Background: Visceral leishmaniasis (VL) is one of the most important neglected tropical diseases. The zoonotic form of VL is endemic in some areas of Iran. We aimed to determine the status of VL identified in humans and canines in different parts of Iran from 2013 to 2022.
Method: A national representative cross-sectional study was conducted in 10 provinces of Iran, including the national leishmaniasis reference lab. We employed the direct agglutination test (DAT) as a reliable serological method to detect anti-Leishmania infantum antibodies in humans and animal reservoir hosts. Additionally, a narrative literature review was conducted to identify relevant studies on VL seroprevalence in Iran from 2013 to 2023.
Results: The results of 21281 human and 5610 canine serum samples from 2013 to 2022 are reported. Altogether, 448 (2.1%, 95%CI: 2.0-2.3) human serum samples showed anti-L. infantum antibody levels of ≥1:3200. Of these samples, 13716 (64.5%) were collected actively, which showed a seroprevalence of 0.6% (95% CI: 0.5-0.8) and 7565 (35.5%) were collected passively, which showed a seroprevalence of 4.8% (95%CI: 4.3-5.3). Overall, 1035 (20.1%, 95%CI: 19.0-21.2) of 5160 domestic dogs (Canis familiaris) samples showed anti-L. infantum antibody levels of ≥1:320. Northwest (2.8%) and northeast (0.96%) regions had the highest human VL seroprevalence, while northwest (21.5%) and south (14.4%) regions had the highest canine VL seroprevalence.
Conclusion: Zoonotic VL, an endemic parasitic disease, is still present in several different distinct areas across Iran. While human VL cases have shown a declining trend over the last decade, the prevalence of canine VL remains significant.

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.
Method: 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.
Result: 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. 

Background: We aimed to design a B and T cell recombinant protein vaccine of Toxoplasma gondii with in silico approach. MIC13 plays an important role in spreading the parasite in the host body. GRA1 causes the persistence of the parasite in the parasitophorous vacuole. SAG1 plays a role in host-cell adhesion and cell invasion.
Methods: Amino acid positions 73-272 from MIC13, 71-190 from GRA1, and 101-300 from SAG1 were selected and joined with linker A(EAAAK)A. The structures, antigenicity, allergenicity, physicochemical properties, as well as codon optimization and mRNA structure of this recombinant protein called MGS1, were predicted using bioinformatics servers. The designed structure was synthesized and then cloned in pET28a (+) plasmid and transformed into Escherichia coli BL21.
Results: The number of amino acids in this antigen was 555, and its antigenicity was estimated to be 0.6340. SDS-PAGE and Western blotting confirmed gene expression and successful production of the protein with a molecular weight of 59.56kDa. This protein will be used in our future studies as an anti-Toxoplasma vaccine candidate in animal models
Conclusion: In silico methods are efficient for understanding information about proteins, selecting immunogenic epitopes, and finally producing recombinant proteins, as well as reducing the time and cost of vaccine design.

Background:  We aimed to characterize Cryptosporidium spp. in rats, cats, pigeons, and crows.
Methods: Fifty-five animal origin Cryptosporidium spp. genome were identified, genotyped and confirmed by nested PCR and of RFLP-PCR analysis as well as sequenced based on 18s rRNA and gp60 genes in Tehran (2012-2019). Finally, the phylogenetic analysis was performed by MEGA software (version 7).
Results: By the molecular method, Cryptosporidium spp. were detected in 24 (15.2%), 15 (15%), 2 (2%) and 13 (13%) cases of wild rats, cat, pigeon, and crow, respectively. Among the identified species by the RFLP pattern, most isolates were identified as C. parvum (24/157) 17.8% in rats, (15/100) 15% in cats, (13/100) 13%in crew and (2/100) 2% in pigeons; and the rest of the cases were C. muris and C. felis. The results of sequencing did not prove the existence of C. parvum, C. felis, C. muris, and rat genotype. Subtyping of C. parvum was indicated that the dominant subtype family belongs to the IId family and the subtype A20G1 was the most common subtype detected in all hosts while A19G1 was detected in one isolate of cat and pigeon.
Conclusion: Free-ranging animals are infected by species/subtype of Cryptosporidium, which can infect humans. This shows by itself the hygienic importance of the free-ranging animals in urban ecosystems. In the transmission of human cryptosporidiosis, the multi-host Cryptosporidium species such as C. parvum, C. felis, and C. muris can be transferred potentially from these animals to humans.

Background: This survey was designed to study the molecular epidemiology and risk factors of Entamoeba gingivalis and Trichomonas tenax in children with underlying malignancies and those on chemotherapy in Lorestan province, West of Iran.
Methods: The present cross-sectional descriptive study was performed on children who suffering from different types of malignancies or receiving treatment by chemotherapy referring to oncology section of hospitals of Lorestan Province, Iran during May 2021 to April 2022. The frequency of oral cavity protozoa was investigated using microscopic and conventional polymerase chain reaction (PCR).
Results: E. gingivalis and T. tenax parasites were found in 23 (25.5%) by microscopic method and 28 (31.1%) using PCR in children with malignancy. Among positive samples, 20 (71.4%) were infected with E. gingivalis; whereas 8 (28.6%) of the participants were positive for T. tenax. In the multivariate model, living in rural regions (OR= 3.437; 95% CI= 1.22-9.63; p=0.019) and using mouthwash (OR= 0.082; 95% CI= 0.018-0.37; p<0.001) were significantly related with the frequency of oral cavity parasites.
Conclusion: Our results showed the high frequency of oral cavity parasites in children who suffering malignancies or receiving treatment by chemotherapy in Lorestan province, Iran. The awareness of the main risk factors for oral cavity parasites particularly using mouthwash is necessary in improving public and oral health strategies in children with cancer. Consequently, oncologist and dental practitioners must be aware to identify and manage oral health concerns in in children who suffering from different types of malignancies to prevent the oral diseases and infections.

Background: We aimed to evaluate the accuracy of genotyping of Leishmania species by the spliced leader mini-exon gene.
Methods: Suspected leishmaniasis patients, referred to Masieh Daneshvary Hospital, Tehran, Iran were included from May 2017 to September 2021. The Leishmania species were genotyped by PCR-RFLP based on the SL mini-exon gene and the ITS1 region of SSU-rRNA gene and compared with the sequencing results. The expressed metabolites of metacyclic promastigotes were evaluated by Proton nuclear magnetic resonance (¹H-NMR).
Results: Out of 66 suspected cases, 36 (54.4%) were positive for Leishmania species based on the PCR assays. In 21 (31.8%) cases, promastigotes grew on culture tubes. Based on the RFLP of SL RNA profile, 13 (19.7%) L. tropica, 9 (13.6%) L. major, 3 (4.5%) L. infantum, and 8 (12.1%) C. fasciculata isolates, isolated from culture media, were identified; however, 3 (4.5%) cases were unidentifiable due to the low number of parasites. Seventeen metabolites were expressed by the metacyclic forms of L. major, L. tropica and C. fasciculata isolates. The top differential metabolites expressed more in C. fasciculata were FAD, p-Methoxybenzyl alcohol and S-b-G-5, 5-G-b-S (A = CH2) (P<0.005) whereas Veratryl glycerols and D-(+)-Mannose were significantly increased in L. major and Betulin, L-Tyrosine in L. tropica (P<0.01).
Conclusion: The invaluable techniques such as sequencing and ¹H-NMR confirmed the results of genotyping of Leishmania species based on the SL mini-exon gene.  SL mini exon gene can be used as a diagnostic tool to differentiate various Leishmania genotypes and detect contamination of culture media with C. fasciculata. 

Background: More than 250 million people are infected by malaria parasites annually while around one million children less than 5 years of age die every year due to malaria. We aimed to assess the seasonal trends and usefulness of capillary and venous blood for rapid diagnosis of malaria.
Methods: This cross-sectional study of 18 months duration was conducted at the National Institute of Child Health (NICH), Karachi. All patients reporting fever as chief complaint were recruited as study subjects. A semi-structured questionnaire was used to collect demographic information, presenting complaints, awareness of caregivers regarding malaria, preventive measures and history of malaria fever. Three ml Venous (2-3ml) as well as peripheral blood (3-4 drops) samples of all patients were collected for microscopy and rapid diagnostic tests (RDTs).
Results: Out of total 477 patients with fever Venous and Capillary Blood RDTs methods detected 33(6.9%) and 30(6.3%) as the malaria positive while Venous and Capillary Blood Microscopy detected 30(6.1%) and 32(6.7%) cases respectively. Plasmodium Vivax infection was the most prevalent (87.9%) and majority (39.39%) of the cases occurred in the quarter, July to September.
Conclusion: July to September is the peak season for malaria and P. Vivax (87.9%) is the predominant strain in Karachi. Venous and capillary blood are equally useful for malaria diagnosis however, convenience and less invasiveness may justify the preference of capillary blood over venous blood for early diagnosis of malaria.

Background: Leishmania is the parasitic protozoan responsible for leishmaniases, a disease that can cause a range of cutaneous, mucosal, and visceral infections. Two subgenera L. Viannia and L. Leishmania are known to infect humans in the tropics and subtropics of the Americas. The aim of the present study was to develop a new pair of primers for the two subgenera and test in clinical samples.
Methods: We designed two new pairs of primers for a PCR method from two conserved genes, cysteine proteinase B (cpb) and N-acetylglucosamine-6-phosfate deacetylase-like protein (nagA), as specific markers for those two respective subgenera. Primers were tested with 16 microscopical positive clinical samples from the Amazon region of Ecuador obtained in 2010-2020 period.
Results: The cpb presented a band of 172 bp and the nagA a band of 300 bp, thus clearly differentiating L. viannia from L. leishmania. Additionally, primers identified and differentiated the clinical samples in the two subgenera.
Conclusion: The new primers targeting different two genes and standardized in a PCR assay could identified and differentiated Leishmania parasites at subgenus level. This protocol could be used for Leishmania genus identification and diagnosis at the subgenus level and for determining the parasite's geographical distribution where different Leishmania subgenera are found in the same area.

Background: A lizard Leishmania has been isolated from a lizard (Agama agilis) in Iran. Its genome sequence has not been determined, so far.
Methods: The study was done at Shahid Beheshti University of Medical Sciences, Tehran, Iran in 2017-2023. Leishmania promastigotes were cultured in RPMI₁₆₄₀ culture medium and collected at logarithmic phase by centrigugation. Parasite RNA was extracted by the Qiagene standard kit and its quantity and quality was determined and sequenced by NGS method with Illumina PE machine at BGI Company (China).
Results: The number of 8316 mRNA, 83 tRNA, 63 rRNA, 83 ncRNA, 5 snRNA, 1039 snoRNA, 36 region, and 3 repeat regions, 8343 CDS, 9597 Exon and 9292 Genes were identified in promastigote of Iranian lizard Leishmania.
Conclusion: Genomic elements of Iranian lizards Leishmania (with unique characteristics) were determined and identified by NGS system.

Background: Paragonimus is a genus of parasitic flatworms known as lung flukes that cause the parasitic disease paragonimiasis in humans and other mammals. We aimed to use bibliometric analysis to identify the global characteristics and temporal trends of published literature about paragonimiasis.
Methods: Using the Web of Science database, we identified all original articles on paragonimiasis 1997 to 2022. After collecting the bibliographic and citation data, keywords, citation networks, and co-citations pertaining to paragonimiasis was carried out using the VOSviewer program.
Results: The study identified 563 paragonimiasis articles published in 250 journals. Publications in paragonimiasis research have been cited 6190 times and 2803 times without self-citations. The years with the most publications were 2013, 2016, and 2021. The minimal threshold for analysis was met by 19 of the 52 countries investigated. The study included 19 items, yielding 170 links between countries. The total strength of these links was discovered to be 104772. The journal with the most publications in this category was Parasitology Research (n=31). The most frequently used terms in paragonimiasis study were "paragonimiasis", "Paragonimus westermanii", and "lung-fluke."
Conclusion: The study concluded by providing an overview of the paragonimiasis research field, including current trends, development, and researcher collaboration. By addressing gaps in this bibliometric analysis and increasing collaboration, stakeholders could strengthen their strategies to effectively combat paragonimiasis and improve public health outcomes.

Background: The pathogen of angiostrongyliasis is the parasite Angiostrongylus cantonensis, and the transcriptome profiling of the male adult was unclear. We aimed to understand how the male adults adapt, so the expression profile of A. cantonensis adult males was analyzed.
Methods: In order to improve the understanding of the transcriptome of adult males, RNA from three groups of male adult A. cantonensis was extracted and reverse transcribed to construct cDNA libraries. After sequencing, annotation of unigenes and transcripts was performed by querying the NR (Non-Redundant Protein Sequence Database), GO (Gene Ontology) and COG/ KOG (Clusters of Orthologous Groups of proteins/euKaryotic Ortholog Groups) databases.
Results: For each group of adults, 43,260,894 raw reads and 43,200,341 clean reads were obtained. After successful assembly, 87,649 unigenes and 146,895 transcripts were obtained. Annotation of the unigenes and transcripts was identical and male adults expressed a series of genes encoding proteins specific to the male gender at the adult stage, such as proteins involved in energy metabolism, energy synthesis and transport. Expression of the ribosome pathway suggests a relationship with the physical activities during the adult male stage.
Conclusion: The transcriptome analysis is a good reference to understand further the expression profile of male adult A. cantonensis.

Background: Immune cells and their secreted cytokines are known as the first barrier against pathogens. Leishmania major as an intracellular protozoan produces anti-inflammatory cytokines that lead to proliferation and survival of the parasite in the macrophages. miRNAs are small non-coding RNA molecules that regulate mRNAs expression. We aimed to investigate the relationship between the expression of TGF-β and a bioinformatically candidate miRNA, in leishmaniasis as a model of TGF-β overexpression.
Methods: The miRNAs that target TGF-β -3´UTR were predicted and scored by bioinformatic tools. After cloning of TGF-β-3'UTR in psi-CHECK ^TM- 2 vector, targeting validation was confirmed using Luciferase assay. After miRNA mimic transfection, the expression of miR-27a, TGF-β, as well as Nitric Oxide concentration was evaluated.
Results: miR-27a received the highest score for targeting TGF-β in bioinformatic predictions. Luciferase assay confirmed that miR-27a is targeting TGF-β-3'UTR, since miR-27a transfection decreased the luciferase activity. After miRNA transfection, TGF-β expression and Nitric Oxide concentration were declined in L. major infected macrophages.
Conclusion: Bioinformatic prediction, luciferase assay, and miRNA transfection results showed that miR-27a targets TGF-β. Since miRNA and cytokine-base therapies are developing in infectious diseases, finding and validating miRNAs targeting regulatory cytokines can be a novel strategy for controlling and treating leishmaniasis.

Splenic infarction is a rare complication of both malaria and COVID-19. We report a splenic infarction case due to COVID-19 and malaria co-infection. A 35‑year‑old male with no known chronical disease tested positive for both COVID-19 and malaria in Turkey in 2022. Oral artemether and lumefantrine treatment was started. On the third day of the treatment, he complained about a severe left upper quadrant pain. A repeated abdominal CT showed splenomegaly and 8 cm diameter hypodense areas throughout the spleen consistent with splenic infarction. The patient was discharged with low molecular weight heparin. A rare complication that can be seen in both diseases developed a more rigorous recommendation for anticoagulant therapy is needed for co-infections of COVID-19 with diseases that may present similar thrombotic complications.

Bovine tropical theileriosis is one of the potentially fatal disease of dairy cattle, which is caused by hemoparasite Theleria annulata. About seven years old cross-bred cow was presented with complaint of pyrexia, inappetance, lacrimation and ocular swelling since last 5 days. The clinical examination showed elevated rectal temperature (39.4 ⁰C), mild enlarged pre-scapular lymph nodes, bilateral bulging of temporal fossa, protruded pale and icteric conjunctivae of the eyes with lacrimation and presence of ticks on body. The case was suspected for haemoprotozoan disease. Blood and serum sample were collected for hematological, blood smear examination and molecular examination (PCR), and biochemical analysis respectively. Microscopic examination of blood smear revealed intra-erythrocytic signet ring shaped periplasm of Theileria annulata. Hemato-biochemical examination revealed anemia, hypoproteinemia, hypoalbuminemia and jaundice. Further, PCR assay was done using T. annulata-specific primer pair, Cyto b1 gene targeting the amplicon of 312 bp showed specific band on Gel-electrophoresis. Therapeutic regimen was started with Buparvaquone @ 2.5 mg/kg body weight IM single dose followed by Oxytetracycline @ 10 mg/kg body weight IV in 500 ml of NS for 5 days and Prednisolone @ 0.25 mg/kg body weight IM for 3 days along with supportive therapy. The cattle well responded to the therapy and complete regression of ocular signs was observed within one week of treatment.

We diagnosed a case report of amoebic meningoencephalitis by Naegleria fowleri. This case represented the first recording in Iraq where it was not recording previously. This case was diagnosed after the death of an 18-year-old girl patient who lived in a rural area of Mosul in Iraq. Genetics detection of N. fowleri showed PCR product was 183bp for 18S rRNA gene. It was registered as the first recording of Iraqi isolate N. fowleri in GenBank with accession number OP380864.1. It is necessary to examine microscopically the cerebral spinal fluid (CSF) to observe the amoeba stages and exclude the bacterial causative. Rapid diagnosis may help in the treatment of amoebic meningoencephalitis. In addition, genetic identification can diagnose amoeba. Avoiding swimming or using freshwater contributes to prevent amoebic meningoencephalitis infection.

Gallbladder is a rare localization for hydatid disease. Complications are even rarer and precise diagnosis is quite difficult even with radiological assistance. We report a rare case of 41-yr-old male patient presenting with the rupture of a gallbladder hydatid cyst with multiple fistulae to intra and extrahepatic bile ducts and pancreas, at Kocaeli State Hospital, Turkey in 2021. The patient had abdominal pain and abdominal CT scan reported a bizarre “contrast enhanced cholangiography” sign - radiopaque contrast substance in gallbladder, intra and extrahepatic bile ducts and pancreatic ducts, with concomitant acute cholecystitis. Surgery was performed and intraoperatively gallbladder hydatid cyst with multiple fistulae was noted. Cholecystectomy with total cyst excision was performed. Endoscopic retrograde cholangio pancreatography (ERCP) was utilized to irrigate and eradicate the parasite in the fistulae tracts localized near pancreas and intra/extrahepatic ducts. Postoperative period was uneventful, antiparasitic treatment was started, and in the yearly follow-up patient had no recurrence. Multidisciplinary and minimal invasive management is crucial in such bizarre, complicated cases.