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Background: Strongyloides stercoralis is one of the soil-transmitted helminths (STH) in tropical and subtropical regions. The role of lipid profile has been investigated in the survival of larval stages of helminths, but there is limited information about the role of lipid profiles and strongyloidiasis. Hence, we aimed to investigate the seroprevalence of S. stercoralis infection in patients with hyperlipidemia is compared with the non-hyperlipidemia. Methods: In 2023, participants were selected from the laboratory of Porsina Hospital in Guilan Province, northern Iran and their lipid profiles including TG, CHOL, LDL, HDL, and VLDL were measured. They were divided into two groups of case and control and matched based on sex and age. S. stercoralis Ab (IgG) was measured by ELISA methods, using the NovaTec kit. Finally, statistical analysis was performed. Results: Each case and control group consisted of 105 participants, from 13 to 80 years old. 56.66% were female and 43.33% were male. The sero-prevalence of S. stercoralis was found 4.76% in the case group compared to 0.95% in the control group. We found an association between TG fall and VLDL with sero-prevalence of S. stercoralis in hyperlipidemia group (P= 0.034), but other lipid profiles did not show a significant association. A significant relationship was found between contact with dogs and sero-prevalence of S. stercoralis (P=0.001). Conclusion: The sero-prevalence of S. stercoralis in the case group was 5 times higher than the control group. A significant association between TG and VLDL fall with S. stercoralis Ab (IgG) was observed, but future studies with more sample sizes are suggested to investigate the anti-atherogenic effect of S. stercoralis. Also, a genetic assessment of S. stercoralis and the host (humane and dogs) is recommended to research zoonotic potential in epidemic areas.

Abstract Leishmaniasis is a disease of poverty that imposes a devastating medical, social, and economic burden on over 1 billion people nationwide. To date, no in-depth study to analyze the major global challenges and needs assessment has been carried out. This investigation aimed to explore a comprehensive narrative review of leishmaniasis’s main challenges and initially highlight obstacles that might impede the implementation of control measures. Also, we propose a specific list of priorities for needs assessment. The presence of socioeconomic factors, multiple clinical and epidemiological forms, various Leishmania species, the complexity of the life cycle, the absence of effective drugs and vaccines, and the lack of efficient vector and reservoir control make this organism unique and sophisticated in playing a tangled role to react tricky with its surrounding environments, despite extensive efforts and implementation of all-inclusive former control measures. These facts indicate that the previous strategic plans, financial support, and basic infrastructures connected to leishmaniasis surveillance are still insufficient. Strengthening the leishmaniasis framework in a context of accelerated programmatic action and intensification of cross-cutting activities along with other neglected tropical diseases (NTDs) is confidently expected to result in greater effectiveness, cost–benefit, and fruitful management. Sensitive diagnostics, effective therapeutics, and efficacious vaccines are vital to accelerating advancement toward elimination, and reducing morbidity/mortality and program costs. Collective actions devoted by all sectors and policy-makers can hopefully overcome technical and operational barriers to guarantee that effective and coordinated implementation plans are sustained to meet the road map for NTDs 2021- 2030 goals.

Background: Visceral leishmaniasis (VL) is a public health issue in endemic countries with poor sanitation facilities. In this study, the seroprevalence rate and associated risk factors of VL were investigated during September 2020 to February 2021 in pregnant women referred to Ostad Mottahari and Peymanieh hospitals in Jahrom county, Fars province, southern Iran. Material and methods: A total of 220 serum samples of pregnant women were assessed for the presence of Anti-Leishmania infantum antibodies by direct agglutination antigen (DAT). The associated risk factors were obtained using questionnaires. Results: The overall seroprevalence of VL in pregnant women was 12.72% (28/220). Considering the antibody titer, titer 1:1600 was detected in 23 samples, titer 1:3200 in 4 samples, and titer 1:6400 in one sample. All 5 women with titer >3200 had mild fever. As such, there was a statistically significant difference regarding the age (≥39 years old with p-value: 0.01). Conclusions: We recommend an appropriate health education program for pregnant women and serological screening of VL before pregnancy in endemic cities. Moreover, we believed a need for more epidemiological studies for better understand the status of VL in pregnant women.

Abstract Background Cutaneous Leishmaniasis (CL) is a parasitic disease with diverse outcomes. Clinical diversity is influenced by various factors such as Leishmania species and host genetic background. The role of Leishmania RNA virus (LRV), as an endosymbiont, is suggested to not only affect the pathogenesis of Leishmania, but also impact host immune responses. This study aimed to investigate the influence of LRV2 on the expression of a number of virulence factors (VFs) of Leishmania and pro-inflammatory biomarkers. Materials and methods Sample were obtained from CL patients from Golestan province. Leishmania species were identified by PCR (LIN 4, 17), and the presence of LRV2 was checked using the semi-nested PCR (RdRp gene). Human monocyte cell line (THP-1) was treated with three isolates of L. major with LRV2 and one isolate of L. major without LRV2. The treatments with four isolates were administered for the time points: zero, 12, 24, 36, and 48 h after co-infection. The expression levels of Leishmania VFs genes including GP63, HSP83, and MPI, as well as pro-inflammatory biomarkers genes including NLRP3, IL18, and IL1β, were measured using quantitative real-time PCR. Results The expression of GP63, HSP83, and MPI revealed up-regulation in LRV2 + isolates compared to LRV2- isolates. The expression of the pro-inflammatory biomarkers including NLRP3, IL1β, and IL18 genes in LRV2- were higher than LRV2 + isolates. Conclusion This finding suggests that LRV2 + may have a probable effect on the Leishmania VFs and pro-inflammatory biomarkers in the human macrophage model.

BackgroundAcanthamoeba keratitis (AK) is a corneal sight-threatening infection caused by the free-living amoebae of the genus Acanthamoeba. Early and appropriate treatment significantly impacts visual outcomes. Mucoadhesive polymers such as chitosan are a potential strategy to prolong the residence time and bioavailability of the encapsulated drugs in the cornea. Regarding the recent administration of miltefosine (MF) for treating resistant AK, in the present study, we synthesized miltefosine-loaded chitosan nanoparticles (MF-CS-NPs) and evaluated them against Acanthamoeba.Methodology/principal findingsChitosan nanoparticles (CNPs) were prepared using the ionic gelation method with negatively charged tripolyphosphate (TPP). The zeta-potential (ZP) and the particle size of MF-CS-NPs were 21.8±3.2 mV and 46.61±18.16 nm, respectively. The release profile of MF-CS-NPs indicated linearity with sustained drug release. The cytotoxicity of MF-CS-NPs on the Vero cell line was 2.67 and 1.64 times lower than free MF at 24 and 48 hours. This formulation exhibited no hemolytic activity in vitro and ocular irritation in rabbit eyes. The IC50 of MF-CS-NPs showed a significant reduction by 2.06 and 1.69-fold in trophozoites at 24 and 48 hours compared to free MF. Also, the MF-CS-NPs IC50 in the cysts form was slightly decreased by 1.26 and 1.21-fold at 24 and 48 hours compared to free MF.ConclusionsThe MF-CS-NPs were more effective against the trophozoites and cysts than free MF. The nano-chitosan formulation was more effective on trophozoites than the cysts form. MF-CS-NPs reduced toxicity and improved the amoebicidal effect of MF. Nano-chitosan could be an ideal carrier that decreases the cytotoxicity of miltefosine. Further analysis in animal settings is needed to evaluate this nano-formulation for clinical ocular drug delivery.

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Background: Phlebotomus papatasi (Diptera: Psychodidae) is the main vector of zoonotic cutaneous leishmaniasis (ZCL) in Iran. The nonstandard use of pesticides against pests, particularly in agriculture, indirectly has caused the de­velopment of resistance and, consequently, the threat of control measures in ZCL endemic areas. Up to 2023, several reports of resistance in Ph. papatasi have been declared in the old world. The purpose of this study was to measure the lethal time (LT50 and LT90) of Ph. papatasi sand flies in the ZCL endemic center of Esfahan to DDT and deltamethrin insecticides. Methods: Sand flies were collected in Borkhar and were tested using WHO adult mosquito test kit against DDT 4% and deltamethrin 0.0002%. The sand fly’s survival was recorded during exposure time in 225, 450|, 900, 1800, and 3600-seconds’ intervals for DDT and Deltamethrin and they were allowed to recover for 24 hours. Then LT50 and LT90 were analyzed using probit software. Phlebotomus papatasi were identified using morphological keys and other sand flies’ species were excluded from the analysis. Results: The insecticide against female Ph. papatasi revealed hundred percent mortality when exposed to DDT 4% and deltamethrin 0.0002%. The LT50 and LT90 were 19.32 and 22.74 minutes for DDT 4% and 39.92 and 51.33 minutes for deltamethrin 0.0002% respectively. Conclusion: Results of this study revealed that Ph. papatasi is still susceptible to DDT and deltamethrin. This data pro­vides valuable knowledge to implement effective control strategies against ZCL main vector and help to manage insec­ticide resistance in the region.

ABSTRACT This study aimed to prepare and assess atovaquone (ATQ)-loaded exosomes (EXO-ATQ) against Toxoplasma gondii to improve their therapeutic activities on acute and chronic phases of murine toxoplasmosis. Exosomes were isolated from a mouse macrophage cell line (J774A.1). The isolated exosomes were loaded with ATQ (EXO-ATQ), applying the co-incubation method. In vitro efficacy of EXO-ATQ against T. gondii infection was assessed in Vero cell culture. An in vivo experiment was carried out in BALB/c mice infected with RH (acute strain) and Tehran (chronic strain) of T. gondii, followed by treatment with EXO-ATQ. Their survival time and parasite load were compared with that in the suspension of ATQ (S-ATQ) as a positive control group. The in vitro study showed that exosomes improved the efficacy of ATQ, and EXO-ATQ significantly reduced intracellular proliferation of T. gondii tachyzoites compared to S-ATQ (P ≤ 0.05). In acute experimental toxoplasmosis, the mice treated with EXO-ATQ showed a significant reduction in tachyzoites count of the peritoneal cavity and a longer survival time (P ≤ 0.05). Furthermore, EXO-ATQ administration significantly decreased the mean number (97.3% cyst reduction) and the average size of the brain cysts of chronically infected mice with T. gondii, Tehran strain (P ≤ 0.05). Besides, the decrease in cyst numbers was confirmed by the down-regulation of BAG1 using a real-time PCR assay. Our results implied that exosomes have the potential to be used as an efficient drug delivery system, and the loading of ATQ into the exosomes ameliorates the effect of ATQ against T. gondii. This study suggests a new strategy for improving the effectiveness of ATQ against acute and chronic phases of T. gondii. IMPORTANCE This study is the first of its kind that suggests exosomes as a nano-carrier loaded with atovaquone (ATQ), which could be considered as a new strategy for improving the effectiveness of ATQ against acute and chronic phases of Toxoplasma gondii.

Background: Toxoplasma gondii infects nearly one-third of the world's population. Due to the significant side effects of current treatment options, identifying safe and effective therapies seems crucial. Nanoparticles (NPs) are new promising compounds in treating pathogenic organisms. Currently, no research has investigated the effects of zinc oxide NPs (ZnO-NPs) on Toxoplasma parasite. We aimed to investigate the therapeutic efficacy of ZnO-NPs against tachyzoite forms of T. gondii, RH strain in BALB/c mice. Methods: In an experiment with 35 female BALB/c mice infected with T. gondii tachyzoites, colloidal ZnO-NPs at concentrations of 10, 20, and 50 ppm, as well as a 50 ppm ZnO solution and a control group, were orally administered four hours after inoculation and continued daily until the mices’ death. Survival rates were calculated and tachyzoite counts were evaluated in the peritoneal fluids of infected mice. Results: The administration of ZnO-NPs resulted in the reduction of tachyzoite counts in infected mice compared to both the ZnO-treated and control group (P

Background: Enzymatic digestion of extra cellular matrix proteins by proteinases of Leishmania promastigotes is a complex process. Hence, studies on functional proteomics of these enzymes can help select these enzymes as possible vaccine candidates or selecting candidates for chemotherapy and immunotherapy. Several proteolytic enzymes are involved in virulence of Leishmania spp. These enzymes are mostly serine, cysteine and metalloproteases. We aimed to detect proteases in Leishmania promastigote exosomes. Methods: Serine, cysteine and metalloproteases were investigated in exosomes and lysate of L. major promastigote using gelatin zymography. The study was carried out in the Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran, in 2021. Results: Zymography findings of metalloproteinases showed transparent bands, including a 63-kDa glycoprotein (GP63). This glycoprotein is a major surface metalloproteinase. In addition, transparent bands belonged to serin proteases and cathepsin were demonstrated in gels associated to Leishmania promastigote lysate and exosomes. Conclusion: Several metalloproteases, serin proteases and cathepsins were shown in promastigote lysate and exosomes of L. major, which could purified and used as fractions for immunodiagnostic.

Background: Recent studies have shown an increasing number of patients with cutaneous leishmaniasis (CL) who do not respond to pentavalent antimonials as the first line of treatment for CL. Nanocarriers such as extracellular vesicles (EVs) are efficient vehicles that might be used as drug delivery systems for the treatment of diseases. Therefore, we aimed to isolate and characterize the EVs of Leishmania major, load them with Amphotericin B (AmB), and investigate the toxicity and efficacy of the prepared drug form. Methods: The EVs of L. major were isolated, characterized, and loaded with amphotericin B (AmB), and the EVs-Amphotericin B (EVs-AmB) form was synthesized. Relevant in vitro and in vivo methods were performed to evaluate the toxicity and efficacy of EVs-AmB compared to the control. Results: The anti-leishmanial activity of the EVs-AmB showed a higher percentage inhibition (PI%) (P = 0.023) compared to the AmB at different concentrations and time points. Obtained data showed a significant increase in the lesion size and parasite load in the lesion, PBS, and EVs mice groups in comparison with EVs-AmB, AmB, and Glucantime groups (P < 0.05), EVs-AmB had a significant decrease in lesion sizes in comparison with AmB (P < 0.05). Results showed that EVs-AmB decreased its toxicity to the kidneys and liver (P < 0.05). Conclusion: EVs-AmB improved the efficacy of AmB in mouse skin lesions and reduced hepatorenal toxicity. Furthermore, EVs could be a promising nanoplatform for the delivery of AmB in CL caused by L. major.

Background: We aimed to verify the susceptibility of Leishmania infantum, L. major and L. tropica, to commercial lectins in order to identify the three Leishmania species. Methods: The degree of agglutination was determined both macroscopically and microscopically and was scored negative (-) to positive (from 1+- 4+) based on their percentage of agglutination. Results: Jacalin and UEA-1 were capable of agglutination of L. infantum isolates in both logarithmic and stationary phases at a concentration of 1000 µg/ml (100%). L. tropica isolates showed agglutination with the lectin UEA-1 in both logarithmic and stationary phases (62.5% and 87.5%). L. major and L. tropica showed 75% agglutination with lectin Jacalin in both logarithmic and stationary phases. L. tropica isolates showed 25% agglutination with the lectin WGA in the logarithmic phase. L. infantum, L. major and L. tropica isolates showed 25, 12.5 and 37.5% agglutination in the stationary phase, however, did not show agglutination in logarithmic phases. L. major isolates showed 12.5% agglutination with the lectin PHA in the stationary phase, however, were incapable of agglutination with the L. tropica and L. infantum in both logarithmic and stationary phases. Conclusion: Despite the fact, that JCA and I-UEA lectins were not able to completely separate L. infantum, L. major and L. tropica. WGA lectin and PHA lectin can help in separating the species of Leishmania parasites.

Background: Cutaneous leishmaniasis (CL) is a parasitic disease that presents a broad spectrum of clinical features. Treatment of CL is problematic. We aimed to compare the field therapeutic efficacy of topical nanoliposomes containing 0.4% amphotericin B (Nano Lip-AmB) alone and in combination with cryotherapy and/or Glucantime® on human CL in the endemic areas of Iran. Methods: This retrospective study was performed based on the results of using Nano Lip-AmB alone or with Glucantime® and/or cryotherapy in the treatment of zoonotic cutaneous leishmaniasis (ZCL) in patients referred to health centers of Isfahan, Golestan and Ilam Provinces of Iran as endemic foci of ZCL caused by Leishmania major besides Mashhad and Bam cities as endemic foci of anthroponotic cutaneous leishmaniasis (ACL) caused by with L. tropica. Results: Two hundred and seventy-eight patients with CL were included in the current study. All of the patients (100%) who received Nano Lip-AmB alone or in combination with Glucantime® and/or cryotherapy based on guideline of Iranian national committee for the treatment of CL. Two patients with 7 skin lesions, who was resident in ACL endemic area and received Nano Lip-AmB plus Glucantime® and another patient was a resident of ZCL endemic area and received Nano Lip-AmB plus cryotherapy showed clinical relapses after treatment. Conclusion: Sina Ampholeish® in combination with other standard protocols of treatment of CL is well tolerated and with acceptable clinical efficacy rate.

Abstract Background In recent years, cases of human visceral leishmaniasis (HVL) have been reported in some districts of Golestan Province, northeastern Iran, particularly in rural areas. Recent epidemiological evidence in Leishmania infantum endemic regions of in Iran indicates approximately 50%–80% of seropositive dogs are asymptomatic for Leishmania infection. Objectives The goal in this study was to determine Leishmania species infecting domestic dogs in Golestan Province, Iran. Methods Between 2015 and 2016, blood samples were obtained from 100 domestic dogs in rural regions of Golestan Province, northeastern Iran. All samples were tested for anti‐Leishmania antibodies using a direct agglutination test (DAT), and for Leishmania spp. kinetoplast DNA (kDNA) using PCR. Results Seven (7%) dogs were antibody positive and 25 dogs (25%) were Leishmania spp. DNA positives by PCR positive for leishmaniasis. Four of the seven (71%) antibody‐positive dogs and 19 of the 25 (76%) PCR‐positive dogs were asymptomatic. The rate of infection detected by PCR was significantly higher in male dogs (21/75, 28%) than that in female dogs (4/25, 16%). The ITS1 PCR‐RFLP assay identified the presence of L. infantum, L. tropica or Crithidia spp. in the 25 PCR‐positive samples. Conclusions The high proportion of asymptomatic dogs in the study areas represent they act as potential reservoirs in the transmission cycle of Leishmania spp. and also Crithidia fasciculata as an emerging agent for the first time. Moreover, our data showed that PCR is a more reliable assay than DAT for detecting Leishmania spp. infection among asymptomatic dogs.

Background: Current medications especially the pentavalent antimonial compounds have been used as the first line treatment of cutaneous leishmaniasis (CL), but they have limitations due to serious side effects such as drug resistance, cardio and nephrotoxicity, and high costs. Hence, the demand to find more usable drugs is evident. Synthesis and devel­opment of natural, effective, biocompatible, and harmless compounds against Leishmania major is the principal priority of this study. Methods: By electrospinning method, a new type of nanofiber were synthesized from royal jelly and propolis with dif­ferent ratios. Nanofibers were characterized by Scanning Electron Microscope (SEM), Transmission Electron Micros­copy (TEM), Thermogravimetric Analysis (TGA), Contact angle, and Fourier-transform infrared spectroscopy (FTIR). The Half-maximal inhibitory concentration (IC50), Half-maximal effective concentration (EC50) and the 50% cytotoxic concentration (CC50) for different concentrations of nanofibers were determined using quantitative calorimetric meth­ods. Inductively coupled plasma-optical emission spectrometry (ICP-OES) and flow cytometry were performed as complementary tests. Results: The results showed that the proposed formulas provide a new achievement that, despite the significant killing activity on L. major, has negligible cytotoxicity on the host cells. Royal jelly nanofibers have significantly shown the best 72 hours results (IC50= 35 μg/ml and EC50=16.4 μg/ml) and the least cytotoxicity. Conclusion: This study presents a great challenge to introduce a new low-cost treatment method for CL, accelerate wound healing, and reduce scarring with minimal side effects and biocompatible materials. Royal jelly and propolis nanofibers significantly inhibit the growth of L. major in-vitro.

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.

Abstract Objective Zoonotic visceral leishmaniasis (VL) is caused by Leishmania infantum, of which dogs are the main reservoir. VL is endemic in the Middle East, also in some parts of Iran. Following reports of new cases of VL in children in Maragheh County, the non‐endemic area of the disease, we encouraged to conduct a preliminary study on domestic dogs and cats to identify their potential role as reservoirs for the disease. Materials and Methods This study was conducted during a period of 3 years from 2018 to 2021. Two hundred ownership dogs and 25 cats from Maragheh County, north‐west of Iran, were randomly screened. Blood samples were collected. A direct agglutination test (DAT) was used for the detection of anti‐L. infantum antibodies. Furthermore, buffy coat samples from the L. infantum seropositive animals were examined to detect parasite presence using polymerase chain reaction. Results Out of the total of 200 ownership dogs evaluated, 170 (85%) were male and 30 (15%) were female with a mean age of 4.3 years. Anti‐L. infantum antibodies (IgG cut‐off ≥ 1:320) were observed in 3.5% of dogs (7/200) by the DAT test. All seropositive dogs were identified in the first year of examination. Regarding molecular approaches in seropositive dogs, two samples were positive for a 565 bp kDNA minicircle gene specific for L. infantum. During the study period, no seropositive case was detected in the cats examined. Conclusions This study shows that the domestic cycle of L. infantum has been established in the studied region. It is necessary to increase the awareness and monitoring of the disease with the study of wild reservoirs periodically.

Background: Cutaneous leishmaniasis (CL) is an endemic infection in the Middle East, including Iran that is also spreading to new foci. We aimed to determine the leishmaniasis species causing CL in Alborz province. Methods: Overall, out of 55-suspected CL patients referred to health centers in Alborz Province, north central Iran in 2019, 40 patients had positive smear for CL based on optical microscopy. The internal transcribed spacer 1 (ITS1) of nuclear ribosomal DNA (rDNA) was amplified by PCR. Leishmania species were identified by PCR–restriction fragment length polymorphism (PCR-RFLP) using BshF I (Hae III) enzyme. Results: Out of the 40 positive patients with CL, 34 cases (85%) had been caused by Leishmania (L) major and six (15%) by L. tropica. Fifteen patients had no history of traveling to the disease endemic areas, of which nine were Iranians. Skin lesions and scars caused by CL were mostly observed on the hands and face. Moreover, more than two skin lesions were observed in 22 cases (55%), all of which were infected with L. major. A single skin ulcer was seen in 18 (45%) of the CL patients. Conclusion: Climate change, reduced rainfall, and demographic changes such as migration into Alborz Province and the increasing marginalization of the population and their entry to settle in new areas might have caused natural transmission of both L. tropica and L. major in this province.

Background: Leishmaniasis is currently considered a re-emerging or emerging infection based on the geographic region. The outcome of leishmaniasis vastly depends on Leishmania-host interaction. This preliminary study aimed to show the association of human leukocyte antigen (HLA) class I and II genes with healed and non-healed cutaneous leishmaniasis (CL), and symptomatic and asymptomatic visceral leishmaniasis (VL) compared with control groups in Iran. Methods: Ninety-five people, including 31 patients versus 64 individuals in the control group, were enrolled. Among them, 20 patients had confirmed CL based on amastigote observation, 10 had improved CL and 10 non-healed CL. Eleven patients were suffering from confirmed VL based on direct agglutination test (Five asymptomatic and six symptomatic VL cases). Besides, they were residents in an endemic area of VL in the northwest of Iran. To select a control group, it was ensured that they had no history of leishmaniasis. Peripheral blood samples were collected from each patient. After DNA extraction, HLA typing was conducted using polymerase chain reaction - sequence-specific priming (PCR-SSP). Subsequently, data were statistically analyzed by SPSS. Results: There was a statistical relationship between the presence of HLA-A26 and CL, healed CL and the existence of the B38 allele, C1 allele and symptomatic VL, as well as B1.4 allele and asymptomatic VL (P˂0.05). Conclusion: This primary finding indicates that several HLA genes have a potential role in the susceptibility of Iranian people to CL and VL.

Abstract Visceral leishmaniasis (VL) has rarely been reported in patients with chronic granulomatous disease (CGD), despite the fact that they seem more susceptible to intracellular infection. We describe a rare presentation of VL and epididymo‐orchitis in a patient with CGD, which has not been seen previously in the literature following inflammatory syndrome.

Abstract 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

Abstract Background Ocular infection with Toxoplasma gondii is a major preventable cause of blindness, especially in young people. The aim of the present study was to assess detection rate of T. gondii DNA in blood samples of clinically diagnosed of ocular toxoplasmosis using uracil DNA glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP) and real-time quantitative PCR (qPCR) based on REP-529 and B1. Methods One hundred and seventeen patients with clinically diagnosed ocular toxoplasmosis (OT) were participated in the study as well as 200 control patients. Peripheral blood samples were assessed using UDG-LAMP and qPCR techniques targeting REP-529 and B1. Results Detection limits of qPCR using REP-529 and B1 were estimated as 0.1 and 1 fg of T. gondii genomic DNA, respectively. The limits of detection for UDG-LAMP using REP-529 and B1 were 1 and 100 fg, respectively. In this study, 18 and 16 patients were positive in qPCR using REP-529 and B1, respectively. Based on the results of UDG-LAMP, 15 and 14 patients were positive using REP-529 and B1, respectively. Results of the study on patients with active ocular lesion showed that sensitivity of REP-529 and BI targets included 64 and 63%, respectively using qPCR. Sensitivity of 62 and 61%, were concluded from UDG-LAMP using REP-529 and B1 in the blood cases of active ocular lesion. qPCR was more sensitive than UDG-LAMP for the detection of Toxoplasma gondii DNA in peripheral blood samples of patients with clinically diagnosed toxoplasmic chorioretinitis. Furthermore, the REP-529 included a better detection rate for the diagnosis of ocular toxoplasmosis in blood samples, compared to that the B1 gene did. Moreover, the qPCR and UDG-LAMP specificity assessments have demonstrated no amplifications of DNAs extracted from other microorganisms based on REP-529 and B1. Conclusions Data from the current study suggest that qPCR and UDG-LAMP based on the REP-529 are promising diagnostic methods for the diagnosis of ocular toxoplasmosis in blood samples of patients with active chorioretinal lesions.

Background: After the earthquake in 2017 a few new cases of visceral leishmaniasis (VL) were reported from SarPol-e-Zahab district of Kermanshah Province, western part of Iran. This study was conducted to determine the seroprevalence in Kermanshah Province. Methods: This descriptive cross-sectional study was conducted on children up to 12 years of age from SarPol-e-Zahab County, Kermanshah Province, western part of Iran in 2021. For each individual, a questionnaire including age, sex, clinical features, history of the disease, and contact with canines as reservoir hosts of VL were completed, separately. To determine VL seroprevalence, blood samples were collected from the children and after centrifugation, the sera samples were separated and tested using Direct Agglutination Test (DAT) for detection of anti-L. infantum antibodies. Statistical analyses were performed using SPSS16. Results: Totally, 13 persons were seropositive; 7 samples with titer 1:800, 3 samples had 1:1600, 2 samples had 1:3200 and 1 sample had 1:6400. None of the seropositive cases had a history of kala-azar. There was no significant difference between males and females at titers of anti-Leishmania specific antibodies. Conclusion: L. infantum infection is being circulated with low prevalence in children up to 12 years old from SarPol-e-Zahab County but it is necessary that the surveillance system is regularly monitored among physicians and public health managers in the studied areas.

Background: The primary aim of this study is to determine infection to Leishmania parasites in the wild population of Phlebotomus caucasicus and Phlebotomus mongolensis using molecular methods in some important zoonotic cutaneous leishmaniasis foci in Iran. Methods: Sand flies were collected from active colonies of rodent burrows from 16 trapping sites using sticky trap pa­per. In order to detect and identify of Leishmania parasites in females Ph. caucasicus and Ph. mongolensis, the Nested–PCR amplification of ITS2-rDNA region was performed to generate amplicon with 245bp for Leishmania major, 206bp for L. gerbilli and 141bp for L. turanica. Results: In the current study we found DNA of different gerbil parasites such as L. major and L. turanica, and mixed infection of L. major/L. turanica in Ph. caucasicus and Ph. mongolensis. It should be noted that, in Iran, natural infec­tion with Leishmania parasites is recorded for the first time in this study in Ph. mongolensis. Conclusion: Both species of Ph. caucasicus and Ph. mongolensis not only may participate in the ZCL transmission cycle between reservoir hosts, but also results of this study support the role of these species as secondary vectors in the transmission of leishmaniasis to humans.

Background: Visceral leishmaniasis (VL) also known as Kala-azar is considered as one of the zoonotic infections in Mediterranean countries. The reservoir of this infection is dogs and sand flies are considered as its vectors. Due to reported sporadic cases of Kala-azar in the past five years in Shahroud County, Semnan Province, Iran, this study aimed to investigate the status of this infection in this area and to determine its seroepidemiology to take required measurements for infection control and treatment. Methods: This descriptive cross-sectional study was conducted on 504 subjects residing in seven villages in Shahroud County, Semnan, Iran. Samples were randomly selected using the cluster sampling method and blood samples were collected from subjects aged up to 13 years old (90%) and adults over 13 years old (10%) from September 2018 to May 2019. After separating sera from whole blood, samples were subjected to direct agglutination test (DAT) to detect anti-Leishmania infantum antibodies. First dilutions were prepared from 1:10 to 1:800 for human samples. Results: Results of serological testing with 1:800 titration indicated that no sample was positive for antibody against Leishmania infantum. After secondary screening, 10 cases (1.98%) showed the antibody titer of 1:100, while four cases (0.79%) showed the antibody titer of 1:400. According to clinical findings, no patient was suffering from fever, weight loss, splenomegaly, hepatomegaly, and cachexia and therefore did not show the symptoms of Kala-azar. Conclusion: The results of the current study indicate that Kala-azar is not prevalent in Shahroud County.

Background: Fleas (Insecta: Siphonaptera) are considered as highly specialized bloodsucking on mammals such as dogs. The existence of three factors, namely a vast distribution area, different hosts, and digestive system with a specific mecha­nism for digesting blood has led to species of fleas who nourish from mammals be introduced as the potential vectors of diseases. The aim of this study was to assess Leishmania infantum natural infection of dog fleas in northwest Iran in 2018. Methods: A total of 20 infested domestic dogs (Canis lupus familiaris) were randomly selected from 5 villages. Fleas were collected using brushing against dog hairs and fine forceps. Then, they were morphologically identified and pre­served in ethanol for molecular assay. The kinetoplast DNA of the parasite was used for detection of Leishmania infan­tum using a semi-nested polymerase chain reaction (PCR) assay. Results: The human flea, Pulex irritans, and the cat flea, Ctenocephalides felis were identified on 40% and 35% of dogs, respectively. The results of PCR indicated that L. infantum was found in the Ctenocephalides canis (75%) and C. felis (66.7%) col­lected from infected dogs. No leishmanial infection was observed in P. irritans. Conclusion: It is concluded that fleas could be infected by Leishmania infantum, but maintenance of the parasite and their vectorial competence needs to be determined.

Background: Various arthropods, including Rhipicephalus sanguineus and Ctenocephalides felis felis have been sug­gested as secondary vectors of Leishmania spp. many years ago. This study was conducted to determine zoonotic cuta­neous leishmaniasis (ZCL) PCR positivity of reservoir hosts and their ectoparasites for Leishmania spp. in Segzi plain in Esfahan Province from October 2016 to October 2017. Methods: Microscopic examination and nested PCR were used to detect and identify Leishmania spp. isolated from rodents' ear and ectoparasites, and then, the results were confirmed by two methods, PCR-restriction fragment length polymorphism and sequencing. Results: Totally, 93 rodents (92 Rhombomys opimus and one Nesokia indica) and nine different species of ectoparasites (n=527) including fleas, mites, and ticks were collected during different seasons in the study area. Fourteen R. opimus were positive for Leishmania spp. by microscopic examination while one N. indica and 77 R. opimus were positive by nested PCR. The infection rate of rodents with Leishmania major and L. turanica was 39.79% (n=37) and 15.05% (n=14), respectively. Mixed natural infections with L. major and L. turanica were seen in rodents. Moreover, 72.22% of fleas (39/54), 75.0% of mites (5/8), and 100% of tick nymph (1/1) were PCR positive for Leishmania parasites. Conclusions: The highest rate of infection with L. major and L. turanica in R. opimus populations was observed in summer and spring, respectively. It is suggested that the role of L. turanica and the probable role of ectoparasites in the epidemiology of disease should be investigated. Xenodiagnostic testing is recommended for future testing.

Abstract Background Circumsporozoite protein (CSP) has a central immune domain that includes short regions of repeating amino acid sequences. This immunodynamic region is an epitope of B cells that can elicit an immune response in human and laboratory animals. The aim of the present study was to express the recombinant PvCSP-VK210 antigen and evaluate it for assaying antibodies obtained during human P. vivax infection by Western blotting and indirect ELISA (enzyme-linked immunosorbent assay). Method Genomic DNA of P. vivax was isolated from a blood sample of an Iranian person with vivax malaria, and by PCR, the fragment of the PvCSP-VK210 gene was amplified. The gene fragment was cut after gel purification by BamHI and HindIII enzymes and then cloned into pET28a expression vector. Finally, the recombinant pET28a was transformed into the E. coli BL21 (DE3) as the expression host. In order to produce His-tagged protein, the expression host was cultured in LB medium. The protein was purified by Ni–NTA columns and immobilized metal affinity chromatography, and after confirmation by Western blotting technique, was used as the antigen in the indirect ELISA test. Results The recombinant protein was expressed and purified as a 32-kDa protein. The sensitivity and specificity of the indirect ELISA test with the recombinant PvCSP-VK210 antigen were 61.42% and 97.14%, respectively, based on OD = 0.313. Between the results of the microscopic test and the indirect ELISA test with the recombinant PvCSP-VK210 antigen there was a Kappa coefficient of 0.586. The positive and negative predictive value and validity of the ELISA test with the recombinant PvCSP-VK210 antigen were 95.55%, 71.57%, 79.28%, respectively. Conclusion The sensitivity of the indirect ELISA method with the recombinant PvCSP-VK210 antigen was 61.42%, which is the first report from Iran.

Background: Acanthamoebae are a causative agent of Acanthamoeba keratitis (AK) in immunocompetent individuals. Since access to propamidine isethionate (Brolene®) as a first-line treatment has been limited in recent years, in the current study, we examined the effects of pentamidine isethionate against trophozoite and cyst forms of Acanthamoeba. Methods: This experimental study was conducted in the Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran, during 2019-2020. Pentamidine isethionate at concentrations of 50, 100, 200, 400, 600, 800, and 1000 µM were tested against trophozoites and cyst stages of T4 genotype, at 24- and 48-hour incubation period, and the viability was determined by trypan blue staining. In addition, the cytotoxic effect of the drug was examined in Vero cells using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Results: The 50% inhibitory concentration (IC50) of pentamidine isethionate on trophozoite after 24 and 48h were 97.4 µM and 60.99 µM. These results on cyst after 24 and 48h were 470 µM and 175.5 µM, respectively. In MTT assay, the drug showed an inhibitory effect on Vero cell growth with IC50 values of 115.4 µM and 87.42 µM after 24h and 48h, respectively. Conclusion: Pentamidine isethionate exhibited an inhibitory effect on trophozoite and cyst. Given that the trophozoicidal activity of the drug is in the safe dose, it could be suggested as an alternative in patients with AK; however, further investigation is needed in an animal model to confirm the data.

Background: Pentavalent antimonial has been a drug of choice against leishmaniasis, despite the emergence of treatment failure. Identification of resistance markers is urgently needed to design new therapeutic strategies. Iron-Superoxide dismutases (Fe-SODs) are antioxidant enzymes contributing to detoxify reactive oxygen species to prevent a cell from oxidative stress. Since antimonial compounds induce oxidative stress, in this survey, the expression of SOD genes was investigated to identify their expression pattern in clinical resistant isolates. Methods: This cross-sectional survey was done in Mashhad City, northeast of Iran during 2014 to 2019. The RNA expression level of mitochondrial (SODA) and glycosomal (SODB) superoxide dismutase was investigated in 25 antimony responsive (n=15) and unresponsive (n=10) anthroponotic cutaneous leishmaniasis (ACL) patients. Total RNA extraction and cDNA synthesis, the qRT-PCR approach was utilized to investigate the relative RNA expression level. Results: The transcript level of SODs was over-expressed in the most resistant isolates. Gene expression analysis demonstrated the over-expression of SODA and B by a factor of 3.8 and 4.81, respectively, in resistance isolates vs. sensitive ones. Conclusion: Aberrant expression of SODA/B in unresponsive parasites could potentially implicate in detoxifying antimony-induced oxidative stress. Moreover, SODs might be considered as potential predictive markers of the response to antimonials in ACL patients in endemic areas.

Background: Infections by Plasmodium falciparum, are becoming increasingly difficult to treat. Therefore, there is an urgent need for novel antimalarial agents’ discovery against infection. In present study, we described a 2’-O-Methyl gapmer phosphorothioate oligonucleotide antisense targeting translation initiation region of 3D7 strain RH5 gene. Methods: The study was conducted in Pasteur Institute of Iran in 2020. ODNs effects were measured by microscopic examination and real time RT-PCR. For microscopy, microplates were charged with 2’-OMe ODNs at different dilutions. Unsynchronized parasites were added to a total of 0.4 ml (0.4% parasitemia, 5% red blood cells), and slides were prepared. Proportion of infected cells was measured by counting at least 500 red blood cells. Results: RH5 genes start codon regions selected as conserved region besed on alignment results. Gap-RH5-As which was complementary to sequence surrounding AUG RH5 start codon significantly reduced parasite growth (>90% at 50 nM) compared to sense sequence control (Gap-RH5-Se) (17%), (P

Background: A variety of haemoprotozoa including Plasmodium, Haemoproteus and Leucocytozoon cause infections in birds and are transmitted by some known vectors. These parasites cause anemia, low appetite, weakness and ultimate­ly death in birds. The present study was aimed to determine these parasites, in birds of Mazandaran and Golestan prov­inces in Iran. Methods: The project was performed on 340 live birds in 2016. The samples were collected from February to Septem­ber 2016, from each bird, two thin and thick blood smears were prepared and the remaining blood about 1ml was kept in EDTA-containing tubes for molecular studies. The slides were stained with 10% Giemsa, then examined microscopical­ly. About ten percent of the negative samples were considered for Polymerase Chain Reaction (PCR) technique, using specific primers to diagnose Plasmodium and Haemoproteus spp. Electrophoresis was done for PCR products and rele­vant bands to the parasites were identified based on the size. The considered birds belonged to ducks, chickens, roosters, and pigeons. Results: From 340 microscopically examined blood samples 32 (9.5%) samples were positive. Twenty-five (7.35%) of them were infected with the genus Haemoproteus. Seven samples (14%) out of 50 microscopically negative samples were found as Haemoproteus or Plasmodium spp when PCR technique was employed. Conclusion: This study revealed the existence of malaria parasites and other haemosporidia in birds in Iran. Employing molecular methods (PCR examination) could detect more infections.

Abstract Objective Canine visceral leishmaniasis (CVL) is the main source of human visceral leishmaniosis (HVL) in Mediterranean region, including Iran and is spread from domestic dogs to Phlebotomine sand flies vectors to humans. To control the transmission of HVL, early and accurate detection of infected dogs is paramount importance despite it remains a confronting challenge. Herein, we evaluated the performance of direct agglutination test (DAT) against gold standard nested polymerase chain reaction (nested-PCR) for CVL diagnosis in symptomatic and asymptomatic domestic dogs from endemic areas of Iran. Results Venous blood samples were collected from dogs without clinical signs (n = 30) and with clinical signs (n = 35) suggestive of Leishmania infantum infection. Among 65 samples examined, Leishmania DNA was detected by nested-PCR in 89.23% (58/65). Furthermore, 86.15% (56/65) nested-PCR positive samples were also DAT positive. The results of the DAT sensitivity test were 96.43% and 96.67% in symptomatic and asymptomatic dogs, respectively, while the specificity was 100.00% and 60.00% in symptomatic and asymptomatic dogs, respectively. The results of this study also pointed out substantial concordance between DAT test and nested-PCR method in both symptomatic dogs (Κ = 0.783; P

Abstract Canine visceral leishmaniasis (CVL) is endemic in the southern parts of Iran. The current study aimed at molecular and serological evaluation of zoonotic visceral leishmaniasis in dogs in Fars province, southern Iran. Blood samples were collected from 60 dogs in the three villages in Fars Province. Serum samples were tested for antibodies against L. infantum by direct agglutination test (DAT). DNA was extracted from each dog's buffy coat and tested by PCR, targeting the Leishmania ITS‐2 region. From a total of 60 studied dogs, 25 (41.7%) were female, and 35 (58.3%) were male. Dogs' age ranged from 1 to 7 years, with a mean age of 2.97 (±1.4) years. Anti‐Leishmania antibodies were detected in sera samples of 28 (46.7%) dogs, (titre ≥ 1:320). Out of 28 seropositive cases, 13 (46.4%) were female, and 15 (53.6%) were male. Association between seropositivity and dogs’ clinical signs was statistically significant (p

Background: Intestinal parasitic infections (IPIs) are still considered a public health problem of mankind, particularly in immunocompromised patients. We aimed to determine the prevalence of IPIs with an emphasis on immunocompromised patients in a referral hospital in Tehran Province, Iran. Methods: In this cross-sectional study, 442 fecal specimens were collected randomly from patients, referred to Baqiyatallah Hospital in Tehran Province, Iran from May to September 2020. The collected specimens were examined using wet-mount, trichrome and modified Ziehl-Neelsen staining, formalin-ether concentration, and agar plate culture. Results: The prevalence of IPIs was found 17.0% (95% CI: 13.6-20.8%). The prevalence of protozoan parasites (16.3%; 95% CI: 13.0-20.1%) was significantly higher than helminthic parasites (0.7%; 95% CI: 0.1-2.0%). Blastocystis spp., Giardia lamblia, and Entamoeba coli were the most common intestinal protozoan with a prevalence of 12.2%, 1.6%, and 1.4%, respectively. E. histolytica/E. dispar/E. moshkovskii, Iodamoeba bütschlii, Cryptosporidium spp., Chilomastix mesnili as protozoan species and Hymenolepis nana, Dicrocoelium dendriticum, and Ascaris lumbricoides as helminthic species were the other detected parasites. Multiple logistic regression revealed a significant association of IPIs infections with stool consistency and the status of immune system. Conclusion: The prevalence of IPIs among the patients who are immunocompromised was significantly higher than immunocompetent patients (P< 0.05). Periodic stool examinations for screening of IPIs should be included as a part of routine medical check-up in these patients.

Background: Detection of Leishmania RNA virus (LRV) in Old World Leishmania species and their possible role in the disease prognosis requires sensitive and specific methods, preferably independent of the viral genome. We aimed to develop an indirect immunofluorescence antibody (IFA) assay to detect LRV in the Old World Leishmania parasites. Methods: Clinical samples were collected from 86 cutaneous leishmaniasis (CL) patients in different endemic areas of CL in Iran, during 2017-2019. For antibody preparation, the viruses were obtained from sediment of an LRV-infected L. major culture-using freeze and thaw cycles followed by gradient cesium chloride centrifugation. The purified viruses were used to immunize a male 3-4 months rabbit. Various dilutions of the LRV-immunized rabbit's serum and a conjugated antibody were deployed to detect LRV in 48 isolates by IFA assay. Results: LRV virus was detected in four of the 48 CL cases using IFA method. Amplification of a partial fragment of RNA-dependent RNA polymerase (RdRp) gene from the isolates confirmed the IFA results. In phylogeny, the generated RdRp sequences from four isolates were grouped with the other Old World LRVs, but separate from L. aethiopica LRVs, which appeared as a highly supported distinct clade. Conclusion: Further optimization of this approach to detect the LRV directly in lesion scrapings can make it a more reliable tool for field studies and disclosing the virus's possible role in disseminating and unusual clinical features.

Background: Cystic echinococcosis (CE) is one of the most important parasitic infections in subgroup seven common neglected diseases of humans and animals. It is in the list of 18 neglected tropical diseases of the WHO. We aimed to analyze the situation of the disease in Iran using Geographical Information System (GIS) and satellite data analysis. Methods: The data obtained from the Ministry of Health and Medical Education, Tehran, Iran and other related centers from 2009 to 2018 were analyzed using GIS. Then, the spatial distribution maps of the disease were generated, and the hot spots of the disease in Iran were determined using spatial analysis of ArcGIS10.5 software. Geographically weighted regression (GWR) analysis in ArcGIS10.5 was used to correlate the variables affecting the disease including temperature, relative humidity, normalized different vegetation index (NDVI) and incidence of hydatidosis. Data analysis was performed by Linear regression analysis and SPSS 21 software using descriptive statistics and chi-square test. Results: Zanjan, Khorasan Razavi, North Khorasan, Chaharmahal Bakhtiari, Hamedan, Semnan, and Ardabil provinces were the hot spots of CE. The results of geographical weighted regression analysis showed that in Khorasan Razavi, North Khorasan, Chaharmahal Bakhtiari, Hamedan, Semnan, Ardabil, Zanjan, Qazvin, and Ilam provinces, the highest correlation between temperature, humidity, vegetation density and the incidence of hydatidosis was observed (P

Background: Visceral leishmaniasis (VL) caused by the Leishmania donovani complex that is transmitted by the bites of female sandflies. Mediterranean type of VL caused by L. infantum. While, Roudbar County of Guilan Province has been introduced as a suspected cutaneous leishmaniasis focus; there are no published data on the seroprevalence of VL in Guilan Province. We aimed to investigate the seroprevalence of this disease in Roudbar County. Methods: This descriptive cross-sectional study was carried out in 2019-2020 among children less than 12 years of age to determine the seroprevalence of VL by direct agglutination test (DAT). Blood samples were randomly collected from 918 children under 12 years of age refers to the public health center in the clusters. Results: Out of 918 children, 14 (1.52%) showed anti-Leishmania antibodies, with 4 (0.43%), 2 (0.21%), 8 (0.87%) anti-L. infantum antibodies at titers 1:800, 1:1600 and ≥1: 3200 respectively. All children with anti-Leishmania antibody titers of ≥1:800 were evaluated by a physician. Clinical manifestation of VL including fever, anemia and hepatosplenomegaly observed in a 6-year-old boy from Defraz village with anti-Leishmania antibody of titers ≥102400.This patient was admitted to the pediatric hospital in Rasht, capital of Guilan province, Iran and was successfully treated. Conclusion: VL is being circulated with low prevalence in children up to 12 years old in Roudbar, northern part of Iran. Accordingly, it is critical to improve the awareness of physicians and public health supervisors about the importance of this fatal disease in Guilan province and especially in Roudbar area.

Background: We aimed to detect the genetic diversity of samples identified morphologically as Fasciola spp. from sheep, cattle and goat from Lorestan Province, western Iran using PCR-RFLP method. Besides, we evaluated the genetic diversity indices, sequencing and phylogenetic analysis using mitochondrial gene (ND1 and CO1). Methods: PCR-RFLP analysis of ribosomal ITS1 fragment by RsaI restriction enzyme to investigate the genetic characteristics of Fasciola species obtained from different hosts (18 sheep, 21 cattle, and 17goats) was conducted. The samples were sequenced. Sequences were evaluated using BLAST software and the parasite species were identified with similarity percentage and overlap with the species registered in the gene bank. Then similarity and diversity of intra-species and intra-species diversity of Fasciola species were calculated. Results: In Lorestan, based on RFLP pattern, 93% (52) of the Fasciola spp. isolates had a RFLP pattern related to F. hepatica and 7% (4) were F. gigantica. No hybrid forms were detected. The CO1 gene could clarify 19 haplotypes against ND1 gene that found 22 haplotypes among livestock. Sequencing results of the mtDNA showed intra-species identity 98. 5%-100% and Intra-species-diversity: 0-1.5% compared to the GenBank sequences. Conclusion: Using PCR-RFLP method, two species of F. hepatica and F. gigantica, were present in Lorestan Province, but F. hepatica was more prevalent. Mitochondrial genes could better test variability indices in different hosts than ribosomal genes, consequently among mitochondrial genes, the ND1 gene could better examine differences and similarities than CO1.

Background: We aimed to investigate the epidemiological, clinical and paraclinical aspects of Visceral Leishmaniasis in Ardabil Province, from 1986 to 2018. Methods: The disease has been diagnosed by a serological validate test as Direct Agglutination Test, parasitological methods, and clinical manifestations. The data were collected and then analyzed. Results: Overall, 2824 out of 69007cases (4.1%) had anti-Leishmania antibodies at titers≥1:3200. The majority of VL cases belonged to Meshkin-Shahr, and then Germi County. The percentages of both male and female cases were 52.8 % and 46.6%, respectively. Approximately 78.1% of cases were less than 5 yr old. The high incidence rate of the disease was recorded in the years 1987 and 2003. 48.6%, 3.2%, and 1.9% of the patients had low titer (1:3200), high titer (1:102400), and suspicious titer (1:1600), respectively. Out of 1200 bone marrow aspiration, 23% were positive, and 77% were negative. Fever, paleness, and hepatosplenomegaly were the main symptoms in patients. Moreover, abdominal swelling, edema, weight loss, lymphadenopathy and jaundice were some other symptoms in the patients. Among biochemistry factors, the levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, triglyceride and cholesterol have been increased. The death rate was estimated to be 2.8%, mainly related to the Ardabil and Germi cases. Conclusion: Kala-Azar is still one of the most important infectious diseases in Ardabil Province, and is responsible for considerable death rate in infants. More attention should be done to parasite control and early diagnosis.

Abstract Background Poor self-care skills and personal hygiene resulted from limitations in learning and understanding, put intellectually disabled individuals at greater risk for intestinal parasitic infections (IPIs). Despite several regional reports in Iran, the overall burden on IPIs among intellectually disabled individuals is poorly understood. Hence, the present study aimed to estimate the pooled prevalence of IPIs among intellectually disabled individuals in Iran. Methods Using the PRISMA guidelines, we conducted a systematic review and meta-analysis of data retrieved from seven electronic databases (PubMed, Web of Science, Scopus, Embase, and ProQuest for English articles, as well as SID and Magiran for Persian) from their inception up to December 2020. Pooled prevalence was estimated using a random-effects model with a 95% confidence interval (CI) and depicted as a forest plot, while heterogeneity was evaluated using Cochran’s Q-test. Results Exactly 1263 of the 3004 intellectually disabled individuals examined by 14 studies across 10 provinces of Iran were positive for IPIs. Overall pooled prevalence estimate was 41% (95% CI 29–53%) with a range of 21% (95% CI 10–32%) to 68% (95% CI 55–80%) across sub-groups. Entamoeba coli (16.2%; 95% CI 10.3–22%), Blastocystis spp. (12.2%; 95% CI 7.2–17.2%), and Giardia duodenalis (11.9%; 95% CI 7.4–16.3%) were the most prevalent protozoan species. In terms of helminthic agents, the most prevalent species were Enterobius vermicularis (11.3%; 95% CI 6.3–16.3%) followed by Strongyloides stercoralis (10.9%; 95% CI 5.0–16.9%) and Hymenolepis nana (2.8%; 95% CI 0.4–5.2%) Conclusion IPIs are highly prevalent among intellectually disabled individuals in Iran. Improving the health status and implementing infectious disease prevention strategies in rehabilitation centers, health promotion interventions to improve personal hygiene of intellectually disabled individuals, as well as utilize sensitive diagnostic methods besides routine stool examination techniques, and treatment of infected individuals will help in the control of these infections among intellectually disabled individuals.

Background: Visceral leishmaniasis (VL) or Kala-azar is an endemic disease in some areas of Iran, including East Azarbaijan, Ardabil, Fars and Bushehr provinces. The disease is sporadic in other parts of the country. According to the reports of numerous cases of the disease from Ahar and Osku counties (cities), East Azarbaijan province and the lack of new study on the prevalence of the disease, this study was designed to determine the seroprevalence of VL in the children of Ahar and Osku counties, in East Azarbaijan Province. Methods: In this descriptive cross-sectional study, blood samples were collected in the test tube without anticoagulants, from children under 8 years from rural areas of Ahar and Osko, in 2018. The samples were evaluated using Direct Agglutination Test (DAT) to measure anti Leishmania infantum antibodies for titer up to 1:3200. Results: Out of 209 children

Abstract Background Leishmania parasites express various essential proteins in different growth phases (logarithmic/stationary) and forms (promastigote/amastigote). Targeting the genes encoding such proteins paves the way for controlling these parasites. Centrin is an essential gene, which its protein product seems to be vital for the proliferation of Leishmania parasites. Herein, this study was contrived to analyze the expression level of the centrin gene in different growth phases and forms of Leishmania infantum (L. infantum) parasites isolated from various endemic areas of canine leishmaniasis (CanL) in Iran. Results All three collected isolates were identified as L. infantum using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Real-time reverse transcription (RT)-PCR revealed a statistically significant up-regulation (3.13-fold) in the logarithmic phase promastigotes compared to stationary ones (p 0.05). Conclusions Centrin gene in Iranian isolates of L. infantum is more expressed in exponential than stationary phases and seems to be considered as a promising target in the development of a genetically modified live attenuated vaccine for CanL control.

Chronic granulomatous disease (CGD) described as an essential immunodeficiency problem of phagocytic cells resulting in a phagocyte dysfunction and inability to kill a spectrum of bacteria and fungi. Despite the fact that CGD patients are more susceptible to intracellular infections, visceral leishmaniasis has been reported rarely in these cases. Here, we report an uncommon case of visceral leishmaniasis in a child with CGD. An 8-yr old boy with CGD presented to the infectious disease ward, Children's Medical Center, Tehran University of Medical Sciences, Iran after the onset of 20 days fever with chronic crusted ulcer approximately 3 cm × 3cm on the left upper limb and a small ulcer measuring 0.5 cm × 0.5 cm on the right knee with moderate secretion. Bone Marrow Aspiration (BMA) and Bone Marrow Biopsies (BMB) of fragmented samples were performed and polymorphic population of hematopoietic cells, Megakaryocytes and Leishman bodies were seen. The patient was treated with meglumine antimoniate (Glucantime®) 20 mg/kg for 28 days and after partial improvement patient discharged and continue the treatment at home. Amphotericin B lipid complex (Ambisome®) (3–5 mg/kg per dose once) was administered every 3 -4 weeks for 18 months as secondary prophylaxis that was well tolerated and effective.

Background: Iran is one of the endemic areas of Mediterranean Visceral Leishmaniasis, a disease caused by Leishmania infantum. In this work, we examined whether Proteína quimérica 10 (PQ10) recombinant protein is suitable for immunological diagnosis of human visceral leishmaniasis. Methods: The study was carried out in Tarbiat Modares University during 2016-2018. The coding sequence of PQ10 recombinant protein was sub-cloned in pET28 expression vector and was commercially synthesized by GENERAY Biotechnology, China. Sequencing with proper primers was done, the expression, optimization of expression and protein purification were performed, and the purified recombinant protein was confirmed by western blot. The efficacy of PQ10 for serodiagnosis was evaluated with 50 positive and 50 negative serum samples, which confirmed by the direct agglutination test and collected from individuals living in the visceral leishmaniasis endemic areas of Iran. ELISA was performed with the PQ10 recombinant protein. Results: The 95% CI sensitivity of ELISA that was evaluated with sera from naturally infected individuals was 84%. The 95% CI specificity value of the ELISA determined with sera from healthy individuals (50 serum samples) and from individuals with other infectious diseases was 82%. The 95% CI positive predictive value (PPV) and negative predictive value (NPV) were exterminated 82.35% and 83.67%, respectively. Conclusion: We have used a recombinant synthetic protein to improve serodiagnosis of human visceral leishmaniasis. PQ10 could be useful for diagnosis of asymptomatic cases, as well as in the early phase of infections.

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.

Objective: To predict future trends in the incidence of malaria cases in the southeast of Iran as the most important area of malaria using Seasonal Autoregressive Integrated Moving Average (SARIMA) model, and to check the effect of meteorological variables on the disease incidence. Methods: SARIMA method was applied to fit a model on malaria incidence from April 2001 to March 2018 in Sistan and Baluchistan province in southeastern Iran. Climatic variables such as temperature, rainfall, rainy days, humidity, sunny hours and wind speed were also included in the multivariable model as covariates. Then, the best fitted model was adopted to predict the number of malaria cases for the next 12 months. Results: The best-fitted univariate model for the prediction of malaria in the southeast of Iran was SARIMA (1,0,0)(1,1,1)12 [Akaike Information Criterion (AIC)=307.4, validation root mean square error (RMSE)=0.43]. The occurrence of malaria in a given month was mostly related to the number of cases occurring in the previous 1 (p=1) and 12 (P=1) months. The inverse number of rainy days with 8-month lag (β=0.329 2) and temperature with 3-month lag (β=-0.002 6) were the best predictors that could improve the predictive performance of the univariate model. Finally, SARIMA (1,0,0)(1,1,1)12 including mean temperature with a 3-month lag (validation RMSE=0.414) was selected as the final multivariable model. Conclusions: The number of malaria cases in a given month can be predicted by the number of cases in the prior 1 and 12 months. The number of rainy days with an 8-month lag and temperature with a 3-month lag can improve the predictive power of the model.

Objective: Leishmania RNA virus (LRV) is a double-stranded RNA (dsRNA) virus that circulates within many species of the Leishmania parasite. In this study, we aimed to investigate the presence of LRV2 circulating in Leishmania isolates in an old focus of ZCL located in northeastern of Iran. Methods: Leishmania isolates were collected from 85 patients that confirmed to have cutaneous leishmaniasis (CL) based on parasitological examination. To identify the Leishmania isolates, species-specific primer sets were applied for molecular identification. The presence of LRV2 was performed by RdRp-semi nested-PCR. The genetic diversity were calculated using MEGA and DnaSP. To assess haplotype diversity, 31 LRV2 strains in different regions were surveyed using analysis a 292-bp section of the RdRp sequences. Results: Out of 85 patients, 83 (97.6 %) were diagnosed with L. major and 2 (2.4 %) with L. tropica. LRV2 virus was detected in 59 (69.4%) of the CL cases. For the first time, LRV2 was reported in one L. tropica strain in Iran. The current LRV2 sequences indicated the highest similarities to an Old World LRV2. Moreover, 10 unique haplotypes were identified based on the analyzed sequences of the RdRp gene. Conclusions: Our results indicated the highest occurrence of Leishmania/LRV2 co-circulation in this known ZCL focus from northeastern Iran. Phylogenetic analyses of LRV2 sequences confirmed that these isolates belong to the order of LRV2 from the Old World. This study offered an insight into LRV2 haplotype that the informative issue can be used for genetic research of LRV2 in other regions.

Abstract Background Direct agglutination test (DAT) as a simple, accurate and reliable method, has been widely used for serodiagnosis of visceral leishmaniasis (VL) during the last three decades. The present study is a systematic review and meta-analysis to evaluate the diagnostic accuracy of DAT for serodiagnosis of human VL. Methods Electronic databases, including MEDLINE (via PubMed), SCOPUS, Web of Science, SID and Mag Iran (two Persian scientific search engines) were searched from December 2004 to April 2019. We determined the pooled sensitivity and specificity rates of DAT for the diagnosis of human VL, calculated positive and negative likelihood ratios (LR+ and LR-), and constructed summary receiver operating characteristic (ROC) curves parameters across the eligible studies. Results Of the 2928 records identified in the mentioned electronic databases and after examining reference lists of articles, 24 articles met inclusion criteria and were enrolled in the systematic review and out of them 20 records qualified for meta-analysis. The pooled sensitivity and specificity rates of DAT was 96% [95% CI, 92–98] and 95% [CI95% 86–99], respectively. The likelihood ratio of a positive test (LR+) was found to be 21 [CI95%, 6.6–66.5] and the likelihood ratio of a negative test (LR−) was found to be 0.04 [(CI95%, 0.02–0.08]. The combined estimate of the diagnostic odds ratio for DAT was high [467 (CI95%, 114–1912]). We found that the summary receiver operating characteristic curve (SROC) is positioned near the upper left corner of the curve and the area under curve (AUC) was 0.98 (95% CI, 0.97 to 0.99). Conclusion Referring to our analysis, we determined that DAT can be considered as a valuable tool for the serodiagnosis of human VL with high sensitivity and specificity. As DAT is a simple, accurate and efficient serological test, it can be recommended for serodiagnosis of human VL particularly in endemic areas.

Abstract Objectives Hard ticks (Acari: Ixodidae) are ectoparasites of medical and veterinary importance. They are obligate blood-feeding vectors with the ability to transmit a wide variety of pathogens. Standard morphological keys are normally used for the identification of tick species. However, considering the importance of accurate species identification and the determination of bio-ecological characteristics of species, relying on morphological keys alone can be questionable. In this study, two DNA fragments (ITS2 and COI) were selected for phylogenetic evaluation of Iranian hard tick species belonging to the genera Dermacentor, Hyalomma, and Rhipicephalus. Results 1229 specimens of Dermacentor marginatus, D. niveus, Hyalomma anatolicum, Rhipicephalus bursa, and R. sanguineus s.l constituting 11 populations were collected from three different climatic and zoogeographical zones in Iran. Morphological studies revealed notable differences in important morphological characteristics between different populations of D. marginatus. The results of ITS2 sequence analysis provided additional evidence which supports the conspecificity of D. niveus and D. marginatus. Contrary to this finding, the sequence analysis of COI and phylogeny favored the separation of the two species. Given the greater importance of COI in identifying and discriminating species, a possibility heterospecificity between the two species should be considered.