A morphology-based method for the diagnosis of red blood cells parasitized by Plasmodium malariae and Plasmodium ovale.

BACKGROUND: The morphology of red blood cells (RBCs) is altered significantly during the maturation stages of malaria parasites, which include ring, trophozoite, and schizont. There is dissimilarity in terms of the morphological characteristics of parasitized RBCs infected by the 4 species of Plasmodium, including falciparum, vivax, malariae, and ovale. This makes the process of diagnosis very difficult, which may lead to a wrong treatment method and substantial damage to the health of the patient. An innovative technique in introduced that accurately defines the shape of parasitized RBCs at each stage of infection as a potential method of diagnosis. METHODS: Giemsa-stained thin blood films were prepared using blood samples collected from healthy donors as well as patients infected with P. malariae and P. ovale. The diameter and thickness of healthy and infected RBCs at each stage of infection were measured from their optical images using Olysia and Scanning Probe Image Processor (SPIP) software, respectively. A shape equation was fitted based on the morphological characteristics of RBCs, and their relative 2-dimensional shapes were plotted using Wolfram Mathematica. RESULTS: At the ring stage, the thicknesses of RBCs parasitized by P. malariae (Pm-RBCs) and P. ovale (Po-RBCs) increased by 42% and 51%, respectively. Both Pm-RBCs and Po-RBCs remained nearly biconcave throughout parasite development even though their volumes increased. CONCLUSIONS: It is proposed that the morphology-based characterization technique introduced here could be used to intensify the accuracy of the Giemsa staining diagnosis method for the detection of the Plasmodium genus and infection stage. Based on the significant morphological alterations induced by different Plasmodium species, the results may also find practical use for faster prediction and treatment of human malaria.

Induction of Artesunate Resistance in Plasmodium falciparum 3D7 Strain Using Intermittent Exposure Method and Comparing P.fk13 Sequence between Susceptible and Resistant Strains.

Background: Resistance to artemisinin has threatened major achievements in malaria control, more investigations is needed about resistant strains and related genes. We aimed to induce resistance to artesunate in the Plasmodium falciparum 3D7 strain using intermittent exposure method and comparing P.fk13 gene sequence between susceptible and resistance strains. Methods: P. falciparum 3D7 strain was cultured according to Trager & Jensen method with some modifications. Serial concentrations between 10-2 mol/l, to 10-7mol/l were prepared, then P. falciparum 3D7 was exposed to each of the dilution to determine IC50 and lethal dose. Sensitivity reduction process was started from the concentration of 10-7mol/l and ended at 10-2mol/l. Exposed parasites were collected after at least 27 days after cultivation in each drug concentration. DNA extraction, PCR and sequencing process were performed to investigate any possible mutations in the P.fk13 gene sequence. Results: Effectiveness of 10-2mol/l concentration of artemisinin was found as a lethal dose. IC50 value was equal to 5×10-4 mol/l. The resistant strain was provided in the lab, sequenced and registered in the gene bank as P.f Art -2, (accession number MH796123. 1). Alignment of this registered sample showed no mutation in P.f kelch13 gene in comparison with standard strain submitted in the GenBank. Conclusion: Resistance to artesunate in malaria parasite may occur but with no mutation in the P.f kelch13 gene. Therefore, whole genome sequencing should be applied to determine mutations in resistant strains.

A recombinant Plasmodium vivax apical membrane antigen-1 to detect human infection in Iran.

In Iran, Plasmodium vivax is responsible for more than 80% of the infected cases of malaria per year. Control interventions for vivax malaria in humans rely mainly on developed diagnostic methods. Recombinant P. vivax apical membrane antigen-1 (rPvAMA-1) has been reported to achieve designing rapid, sensitive, and specific molecular diagnosis. This study aimed to perform isolation and expression of a rPvAMA-1, derived from Iranian patients residing in an endemic area. Then, the diagnostic efficiency of the characterized Iranian PvAMA-1 was assessed using an indirect ELISA method. For this purpose, a partial region of AMA-1 gene was amplified, cloned, and expressed in pET32a plasmid. The recombinant His-tagged protein was purified and used to coat the ELISA plate. Antibody detection was assessed by indirect ELISA using rPvAMA-1. The validity of the ELISA method for detection of anti-P. vivax antibodies in the field was compared to light microscopy on 84 confirmed P. vivax patients and compared to 84 non-P. vivax infected individuals. The ELISA cut-off value was calculated as the mean+2SD of OD values of the people living in malaria endemic areas from a south part of Iran. We found a cut-off point of OD=0.311 that showed the best correlation between the sera confirmed with P. vivax infection and healthy control sera. A sensitivity of 81.0% and specificity of 84.5% were found at this cut off titer. A good degree of statistical agreement was found between ELISA using rPvAMA-1 and light microscopy (0.827) by Kappa analysis.

Interaction between Chitosan and Chloroquine against Plasmodium berghei and P. falciparum Using In-Vivo and In-Vitro Tests.

BACKGROUND: The use of antimalarial drugs with number of compounds in combination form may potentiate each other's activity. METHODS: This study was conducted in the School of Public Health, Tehran University of Medical Sciences, Tehran, Iran in 2018. It was based on two methods including in vivo and in vitro tests with aim of considering interaction between chitosan and chloroquine against Plasmodium berghei and P. falciparum parasites using different ratios of the agents with ED50s and IC50s baselines. RESULTS: Administrating 10 and 20 mg/kg (mouse body weight) of chitosan alone to the P. berghei -infected mice up to 4 successive days resulted in 37% and 45% inhibition of P. berghei respectively, while employing the compound with chloroquine in combination form with ratios of 90/10 and 70/30 (chloroquine/chitosan) had a considerable potentiation including 71.58% and 83.85% inhibition effectiveness against P. berghei. Moreover, 20 mg/L (CCM) concentration of chitosan alone could eliminate 69.55% of P. falciparum in culture medium while in combination with chloroquine in ratios of 90/10 (chloroquine/chitosan) had considerable potentiation including 79.14% inhibition effectiveness. Mean survival time of those mice received combination therapy in ratios of 90/10 and 70/30 (chloroquine/chitosan) was longer than those took up mono therapy of either chloroquine or chitosan based on their ED50s doses. CONCLUSION: Interaction between chloroquine and chitosan showed considerable potentiation in combination form against either P. berghei or P. falciparum using in vivo and in vitro tests respectively. Meanwhile, interaction between the above mentioned agents resulted in a notable survival time for those P. berghei-infected mice treated with the combination.

Application of High-Resolution Melting (HRM) Technique towards the Detection of Asymptomatic Malaria in a Malaria Endemic Area of Southeastern Iran under Elimination Program.

BACKGROUND: Asymptomatic malaria, which usually exists in low parasitemia, acts as the Plasmodium species reservoirs contributing towards malaria transmission. This situation hinders malaria elimination programs in endemic areas, thus necessitating an active case detection with a high sensitive method and treatment of cases. This is why we used a High Resolution Melting (HRM) assay to monitor the trend of asymptomatic malaria in a malaria endemic area of Iran which is under elimination program. METHODS: The peripheral blood was sampled from 271 clinically approved non-febrile individuals from a malaria endemic zone of southeastern Iran for asymptomatic malaria prevalence detection by microscopy, Rapid Diagnostic Tests (RDTs) and HRM methods. The HRM assay was done based on the amplification of 18S SSU rRNA gene. RESULTS: The HRM assay revealed infections from three individuals out of 271 (1.1% asymptomatic malaria prevalence) from the participants, two Iranian natives with Plasmodium vivax infection and one Pakistani immigrant with P. falciparum infection. Neither microscopy nor RDTs detected Plasmodium spp infections from the 271 non-febrile individuals. The nucleotide sequencing analysis of the positive controls used in this study showed a close homology with the reference gene bank sequences of P. falciparum 3D7 (CPO16995.1) and P. vivax Sal-1(UO3079.1). CONCLUSION: This study revealed a low frequency of asymptomatic malaria trend within malaria endemic areas of southeastern Iran which are under intense elimination program and also the ability of HRM assay in detecting low Plasmodium spp parasitemia beyond the limits of microscopy and RDTs.

Isolation of Balamuthia mandrillaris from urban dust, free of known infectious involvement.

The free-living amoeba Balamuthia mandrillaris can cause fatal encephalitis in humans and other mammals. The organism is associated with soils, and soil exposure has been identified as a risk factor for this pathogen. However, B. mandrillaris has been isolated only once from soils believed to be the source of the infection in child from California, USA who died of Balamuthia amoebic encephalitis and once from another unrelated soil source. We report for a third time the isolation of B. mandrillaris from the environment and for the second time its isolation from a sample not known to be involved with pathogenicity. We have established the new clonal B. mandrillaris strain (ID-19) in axenic media. The identity of our isolate was originally by morphology using a light microscope and this has been confirmed by 16S rRNA gene PCR. The new strain ID-19 groups with others of the species. The fact that our isolate came from dust particles deposited on surfaces from the air in an urban environment may suggest that it is not just soil exposure that constitutes a risk factor for Balamuthia infection. This is the first report of this organism from Iran.

Delayed Hemolytic Anemia after Treatment with Artesunate: Case Report and Literature Review.

BACKGROUND: In recent years, few cases of post-artemisinin delayed hemolysis have been reported. OBJECTIVE: All cases of post-artemisinin delayed hemolysis were reported from non-middle east areas. No case of post-artemisinin delayed hemolysis has yet been reported from this region. In this paper, we describe a case of post-artemisinin delayed hemolysis in an Iranian female. Moreover, previous reports have been reviewed. METHODS: Patient's data including demographic characteristics, past medical, drug and travelling history, present illness, vital signs, laboratory data, clinical course of current illness and follow-up findings were considered. RESULTS: A 27-year-old female with a recent travel history to Ghana admitted with severe falciparum malaria. She was successfully treated with parenteral artesunate. However after 12 days of post artesunate treatment, she returned with dark urine, malaise and fatigue. CONCLUSION: Considering the clinical course and base on a reliable causality assessment scale, post artesunate delay hemolytic anemia was possible.

Genetic polymorphism of Plasmodium vivax Duffy Binding Protein in malarious areas in southeastern of Iran.

Plasmodium vivax parasite causes the largest number of malaria infection in some malarious areas of the world including Iran. Considering transfer and genetic dynamics of the parasite population in a specific area can help us to predict the spread of the infection either emergence of new cases or drug resistance in the context of elimination program in the malarious areas. Study on the genetic diversity of common alleles in a given geographical area, for vaccine and immune level studies can be important. The purpose of this study was to know the status of P. vivax Duffy Binding Protein (PvDBP) polymorphism in patients infected with the parasite in malaria endemic southeastern Iran. The fragment of gene corresponding to PvDBP of thirty P. vivax malaria infected individuals was amplified. A 1176 bp band related to this fragment was purified and PCR-RFLP method was employed using enzymatic digestion with PstI and RsaI restriction enzymes. Ten percent of samples were sent for sequencing. PCR-RFLP showed that 99.7% of the samples were cut as the same together, either the PstI enzyme or the enzyme of RsaI. In each case, only 2 isolates were unlike others. Findings revealed that there is at least 96% identity among isolates in the nucleotide level. Amino acid pattern of PvDBP in Iranian isolates showed little discrepancies with those PvDBP genes that have been recorded in GenBank. Sequencing of PvDBP isolates of Iranian P. vivax infected patients showed low level of genetic polymorphism among them. Results of this study can prepare valuable information for malaria policy makers to intend them in their malaria control program.

Antimalarial and cytotoxic activities of roots and fruits fractions of Astrodaucus persicus extract.

OBJECTIVES: Astrodaucus persicus (Apiaceae) is one of the two species of this genus which grows in different parts of Iran. Roots of this plant were rich in benzodioxoles and used as food additive or salad in Iran and near countries. The aim of present study was evaluation of antimalarial and cytotoxic effects of different fractions of A. persicus fruits and roots extracts. MATERIALS AND METHODS: Ripe fruits and roots of A. persicus were extracted and fractionated by hexane, chloroform, ethyl acetate and methanol, separately. Antimalarial activities of fractions were performed based on Plasmodium berghei suppressive test in mice model and percentage of parasitemia and suppression were determined for each sample. Cytotoxicity of fruits and roots fractions were investigated against human breast adenocarcinoma (MCF-7), colorectal carcinoma (SW480) and normal (L929) cell lines by MTT assay and IC50 of them were measured. RESULTS: Hexane fraction of roots extract (RHE) and ethyl acetate fraction of fruits extract (FEA) of A. persicus demonstrated highest parasite inhibition (73.3 and 72.3%, respectively at 500 mg/kg/day) which were significantly different from negative control group (P<0.05). In addition, RHE showed potent anticancer activities against MCF-7 (IC50 of 0.01 µg/ml), SW480 (IC50 of 0.36 µg/ml) and L929 (IC50 of 0.70 µg/ml) cell lines. CONCLUSION: According to the results, RHE and FEA fractions of A. persicus could be introduced as excellent choice for antimalarial drug discovery. In addition, cytotoxic activity of RHE was noticeable.

Determination of the axial and circumferential mechanical properties of the skin tissue using experimental testing and constitutive modeling.

The skin, being a multi-layered material, is responsible for protecting the human body from the mechanical, bacterial, and viral insults. The skin tissue may display different mechanical properties according to the anatomical locations of a body. However, these mechanical properties in different anatomical regions and at different loading directions (axial and circumferential) of the mice body to date have not been determined. In this study, the axial and circumferential loads were imposed on the mice skin samples. The elastic modulus and maximum stress of the skin tissues were measured before the failure occurred. The nonlinear mechanical behavior of the skin tissues was also computationally investigated through a suitable constitutive equation. Hyperelastic material model was calibrated using the experimental data. Regardless of the anatomic locations of the mice body, the results revealed significantly different mechanical properties in the axial and circumferential directions and, consequently, the mice skin tissue behaves like a pure anisotropic material. The highest elastic modulus was observed in the back skin under the circumferential direction (6.67 MPa), while the lowest one was seen in the abdomen skin under circumferential loading (0.80 MPa). The Ogden material model was narrowly captured the nonlinear mechanical response of the skin at different loading directions. The results help to understand the isotropic/anisotropic mechanical behavior of the skin tissue at different anatomical locations. They also have implications for a diversity of disciplines, i.e., dermatology, cosmetics industry, clinical decision making, and clinical intervention.

Measurement of the axial and circumferential mechanical properties of rat skin tissue at different anatomical locations.

Skin tissue is not only responsible for thermoregulation but also for protecting the human body from mechanical, bacterial, and viral insults. The mechanical properties of skin tissue may vary according to the anatomical locations in the body. However, the linear elastic and nonlinear hyperelastic mechanical properties of the skin in different anatomical regions and at different loading directions (axial and circumferential) so far have not been determined. In this study, the mechanical properties during tension of the rat abdomen and back were calculated at different loading directions using linear elastic and nonlinear hyperelastic material models. The skin samples were subjected to a series of tensile tests. The elastic modulus and maximum stress of the skin tissues were measured before the incidence of failure. The nonlinear mechanical behavior of the skin tissues was also computationally investigated through a constitutive equation. Hyperelastic strain energy density function was calibrated using the experimental data. The results revealed the anisotropic mechanical behavior of the abdomen and the isotropic mechanical response of the back skin. The highest elastic modulus was observed in the abdomen skin under the axial direction (10 MPa), while the lowest one was seen in the back skin under axial loading (5 MPa). The Mooney-Rivlin material model closely addressed the nonlinear mechanical behavior of the skin at different loading directions, which can be implemented in the future biomechanical models of skin tissue. The results might have implications not only for understanding of the isotropic and anisotropic mechanical behavior of skin tissue at different anatomical locations but also for providing more information for a diversity of disciplines, including dermatology, cosmetics industry, clinical decision making, and clinical intervention.