Iranian Hydatid Disease Registry: Establishment and Implementation of a Neglected Tropical Disease Registry.

BACKGROUND: Cystic echinococcosis (CE) or hydatid disease is a global public health concern which imposes considerable economic costs on the communities in endemic regions. CE surveillance data are not adequately reliable. The present study reports the development and outcomes of a CE registry in Iran. METHODS: Hydatid Registry (HydatidReg) was initially established as a single-center registry in 2014 after the ethical approval of KMU. Following a call from MoHME to promote registry of different diseases and health outcomes, a call for participation was announced and all the Iranian Universities of Medical Sciences were requested to contribute to the registry. Subsequently, a nation-wide registry of hydatid disease was established in 2016. With a global perspective, HydatidReg joined the European Register of Cystic Echinococcosis (ERCE). A data collection form based on minimum dataset was designed and standard operating procedures (SOPs) were prepared to ensure standardized patient enrolment in the registry. A biobank system with two-dimensional barcoding was established along with HydatidReg for management and organization of biological specimens. RESULTS: As of March 2021, a total of 690 patients were enrolled in the registry. HydatidReg registered 362 (17.3%) out of the total 2097 patients enrolled in ERCE. Quality control (QC) of the data demonstrated 91.2% completeness and 80% timeliness. In the biobank, 322 biological specimens from 184 CE patients have been deposited including 70 blood, 96 sera and 156 parasite materials. CONCLUSION: High-quality data in the HydatidReg registry provided opportunities for health professionals to improve quality of care and organize meaningful research.

Hypocalcemia in Covid-19: A Prognostic Marker for Severe Disease.

BACKGROUND & OBJECTIVE: Previous studies have addressed the electrolyte abnormalities such as hypocalcemia in COVID-19 patients. We aimed to compare the laboratory findings especially the electrolyte levels among COVID-19 patients and healthy controls and evaluate their prognostic values. METHODS: This case-control study included 91 COVID-19 patients and 169 healthy individuals. Their laboratory parameters including electrolytes, albumin, liver enzymes, complete blood count, vitamin D, and parathyroid hormone (PTH) were compared. We also analyzed the association between these markers and the major outcomes including severity, mortality and hospitalization. RESULTS: Among patients with COVID-19, 59.3% of the patients had hypocalcemia on admission while in control group only 32.5% had low calcium level (OR=3.02, 95% CI: 1.79-5.13, P<0.001). The rates of death and ICU admission were significantly higher among the patients in hypocalcemic group than those of eucalcemic group (85.7% vs 14.3% and 33.3% Vs 9.1%, respectively). However, there was no significant difference in the mean PTH and vitamin D levels between the two groups. In terms of the severity of the infection, 74.1% of patients in hypocalcemic group had a severe infection while 24.3% of the patients in eucalcemic group were diagnosed with severe infection (OR=8.89, 95% CI: 3.38-23.37, P<0.001). CONCLUSION: Patients with COVID-19 may present with considerable laboratory abnormalities including hypocalcemia. The hypocalcemia would be also associated with worse major clinical outcome and higher mortality risk.

Transient Down-Regulation of Nucleoside Transporter 3 Gene Expression as a Drug Target in Leishmania major Using Antisense RNA Technology.

BACKGROUND: This study was aimed to silencing the Nucleoside transporter 3 (NT3) permease nucleobases involved in the salvage pathway of Leishmania in order to disrupt purine nucleotide uptake in the parasite and consequently, destruction of the parasite. METHODS: Overall, 502 bp fragment of the NT3 gene sequence was designed to produce an antisense transcript upon entry of the vector into the parasite. The NT3 construct was transfected into L. major promastigotes and NT3 gene expression was studied in vivo and in vitro conditions. RESULTS: Relative expression NT3 gene in transgenic Leishmania was decreased in tenth day. The percentages and the number of amastigotes infected macrophages with transgenic L. major were less than infected macrophages with wild-type strain. Our results in two groups of BALB/c female mice (wild-type strain and mutant, n=4 each group) were showed that size and number of ulcers in BALB/c mice infected with transgenic Leishmania promastigotes were less than the BALB/c mice infected with wild-type parasites. CONCLUSION: The results indicate the use of antisense RNA reduces of NT3 gene expression in L. major. More studies are required to obtain a new approach for treating Leishmania infection.

In Vitro and In Vivo Antileishmanial Activities of Pistacia vera Essential Oil.

This study aims to evaluate the in vitro and in vivo antileishmanial activities of Pistacia vera essential oil and compare their efficacy with a reference drug, meglumine antimoniate (Glucantime®). This essential oil (0-100 µg/mL) was evaluated in vitro against the intracellular amastigote forms of Leishmania tropica (MHOM/IR/2002/Mash2) and then tested on cutaneous leishmaniasis of male BALB/c mice by Leishmania major (MRHO/IR/75/ER). In the in vitro assay, it could be observed that P. vera essential oil significantly (p < 0.05) inhibited the growth rate of amastigote forms (IC50 of 21.3 ± 2.1 µg/mL) in a dose-dependent response compared with the control drug. Meglumine antimoniate also demonstrated antileishmanial effects with an IC50 value of 44.6 ± 2.5 µg/mL for this clinical stage. In the in vivo assay, the results indicated that 30 mg/mL of the essential oil had potent suppression effects on cutaneous leishmaniasis in BALB/c mice (87.5% recovery), while 10 and 20 mg/mL of the essential oil represented the suppression effects as weak to intermediate. The mean diameter of the lesions decreased about 0.11 and 0.27 cm after the treatment of the subgroups with the essential oil concentrations of 10 and 20 mg/mL, respectively. In contrast, in the subgroup treated with the essential oil concentration of 30 mg/mL, the mean diameter of the lesions decreased about 0.56 cm. In the control subgroups, the mean diameter of the lesions increased to 1.01 cm. The main components of P. vera essential oil were limonene (26.21%), α-pinene (18.07%), and α-thujene (9.31%). It was also found that P. vera essential oil had no significant cytotoxic effect on J774 cells. The present study found that P. vera essential oil showed considerable in vitro and in vivo effectiveness against L. tropica and L. major compared to the reference drug. These findings also provided the scientific evidence that natural plants could be used in traditional medicine for the prevention and treatment of cutaneous leishmaniasis.

Chemical composition along with anti-leishmanial and cytotoxic activity of Zataria multiflora.

CONTEXT: Natural products and their compounds are some of the most interesting sources of new drugs. Reviews have reported various pharmacological properties such as antimicrobial effects of Zataria multiflora Boiss (Lamiaceae). OBJECTIVE: The present study investigates the chemical composition of Z. multiflora essential oil and evaluates its cytotoxic effects and anti-leishmanial activities against Leishmania tropica in an in vitro model. MATERIALS AND METHODS: The components of Z. multiflora oil were identified by gas chromatography/mass spectroscopy (GC/MS) analysis. Anti-leishmanial effects of the essential oil (0-100 µL/mL) and methanol extract of Z. multiflora (0-100 µg/mL) on promastigote forms as well as their cytotoxic activities against J774 cells were evaluated using MTT assay for 72 h. The leishmanicidal activity against amastigote forms of L. tropica was evaluated at the concentrations of 0-50 µg/mL in a macrophage model for 48 h. RESULTS: The chemical analyses demonstrated that the main components of essential oil were thymol (41.81%), carvacrol (28.85%), and p-cymene (8.36%). Regarding leishmanicidal activity, the IC50 values for the essential oil and methanol extract were 3.2 µL/mL and 9.8 µg/mL against promastigote forms and 8.3 µL/mL and 34.6 µg/mL against amastigote forms, respectively. Essential oil (CC50 89.3 µL/mL) indicated a higher cytotoxic effect than the methanol extract (CC50 591.6 µg/mL) of Z. multiflora. CONCLUSION: The present study revealed the chemical composition of Z. multiflora that might be a natural source of new anti-leishmanial agents in terms of use against cutaneous leishmaniasis.

Designing and Cloning Molecular Constructs to Knock Out N-Acetylglucosamine Phosphatidylinositol De-N-Acetylase (GPI12) Gene in Leishmania major (MRHO/IR/75/ER).

BACKGROUND: Leishmaniasis represents a major public health concern in tropical and sub-tropical countries. At present, there is no efficacious vaccine against the disease and new control methods are needed. One way to access this important goal is to knock out genes of specific macromolecules to evaluate the effect of deletion on the growth, multiplication, pathogenesis and immunity of the parasite. The aim of this study was to design and clone molecular constructs to knock out N-acetylglucosamine phosphatidylinositol de-N-acetylase (GPI12) gene in Leishmania major. METHODS: For designing and making molecular constructs, we used pLEXSY-neo2 and pLEXSY-hyg2 vectors. The molecular constructs were cloned in E. coli strain Top10. The molecular constructs were transfected by electroporation into L. major in two stages. RESULTS: The molecular constructs were confirmed by Colony PCR and sequencing. The recombinant strains were isolated by selective antibiotics, after which they were confirmed by PCR, Southern and Western blots. CONCLUSION: Recombinant parasites were created and examined for subsequent study. With the use of molecular constructs, it was possible to remove and study gene GPI12 and to achieve a live recombinant Leishmania parasite that maintained the original form of the antigenic parasites. This achievement can be used as an experimental model for vaccine development studies. Further investigations are essential to check this model in a suitable host.

Comparison of Scolicidal Effects of Amphotricin B, Silver Nanoparticles,_and Foeniculum vulgare Mill on Hydatid Cysts Protoscoleces.

BACKGROUND: Surgery is the preferred treatment for hydatid cyst (cystic echinococcosis, CE). At present, various scolicidal agents have been used for inactivation of protoscoleces during surgery, but they are associated with adverse side effects. The aim of the present study was to evaluate the scolicidal effects of amphotricin B, Silver nano particles, Foeniculum vulgare Mill, essential oil and hypertonic saline against protoscoleces of hydatid cyst on an in vitro model. METHODS: Protoscoleces were aseptically aspirated from the naturally infected livers of sheep and goats. Various concentrations of AmB (2.5-20 mg/ml), Ag-NPs (0.5-4 mg/ml), F. vulgare essential oil (0.125-1 mg/ml) and hypertonic saline (10-20%) were used for 5-60 min. Eosin exclusion test was used to determine the viability of protoscoleces. RESULTS: Maximum protoscolicidal effect of AmB and Ag-NPs was found at concentrations of 20 and 4 mg/mL, resulting in only 82.3% and 71.6% of the protoscoleces after 60 min of incubation, respectively. In contrast, F. vulgare essential oil at concentration of 1 mg/ml and hypertonic saline 20% killed 100% protoscoleces after 5 and 10 min of exposure, respectively. CONCLUSION: The results indicated weak scolicidal activity of AmB and Ag-NPs; whereas F. vulgare essential oil had potent scolicidal activity against protoscoleces of hydatid cyst that revealed the potential of F. vulgare as a natural source for the production of new scolicidal agent for use in hydatid cyst surgery. However, further studies will be needed to confirm these results by checking the essential oil and its active component in the in vivo model.

In Vitro and In Vivo Antileishmanial Effects of Pistacia khinjuk against Leishmania tropica and Leishmania major.

The present study aims to evaluate the in vitro and in vivo antileishmanial activities of Pistacia khinjuk Stocks (Anacardiaceae) alcoholic extract and to compare its efficacy with a reference drug, meglumine antimoniate (MA, Glucantime), against Leishmania tropica and Leishmania major. This extract (0-100 µg/mL) was evaluated in vitro against promastigote and intracellular amastigote forms of L. tropica (MRHO/IR/75/ER) and then tested on cutaneous leishmaniasis (CL) in male BALB/c mice with L. major to reproduce the antileishmanial activity topically. In vitro, P. khinjuk extract significantly (P < 0.05) inhibited the growth rate of promastigote (IC50 58.6 ± 3.2 µg/mL) and intramacrophage amastigotes (37.3 ± 2.5 µg/mL) of L. tropica as a dose-dependent response. In the in vivo assay, after 30 days of treatment, 75% recovery was observed in the infected mice treated with 30% extract. After treatment of the subgroups with the concentration of 20 and 30% of P. khinjuk extract, mean diameter of lesions was significantly (P < 0.05) reduced. To conclude, the present investigation demonstrated that P. vera extract had in vitro and in vivo effectiveness against L. major. Obtained findings also provide the scientific evidences that natural plants could be used in the traditional medicine for the prevention and treatment of CL.

Antifungal, Antileishmanial, and Cytotoxicity Activities of Various Extracts of Berberis vulgaris (Berberidaceae) and Its Active Principle Berberine.

In this study, in vitro antidermatophytic activity against Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis, and Microsporum gypseum was studied by disk diffusion test and assessment of minimum inhibitory concentration (MIC) using CLSI broth macrodilution method (M38-A2). Moreover, antileishmanial and cytotoxicity activity of B. vulgaris and berberine against promastigotes of Leishmania major and Leishmania tropica were evaluated by colorimetric MTT assay. The findings indicated that the various extracts of B. vulgaris particularly berberine showed high potential antidermatophytic against pathogenic dermatophytes tested with MIC values varying from 0.125 to >4 mg/mL. The results revealed that B. vulgaris extracts as well as berberine were effective in inhibiting L. major and L. tropica promastigotes growth in a dose-dependent manner with IC50 (50% inhibitory concentration) values varying from 2.1 to 26.6 µ g/mL. Moreover, it could be observed that berberine as compared with B. vulgaris exhibited more cytotoxicity against murine macrophages with CC50 (cytotoxicity concentration for 50% of cells) values varying from 27.3 to 362.6 µ g/mL. Results of this investigation were the first step in the search for new antidermatophytic and antileishmanial drugs. However, further works are required to evaluate exact effect of these extracts in animal models as well as volunteer human subjects.

Protoscolecidal Effect of Berberis vulgaris Root Extract and Its Main Compound, Berberine in Cystic Echinococcosis.

BACKGROUND: Cystic echinococcosis (CE), a zoonotic parasitic infection caused by the metacestode (larvae) stage of dog tapeworm Echinococcus granulosus and recognized as a major economic and public health concern in the world. This study aimed to investigate the in vitro scolicidal effect of methanolic extract of Berberis vulgaris L. roots and its main compound, berberine against protoscoleces of hydatid cysts. METHODS: For this purpose, protoscoleces were aseptically aspirated from sheep livers having hydatid cysts. Various concentrations of the methanolic extract (0.25-2 mg/ml) and berberine (0.062- 0.5 mg/ml) were used for 5 to 30 min. Viability of protoscoleces was confirmed by eosin exclusive test. RESULTS: In the present study, all of the various concentrations of the B. vulgaris methanolic extract (0.25, 0.5, 1 and 2 mg/ml) and berberine (0.062, 0.125, 0.25 and 0.5 mg/ml) revealed significant (P<0.05) scolicidal effects against protoscoleces of E. granulosus in a dose-dependent manner. Both berberine and methanolic extract exhibited 100% inhibition against protoscoleces of E. granulosus at the concentration of 2.0 and 0.5 mg/ml after 10 min incubation, respectively. CONCLUSION: According to the results, both B. vulgaris methanolic extract and berberine alone demonstrated high scolicidal activities against protoscoleces of hydatid cysts in low concentration and short exposure time on in vitro model. However, in vivo efficacy of B. vulgaris and berberine also requires to be evaluated using an animal model with hydatid infection.