human Cryptosporidiosis

Background: Cryptosporidium parvum is an important coccidian parasite infecting many mammals, including human. This parasite can manifest as chronic severe diarrhea in immunocompromised individuals, especially those with AIDS. The present study reports the recombinant production of recombinant (r)P2 and rP23 antigens of C. parvum as antigens for detecting human cryptosporidiosis using indirect ELISA tests. Methods: The coding sequences of rP2 and rP23 proteins were codon-optimized, commercially synthesized and sub-cloned in the pET28a expression vector. The expressed proteins were purified by Ni-NTA column chromatography and confirmed by Western blotting. The efficacy of rP2/rP23 proteins for serodiagnosis was evaluated by positive (n = 20) and negative (n = 20) human sera, confirmed by the Ziehl-Neelsen staining as the gold standard test. Results: In ELISA test, the sera from C. parvum-infected patients reacted strongly to rP2/rP23. The sensitivity and specificity related to the diagnostic potential of rP2/rP23 in the ELISA assay were 100%. Conclusion: Our results showed that combination of rP23 and rP2 antigens in ELISA significantly increases the performance of C. parvum serodiagnosis in human cryptosporidiosis.

Identification of Knockdown Resistance Mutations in the Cimex hemipterus (Hemiptera: Cimicidae) in Iran.

The worldwide resurgence of tropical bed bug Cimex hemipterus beginning in the late 1990s has led to growing concern. Molecular data on pyrethroid resistance, which is essential for the control strategies, is unknown for C. hemipterus in Iran. The current study evaluated the deltamethrin resistance status of C. hemipterus by bioassay and molecular tests. Live bed bugs were collected from sleeping quarters (dormitories) in the city of Tehran and used for insecticide bioassay tests. For bioassay evaluation, mixed-sex pools of adult bugs were exposed to deltamethrin (0.025%)-treated paper. Polymerase chain reaction assay evaluated resistance-related mutations in the voltage-gated sodium channel gene (VGSC) gene of studied populations. On the basis of the bioassay test within the 48-h exposure to deltamethrin, C. hemipterus were determined to be resistant. Knockdown time ratios (KR50) in the studied populations of C. hemipterus was 5.5-fold compared with those of the C. lectularius Teh strain. DNA sequencing of the VGSC gene revealed the presence of mutations at M918I and L1014 in C. hemipterus. According to the bioassay and molecular results of current study, C. hemipterus showed a high degree of pyrethroid resistance. The application of multiple approaches including physical, biological, and chemical tests should be regarded in future bed bug control strategies.

Evaluation of Multiplex/Nested Polymerase Chain Reaction and Loop-Mediated Isothermal Amplification for Malaria Diagnosis in Southeastern Iran.

Malaria is one of the most serious health problems in many countries, including Iran. Accurate diagnosis is important regardless of the elimination status of a country. A cross-sectional study was performed on 105 people who were suspected to be positive for malaria infection in Sistan and Baluchistan, Iran. Blood smears (thin and thick films) were stained with 10% Giemsa. DNA was extracted from the prepared thin and thick films for molecular methods. Multiplex/nested polymerase chain reaction (mn-PCR), loop-mediated isothermal amplification (LAMP), and light microscopy (LM) were compared with nested PCR (nPCR) as a gold standard. Of 105 subjects, 52 (49.5%), 58 (55.2%), 58 (55.2%), and 63 (60%) were positive for malaria by LM, nPCR, mn-PCR, and LAMP, respectively. The sensitivity, specificity, and kappa were 92.1%, 100%, and 0.9 for LAMP and 100%, 100%, and 1 for mn-PCR, respectively. Eight cases of coinfection (Plasmodium vivax and Plasmodium falciparum) that were not detected by LM method were diagnosed by mn-PCR and LAMP. In the present study, the high sensitivity and specificity of LAMP and mn-PCR indicate that these two tests are good alternatives to nPCR for malaria diagnosis.

Morphological and Molecular Descriptions of Macracanthorhynchus ingens (Acanthocephala: Oligacanthorhynchidae) Collected from Hedgehogs in Iran.

Aim: Limited data exist on acanthocephalan infections of hedgehogs in the world. Our objective was to investigate the prevalence and distribution of Macracanthorhynchus ingens infection in hedgehogs between August 2021 and March 2022 (n = 30) in the east of Iran. Methods: At first, infection with M. ingens was diagnosed based on morphologic features of the adults such as body length, proboscis, and hooks. Spindle-shaped eggs (mean length, 99.1 microns; mean width, 60.1 microns) were obtained from the body cavity of gravid female specimens. Results: The molecular analysis based on 18S rDNA and COX 1 genes confirmed the morphological identification of isolated M. ingens. The prevalence of M. ingens in our sample was 13.3% with 1-10 worms per infected host. Conclusion: In this study, we identify M. ingens as zoonotic species in hedgehog carcasses for the first time that passed eggs and adult worms, indicating parasite maturation and reproduction. There are a few studies on acanthocephalans in Iran. Therefore, more comparative studies are needed to determine the status of these species.

Giant Brain Hydatid Cyst in an Adult: A New Case Report

Cystic echinococcosis is an important zoonotic disease that occurs in humans and mammals in general, which causes considerable economic loss and poses health concerns in different parts of the world. The patient involved in this case report was a 28-year old man living in Birjand city who had been suffering from intermittent headache, nausea and vomiting for the past two weeks. The other symptoms presented by the patient were dizziness, blurred vision, seizures and imbalance. The patient only complained of headache in the last two weeks and had no symptoms of visual or speech impairment. He had a history of consuming raw vegetables, but did not have canine contact. In brain computed tomography and magnetic resonance imaging, a large cyst was evident in his brain. The patient was admitted to Razi Hospital in Birjand and followed-up by surgical treatment of the hydatid cyst, with no complications observed.

Genotyping of the Echinococcus granulosus in Paraffin-Embedded Human Tissue Samples from Iran.

PURPOSE: Cystic Echinococcosis (CE) is a medically important disease that is caused by the metacestodes of Echinococcus granulosus. Human hydatid is considered an endemic disease in specific regions of Iran. The goal of the present study was to determine the genetic diversity of E. granulosus from the paraffin-embedded human tissue samples which were collected from the endemic regions of Iran. METHODS: Fifty-five formalin-fixed and paraffin-embedded hydatid cysts (FFPE) of humans, which had been removed surgically, were obtained from the South Khorasan and Sistan and Baluchistan provinces. These regions are related to the East and Southeast regions of Iran, respectively. The cox1 and nad1 genes from mitochondria were amplified from the extracted DNA and sequenced. The sequences were edited using the BioEdit software. Furthermore, phylogenetic and genetic diversity analyses were performed. RESULTS: Sequencing of the cox1 and nad1 genes from the 44 CE samples was done successfully. Genetic analysis revealed that 38 (86.3%) and 6 (13.6%) of the isolates were G1- and G6-genotypes, respectively. In general, eight and six haplotypes were identified by cox1 and nad1 genes analysis, respectively. For G1 strains, the haplotype diversity index was higher for the cox1 gene (0.6 ± 0.07) in comparison with the nad1 gene (0.4 ± 0.09). CONCLUSION: The findings of the present study showed that the sheep strain (G1) and the less important camel strain (G6) play the main roles in the transmission cycle of CE in the East and Southeast regions of Iran. Therefore, these results could be useful for managing the hydatid disease control programs in the studied and other similar areas.

The Efficacy of Hydroalcoholic Extracts of Prosopis farcta Against Leishmania major

Objective: Cutaneous leishmaniasis caused by Leishmania major (L. major) is an endemic disease in Iran. The current reference drugs, including Glucantime, possess high toxicity in addition to some side-effects. Therefore, there is a growing interest in exploring biomedical plants. The goal of the present study was to evaluate the anti-leishmanial activity and cytotoxicity of hydroalcoholic extracts from Prosopis farcta (P. farcta) over promastigote and amastigote forms. Methods: This study was performed at the Iran Birjand University of Medical Sciences, during the year 2019. In this study, the hydroalcoholic extracts of the stems, leaves (LE) and fruits (FE) of P. farcta were obtained. The anti-leishmanial activity was assessed against leptomonad promastigotes and intracellular amastigotes of L. major. The cytotoxicity of these extracts was determined in murine macrophages. Results: The FE and LE of P. farcta demonstrated a significant leishmanicidal effect against L. major promastigotes with an IC50 of 0.9 mg/mL and 1.1 mg/mL, respectively. The FE showed the most anti-leishmanial activity and presented with the highest index of selectivity (SI=14.6) as an anti-leishmanial product. Infected macrophages treated using the FE showed a reduction in parasite burden by 97.3%. Conclusion: The results of the present study demonstrated the leishmanicidal activity of P. farcta on both promastigotes and intracellular amastigotes. There is a need for performing comprehensive studies on relevant animal models and to access the effects of active components of P. farcta extract on the growth of L. major.

Thermotolerant Acanthamoeba spp. isolated from recreational water in Gorgan City, north of Iran.

Acanthamoeba as free-living parasites are scattered ubiquitously, throughout the world. This study was aimed to evaluate the presence of Acanthamoeba spp. genotypes in the recreational water sources in Gorgan County, the capital of Golestan Province using both morphological and molecular approaches. Thirty water samples were collected from different recreational waters in Gorgan, the capital of Golestan Province, northern Iran during 2015-2016. Samples were filtered and followed by culture in non-nutrient agar. Acanthamoeba were identified both by morphological and molecular analysis. The pathogenical potential of positive cloned samples were also determined using tolerance test. Twenty-six percent of recreational water were identified as Acanthamoeba spp. based on the morphological analysis and from these positive samples, five samples were successfully sequenced after molecular studies. Phylogenetic analysis showed the clustering of four samples in T4 genotype group and only one sample as T15 genotype. Thermotolerance test revealed that all cloned samples were highly positive. Since the attractiveness of recreational places for people is increasing, the potential risk of this water should be monitored routinely in each region. More studies are needed to better evaluate the risk of this ubiquitous parasite for the human.

Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods

Background: Dirofilaria immitis is a cosmopolitan zoonotic, vector-borne parasite of carnivorous animals causing dirofilariasis in human beings. Common commercial serodiagnostic tests for canine dirofilariasis usually lead to different results in their sensitivity and specificity. The present study reports development of recombinant DgK (rDgK) antigen of D. immitis for accurate immunodiagnosis of D. Immitis-infected dogs using indirect ELISA test. Methods: The rDgK coding sequence was successfully sequenced, codon optimized and cloned into pET-24a(+) expression vector and then expressed in Escherichia coli. The recombinant DgK was affinity purified using Ni²+-charged HiTrap chelating column, followed by testing in Western blotting and enzyme-linked immunosorbent assays (ELISA) with dog sera from a dirofilariasis endemic area. The performance of rDgK ELISA was evaluated using 60 sera collected from suspected dogs, while molecular technique was used as a reference test. Results: Sera from positive control D. immitis infection produced a strong IgG antibody response to rDgK both in ELISA and Western blotting tests. The sensitivity and specificity related to diagnostic potential of rDgK for ELISA were 92.5% and 87.5%, respectively. The results of rDgK ELISA showed a high agreement (0.764) with molecular identification. Conclusion: The findings revealed that the developed new rDgK antigen is sensitive and specific for immunodiagnosis of canine dirofilariasis using ELISA test.