Molecular Identification and Phylogenetic Analysis of Leishmania major Isolated from Zoonotic Cutaneous Leishmaniasis Foci in Northeast Iran.

Background: Epidemiological studies, classification and genetic studies of Leishmania species are effective in treatment, control and prevention in endemic areas. We aimed to investigate the genetic diversity and phylogeny of Leishmania in Zoonotic foci located in northeastern Iran using nagt gene for the first time. Methods: DNA of 100 confirmed positive slides collected from the health centers of Sarkhes, Darghez, Fariman, Esfarayen, and Sabzevar were extracted during 2020-2021. The partial sequence of kDNA was amplified to identify the species. Twenty-five DNA samples were randomly subjected to amplify by nagt gene primes and were sequenced. The sequences were aligned with reference sequences in National Center for Biotechnology Information (NCBI). Then, the genetic similarities of the sequences were checked using Clustalx2.1 software and the phylogenetic tree was drawn by Mega 7 software. Results: All the positive samples were diagnosed as L. major. Approximately, half of the sequences of species were similar to two reference genes JX103550.1:404-712 L. major Esfahan and KX759012.1:568-807 L. Major Ilam (more than 90% similarity). According to the results of the phylogeny tree, the closest genotype to our study samples was JX103550.1:404-712 L. major Esfahan. Conclusion: The most causative agent CL in these areas was L. major. The genetic diversity of L. major was high such as other zoonotic foci in Iran. Due to the high similarity of the strains in the study areas with the strains of Isfahan and Ilam, similar control and prevention methods is suggested in these areas.

Validation of a mixture of rK26 and rK39 antigens from Iranian strain of Leishmania infantum to detect anti-Leishmania antibodies in human and reservoir hosts.

Mediterranean type of visceral leishmaniasis (VL) is a zoonotic parasitic infection. Some provinces of Iran are endemic for VL while other parts are considered as sporadic areas. This study aimed to assess a combination of recombinant K26 and rK39 antigens as well as crude antigen (CA), derived from an Iranian strain of L. infantum, compared to direct agglutination test (DAT) for the detection of VL in humans and domestic dogs as animal reservoir hosts of the disease. A combination of rK26 and rK39 antigens and also CA was evaluated using indirect ELISA on serum samples of 171 VL confirmed humans (n = 84) and domestic dogs (n = 87) as well as 176 healthy humans (n = 86) and domestic dogs (n = 90). Moreover, 36 serum samples of humans (n = 20) and canines (n = 16) with other potentially infectious diseases were collected and tested for finding cross- reactivity. The results of ELISA were compared to DAT, currently considered as gold standard for the serodiagnosis of VL. The sensitivity and specificity, positive predictive and negative predictive values were calculated compared to DAT. The positive sera had previously shown a positive DAT titer ≥ 1:800 for humans and ≥ 1:80 for dogs. Analysis was done by MedCalc and SPSS softwares. Using the combination of rK26 and rK39 in ELISA, a sensitivity of 95.2% and a specificity of 93.0% % were found in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 98.9% and specificity of 96.7%% were found at a 1:80 (cut-off) titer compared with DAT. A good degree of agreement was found between the combined rK39 and rK26-ELISA with DAT in human (0.882) and dog serum samples (0.955) by kappa analysis (p < 0.05). The ELISA using the CA test showed 75% sensitivity in human and 93.1% in dog serum samples as well as 53.5% specificity in human and 83.3% in dog,s sera, respectively. The combination of rK26 and rK39 recombinant antigen prepared from Iranian strain of Leishmania infantum showed high accuracy for the serodiagnosis of VL in human and domestic dogs. Further extended field trial with a larger sample size is recommended.

A systematic review and meta-analysis of Hymenolepis nana in human and rodent hosts in Iran: A remaining public health concern.

Hymenolepis nana, as a neglected zoonotic helminth, naturally occurs in both humans and rodents. Herein, a systematic review and meta-analysis was carried out to estimate pooled prevalence of H. nana infection among human and rodent hosts for the first time in Iran. PubMed, Web of Science, Scopus, and Embase databases (English articles) and SID and Magiran databases (Persian articles) were systematically searched for relevant studies published from inception till May 24, 2020. Chi2 and I2 index were used to assess the heterogeneity of the included studies. Publication bias was assessed using Egger's intercept and visual inspection of the funnel plot. Pooled prevalence was estimated using random-effects model with 95 % confidence interval (CI) and depicted as a forest plot. STATA software was used for analysis. The overall pooled prevalence estimate of H. nana in humans included 1.2 % (95 % CI = 1.0-1.4%). Subgroup analysis revealed 2.2 % (95 % CI = 1.8-2.6%) and 0.5 % (95 % CI = 0.3 to 0.8 %) of H. nana infection among school children and food handlers, respectively. In terms of rodent hosts, the overall pooled prevalence of H. nana infection included 13 % (95 % CI = 9.3-16.6%). Subgroup analysis revealed the highest rate of the infection was in Rattus spp. (19.6 %; 95 % CI = 10.6-28.6%), a genus with synanthropic behavior. It seems, despite the advancement in sanitation infrastructure and hygiene status, the H. nana infection compares to other helminthic diseases remains a challenging public health problem in Iran.

Study of Lophomonas blattarum Infection in Kidney Transplant Patients in Mashhad City, Iran.

BACKGROUND: Lophomonas blattarum is a flagellate protozoan which is known as an emerging parasite in the human respiratory system. Organ transplant recipients are considered as immunocompromised patients due to prescription of immunosuppressive drugs. This group of patients is susceptible to opportunistic infection as well as lophomoniasis. This study aims to investigate the prevalence and clinical manifestation of pulmonary infections caused by L. blattarum in kidney transplant recipients. METHODS: This is a case-control study including 50 kidney transplant recipients and 50 controls. The sputum samples were collected from 50 kidney transplant recipients with bronchopulmonary infection signs suspected to lophomoniasis admitted in Montaserieh and Imam Reza hospitals, Mashhad, Iran. 50 healthy individuals as the control group were matched for sex and age with case ones. The consent form, checklist, and required information were provided for each patient. All samples were microscopically examined for the flagellated protozoan, L. blattarum, using direct smear. RESULTS: Among 50 kidney transplant recipients suspected to lophomoniasis, L. blattarum was identified in sputum samples of 4 (8%) participants of the case group including one female and three males. None of the samples were positive among the control group. Symptoms in patients of this study were high fever (4 out of 4 patients), cough (3 out of 4 patients), and dyspnea (2 out of 4 patients). Three patients showed a positive response to metronidazole treatment. CONCLUSION: The results of this study suggest that L. blattarum should be considered as a pathogenic agent in kidney transplant recipients. It is necessary to examine sputum samples in posttransplant pneumonia patients, especially in those resistant to antibacterial therapy.

Generation of a CRISPR/Cas9-Based Vector Specific for Gene Manipulation in Leishmania major.

BACKGROUND: Gene manipulation strategies including gene knockout and editing are becoming more sophisticated in terms of mechanism of action, efficacy and ease of use. In classical molecular methods of gene knockout, homologous arms are designed for induction of crossing over event in double strand DNA. Recently, CRISPR/Cas9 system has been emerged as a precise and powerful tool for gene targeting. In this effort, we aimed to generate a CRISPR/Cas9-based vector specific for targeting genes in Leishmania parasites. METHODS: U6 and DHFR promoters and neomycin-resistance gene were amplified from genome of L. major (MHRO/IR/75/ER) and pEGFP-N1, respectively. U6 promoter was cloned in pX330 vector which is named as pX330-U6. DHFR promoter and neo resistance gene sequence fragments were fused using a combination of SOE (Splicing by overlap extension)-PCR and T/A cloning techniques. To generate pX-leish, fused fragments su-bcloned into the pX330-U6. Two sgRNAs were designed to target the gp63 gene and cloned in pX-leish. RESULTS: The pX-leish vector was designed for simultaneous expression of cas9 and G418 resistance proteins along with a self-cleaving 2A peptide for efficient separation of the two proteins. In this study pX-leish was designed with 3 features: 1) Compatible promoters with Leishmania parasites. 2) Insertion of antibiotic selection marker 3) Designing an all-in-one vector containing all components required for CRISPR/Cas9 system. CONCLUSION: This modified system would be valuable in genome manipulation studies in Leishmania for vaccine research in future.

Immunization against Leishmania major infection in BALB/c mice using a subunit-based DNA vaccine derived from TSA, LmSTI1, KMP11, and LACK predominant antigens.

OBJECTIVES: To design a multivalent DNA vaccine encoding the most immunogenic regions of the Leishmania major antigens including TSA (Thiol-specific antioxidant protein), LmSTI1 (Leishmania major stress-inducible protein1), LACK (Leishmania homologue of receptors for activated C Kinase), and KMP11 (kinetoplastid membrane protein-11) on BALB/c mice. MATERIALS AND METHODS: The chimeric construct was generated including the most immunogenic epitopes containing a combination of domains and oligopeptides of the aforementioned genes. The construct was cloned into pcDNA 3.1 plasmid and named "pleish-dom." Following intramuscular injection of mice, the capability of the vector pleish-dom alone and with pIL-12 (expressing murine IL-12) to raise protective cytokines and parasite burden was evaluated in the BALB/c mice as a susceptible animal model against L. major. RESULTS: The immunized mice with pleish-dom/pIL-12 showed the highest and the lowest levels of interferon-gamma (IFN-γ) and interleukin-10 (IL-10), as well as the lowest leishmanin skin test (LST) reactions, were found through 8 weeks post-infection. CONCLUSION: Although the obtained DNA vaccine from the immunogenic chimeric protein of L. major antigens was able to induce a high level of IFN-γ, it partially protected mice against L. major. However co-administration with pIL-12 led to shift immune response to Th1 phenotype, granuloma formation, and lowered parasite burden, and consequently distinct protection was found.

Prevalence of Intestinal Coccidial Infections among Different Groups of Immunocompromised Patients.

BACKGROUND: Cryptosporidium and Isospora are known as one of the main cause of diarrhea in both immunocompetent and immunocompromised subjects, all over the world. Incidence of enteropathogens such as Cryptosporidium spp. and Isospora belli considerably has increased, since immunodeficiency virus (HIV) rapidly disseminated. In addition, cancer patients are highly susceptible to opportunistic infections. This study aimed to estimate the prevalence of cryptosporidiosis and isosporiasis in immunocompromised patients in Tehran. METHODS: This study carried out on patients admitted to Imam Khomeini hospital during 2013-2014. Stool samples collected from 350 immunocompromised patients. Formol-ether concentration was performed for all stool samples. Zeil-Neelsen technique was applied to stain the prepared smears and finally, all slides were examined by light microscope. RESULTS: Out of 350 patients, 195 (55.7%) and 155 (44.3%) were male and female, respectively. Cryptosporidium oocysts were detected in 3 (0.9%) samples including one sample from HIV+/AIDS patients and 2 samples from organ transplant recipients. Isospora oocysts were detected in 4 (1.1%) samples consisting 2 HIV+/AIDS patients, one patients suffering from malignancy and one patients with other immunodeficiency diseases. CONCLUSION: Cryptosporidium sp, and I. belli are the most prevalent gastrointestinal parasitic protozoans that infect a broad range of individuals, particularly those patients who have a suppressed or deficient immunity system.