Cytotoxicity and Anti-Plasmodium berghei Activity of Emodin Loaded Nanoemulsion.

Background: Malaria parasites cause a tremendous burden of disease in both the tropics and subtropics areas. Growing of drugs resistance in parasites is one of the most threats to malaria control. The aim of study was to investigate the anti-malarial activity of nano-emodin isolated from Rhamnus cathartica on Plasmodium berghei in mice to evaluate parasites inhibition rate using in-vivo test. Methods: The study was conducted in the School of Public Health, Tehran University of Medical Sciences, during 2020. Nano-emodin particles were prepared from Rhamnus cathartica, and confirmed by Zeta Potential Analyzer, DLS and electron microscopy techniques. Mice were infected with P. berghei and treated by emodin nano-particles. Parasitemia was evaluated in each group in comparison with control group. Toxicity test was done using twice the highest concentration of emodin extract on a separate group of mice and ED50 was calculated. Results: Emodin extract was significantly effective in all concentrations on D4 (P<0.05). The most effective on parasitemia was observed in 400 mg/kg of Liquid Nano-emodin and solid (non-Nano) emodin. ED50 for emodin extract was determined 220 mg/kg. Toxicity test showed no toxic effect on the subjects. Conclusion: The emodin extract is safe, lack of side effects. So, it can be used for more and longer period of time and in higher doses. Emodin extract, either in form of liquid and nanoparticle or in a solid form, has the same therapeutic effect on P. berghei in infected Balb/c mice.

Detection of Malaria Parasites and Other Haemosporidia in Migratory and Native Birds in Mazandaran and Golestan Provinces, Iran.

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.

Serological responses to a soluble recombinant circumsporozoite protein-VK210 of Plasmodium vivax (rPvCSP-VK210) among Iranian malaria patients.

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.

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.

Genotyping and Phylogenetic Analysis of Plasmodium vivax Circumsporozoite Protein (PvCSP) Gene of Clinical Isolates in South-Eastern Iran.

BACKGROUND: Circumsporozoite protein (CSP) is one of the most important surface sporozoite antigens in malaria, recently considered as a candidate for vaccination. Considering the importance of CSP, this study was conducted to investigate the polymorphism and genetic diversity of Plasmodium vivax Circumsporozoite Protein (Pvcsp) in the southeastern region of Iran during 2015-2016. METHODS: To investigate polymorphism and genetic diversity, 20 blood samples were collected from patients with P. vivax, then DNA was extracted and amplified using partial sequence of CSP gene. Polymerase chain reaction (PCR) products were sequenced and compared to sequences from genomic databases using BLAST. Genetic evaluation and phylogenic analysis were performed using MEGA7 and DnaSP5 software's on 38 sequences include 20 sequences of our study and 18 sequences of Gene Bank. RESULTS: Eleven isolates were VK210 genotype and 9 isolates contained VK247. The result of variable segregation nucleotide site indicated that the differentiation of sequences in CSP were 25.67% in our 20 samples which are less than the 38 samples with a value of 26.67%. Comparing the ratio of dN/dS regions in the CSP gene indicates that the CSP varies more synonymously and amino acid has lower variation. Out of 38 samples, 35 unique haplotypes were identified based on 1042 nucleotide sequences in CSP, showing a variation percentage of 99.4%. CONCLUSION: The Tajima D analyses showed that CSP gene in P. vivax had a positive number in the total analyzed sequences, which means that the P. vivax mutations are in order to select positive evolution.

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.

Situation of Asymptomatic Malaria among Iranian Native and Afghan and Pakistani Immigrants in a Malarious Area under the National Malaria Elimination Program of Iran.

BACKGROUND: This study was designed to detect, if there are asymptomatic malaria infections amongst native and immigrant population from Afghanistan and Pakistan countries in Sistan & Baluchistan Province of Iran, where is under the national malaria elimination program. METHODS: This cross-sectional study was performed among native individuals and resident immigrants in the southeastern province of Sistan & Baluchistan from May 2016 to Jul 2017. A total of 271 individuals were considered in this cross-sectional study based on microscopical method, Rapid Diagnostic Tests (RDTs) and PCR techniques. Out of 271 native and immigrant participants 140 (52%) and 131 (48%) were male and female, respectively. RESULTS: None of the prepared samples was diagnosed as malaria positive case when was considered via above mentioned three techniques. CONCLUSION: Neither native nor immigrant individuals had asymptomatic malaria, hinting that national malaria elimination program is performed according to planned schedule in the studied areas.

Identification of Alleles in the MSP1 Gene Related to Complicated Malaria in Patients Infected with Plasmodium falciparum in Southeast of Iran.

BACKGROUND: To overcome human malaria problem several solutions have been employed including extensive studies in the field of Plasmodia relevant antigens. The aim of this study was to determine allelic variation in the MSP1 gene of Plasmodium falciparum among some falciparum malaria-infected patients in Southeastern Iran. METHODS: Twenty P. falciparum positive cases were enrolled from Sistan and Baluchistan Province, southeastern Iran in 2013-15. From each case, 1.5ml of peripheral blood was collected into EDTA contained tubes. Thick and thin blood smears were stained with standard Giemsa stain and were checked with conventional microscopical method. DNA was extracted from blood samples and amplification of block 2 MSP1 was performed using specific primers. Gel electrophoresis was done and results showed some amplification fragments corresponding to block 2 regions of Pf MSP1 gene. Finally, four samples from different allelic types were sent for sequencing process. RESULTS: Fragments were different in size, so classified into six allelic types as kinds of 1-6 based on happening frequencies. Digestion of PCR products revealed two sub allelic types (A and B) within allelic types 2 and 3, but not in allelic types 1, 4, 5 and 6. Twenty percent of samples were sent for sequencing. Sequence alignment showed 78.95% to 91.83% identity between samples. CONCLUSION: Identity between samples and phylogenetic tree revealed that there is an extensive diversity range among isolates. Fifty percent of the isolates were under the risk of complicated malaria. Two of these patients (10%) needed special care and recovery was obtained after getting hospital services.

Idiopathic granulomatous mastitis: Looking for the most effective therapy with the least side effects according to the severity of the disease in 374 patients in Iran.

The goal of this study is to compare the response rate and the recurrence rate of available therapeutic modalities in the treatment of Idiopathic Granulomatous Mastitis (IGM). 374 patients with pathologically confirmed IGM, were included. They were subdivided into three levels of severity. Close observation had the best response rate with the lowest recurrence rate in mild to moderate cases. Severe cases were mostly treated by prednisolone or underwent surgery. The outcome of prednisolone use in severe cases was comparable to NSAIDs. Overall 9% were resistant to treatment and surgical intervention is still an option among them.

In Vivo Anti-Malarial Activity of Heracleum persicum Fruit Extract, in Combination with Chloroquine against Chloroquine-Sensitive Strain of Plasmodium berghei.

BACKGROUND: We evaluated the anti-malarial activity of Heracleum persicum individually and in combination with chloroquine. METHODS: This study was conducted at the School of Public Health, Tehran University of Medical Sciences, Tehran, Iran in 2015-2016. The Peter's method was used for determining fifty percent effective dose (ED50) of the H. persicum extract and chloroquine individually against chloroquine sensitive P. berghei in small white mice. Six experimental groups for H. persicum and 6 groups for chloroquine and two control group (positive and negative) were considered for determination of ED50. Interaction between H. persicum and chloroquine also was evaluated based on fixed ratios method. Ratios of 0/100, 20/80, 40/60, 60/40, 80/20, 100/0 of ED50 of chloroquine and H. persicum respectively were tested against the parasite. Then inhibitory effects of two drugs were calculated and plotted in the relevant graphs. RESULTS: Overall, 1500 mg/kg, and 1000 mg/kg concentrations of H. persicum against P. berghei resulted in ED50 and ED74 respectively. ED50 of chloroquine against the parasite was obtained as 1.4 mg/kg of mouse body weight. Moreover, combination of H. persicum and chloroquine showed a weak potentiation in ratios of 40/60 (chloroquine +H. persicum) with 64% inhibition, but not in other ratios. CONCLUSION: Although H. persicum individually showed a reasonable antimalarial efficacy against chloroquine sensitive P. berghei, in combination with chloroquine it showed additive or antagonism result except in ratios of 40%CQ+60%HP.

Monitoring the Response of Plasmodium vivax to Chloroquine and Uncomplicated P. falciparum to Artesunate-fansidar Antimalarials in Southeastern Iran.

BACKGROUND: For many years, malaria was a major life-threatening parasitic infection in Iran. Although malaria elimination program is implementing in the country, still some cases annually are reported from malaria-endemic areas. METHODS: This study was conducted in five malaria endemic districts of Sistan and Baluchistan Province, southeastern Iran, neighboring Afghanistan and Pakistan countries. Overall, 170 and 38 vivax malaria and falciparum malaria infected patients were enrolled in the study from 2013-2014. All the cases were selected according to criteria of the WHO guideline for in vivo drug sensitivity tests in malaria parasites. Evaluation of drug sensitivity test was conducted with some modifications. RESULTS: The patients with vivax malaria responded to the regimen of chloroquine in 37.4(±15.9), 40(±13.8) and 42(±17.7) h for Pakistani, Iranian and Afghani nationalities respectively based on MPCT evaluation. The results showed a considerable difference between them and Iranian subjects. MPCT for the patients with falciparum malaria was calculated as 28(±18.05), 26(±12.03) and 36(±16.9) h for Iranian, Pakistani and Afghani nationalities respectively. There was a marginally significant difference between Afghani and other nationalities and between males and females. CONCLUSION: Treatment of all the patients resulted in ACPR and MPCT of P. vivax showed that the parasite became more sensitive to chloroquine than previous years in studied areas.

Experimental Study on Plasmodium berghei, Anopheles Stephensi, and BALB/c Mouse System: Implications for Malaria Transmission Blocking Assays.

BACKGROUND: Plasmodium berghei is a rodent malaria parasite and has been very valuable means in the progress of our understanding of the essential molecular and cellular biology of the malaria parasites. Availability of hosts such as mice and vectors such as Anopheles stephensi has made this parasite a suitable system to study the parasite-host and vector-parasite relationships. METHODS: This study was performed at Medical Entomology and Parasitology laboratories of the School of Public Health, Tehran University of Medical Sciences, Iran in 2016. The investigation was carried out to describe life cycle and parameters influencing maintenance of the parasite within the mice or the mosquito. RESULTS: Results have revealed details and addressed some parameters and points influence maintenance of various life stages of the parasite including merozoites, macrogametocytes, ookinetes, oocysts and sporozoites in the laboratory model P. berghei-A. stephensi-BALB/c mouse. Injection of fresh infected blood results in higher gametocytemia in the animals. The more injected parasites result in earlier and higher parasitemia and exfelagellation centers in the mice blood. However, the highest number of infected mosquitoes and oocysts formation were observed when the parasitemia and exflagellation centers per microscopic field were 9% and 3.6 in the infected mice respectively. The infected mosquitoes should be maintained on 8% (w/v) fructose, 0.05% (w/v) PABA at 20±1 °C and 50%-80% relative humidity. CONCLUSION: This study helps to understand the biology of vertebrate-parasite and mosquito-malaria interactions that may aid in the development of a new generation of drug/vaccine and vector-based measures for malaria control.

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.

Antimalarial efficacy of low molecular weight chitosan against Plasmodium berghei infection in mice.

BACKGROUND & OBJECTIVES: Despite continuous global attempts to fight parasitic infections, malaria still remains one of the major human life threatening diseases. Difficulty of producing efficient antimalaria vaccines and increasing drug-resistant strains, highlight the urgent need to search for a new alternative antimalaria drug. The aim of this study was to find a new agent against malaria parasite with maximum efficacy and minimum range of side-effects. For this, the antiplasmodial activity of commercial chitosan, a natural carbohydrate polymer, was evaluated on Plasmodium berghei via in vivo experiments. This is the first report that to highlight antimalarial effects of low molecular weight chitosan against P. berghei in vivo. METHODS: Low molecular weight chitosan with 95% degree of deacetylation was melted in normal saline with 1% (w/v) acetic acid for preparing 10, 20, 40 and 80 mg/kg concentrations of chitosan, which were then examined for their antimalarial efficacy in P. berghei infected mice. RESULTS: The study showed that differrent concentrations of chitosan exhibited significant antimalarial effect (p= 0.002) when compared with the control group. Also, analysis of mice survival time showed significant differences between 20 and 80 mg/kg concentrations of used chitosan in comparison to negative control group. INTERPRETATION & CONCLUSION: The results of this study showed that the chitosan has potent antimalarial activity and could be suggested as an alternative antimalarial drug component.

Lectin-carbohydrate recognition mechanism of Plasmodium berghei in the midgut of malaria vector Anopheles stephensi using quantum dot as a new approach.

Potential targets of Plasmodium ookinetes at the mosquito midgut walls were investigated in relation to interfering malarial transmission. In this study, the essential application of Quantum Dots (QDs) was used to examine the interaction between Plasmodium berghei ookinetes and the Anopheles stephensi midgut, based on lectin-carbohydrate recognition. Two significant lectins were utilized to determine this interaction. Two QDs, cadmium telluride (CdTe)/CdS and cadmium selenide (CdSe)/CdS, were employed in staining Plasmodium ookinete to study its interaction in the midgut of the mosquito vector in vivo. Concurrently, two lectins, wheat germ agglutinin (WGA) and concanavalin A (Con A), were inadvertently exploited to mask lectin binding sites between ookinetes and mosquito midgut cells. The numbers of ookinetes in both lumen and epithelial cells were eventually counted, following adequate preparation of wax sections extracted from whole midgut, and subsequent examination using a differential interference contrast a fluorescence microscopic technique. Interestingly, we detected that neither of the QDs mutated ookinete invasion into the midgut cells of the investigated mosquitoes. QD staining of ookinetes remained permanent despite the effective embedding procedure. The massive binding potency of ookinetes to midgut cells of the cross-examined mosquitoes undoubtedly revealed that Con A did not interrupt ookinete penetration into the midgut wall. In contrast, WGA inhibited ookinete invasion into the midgut cells. The results proved that QD nanoparticles are biocompatible, non-toxic to P. berghei and stable to photobleaching. The QDs staining, which was successfully implemented for ookinete labelling, is a simple and effective tool which plays a crucial role in bioimaging including the study of parasite-vector interactions.

Determination of Asymptomatic Malaria among Afghani and Pakistani Immigrants and Native Population in South of Kerman Province, Iran.

BACKGROUND: This study was proposed to monitor the situation of asymptomatic malaria among the native population and Afghani and Pakistani immigrants in Kahnooj and Ghale-Ganj districts from Kerman Province, Southeastern Iran. METHODS: A number of 180 and 120 individuals from Kahnooj and Ghale-Ganj respectively were registered and considered based on a cross-sectional surveillance method. From 300 registered cases, 200 individuals (66.7%) were selected among Afghani and Pakistani immigrants and the rest (33.3%) were native resident individuals. All samples were processed with employing microscopical examination, Rapid Diagnostic Tests (RDTs) and Semi- nested Multiplex PCR techniques. RESULTS: None of the samples collected from native residents showed any malaria parasite, but among Afghani immigrants, one asymptomatic vivax malaria was detected in a 12 yr old girl with 280 parasites per microliter of blood. Moreover, one symptomatic vivax malaria was detected from a Pakistani immigrant with 47560 parasites per microliter of blood. All results obtained via microscopical method, confirmed by RDTs and PCR techniques. CONCLUSION: To achieve the malaria elimination program different studies are needed that to be performed. Monitoring the asymptomatic malaria in all over the malaria endemic areas especially among the immigrant individuals is the most crucial necessity.

Endoparasites of Wild Rodents in Southeastern Iran.

BACKGROUND: This study was aimed to collect wild rodents for endoparasites determination in some parts of Sistan and Baluchistan Province, southeastern Iran nearby Pakistan and Afghanistan countries. METHODS: A total of 100 wild rodents were captured alive with cage traps. Various samples were collected from blood and feces, also impression smear prepared from different organs. The samples were prepared by formalin-ether or stained with Giemsa, after that were examined under microscope. RESULTS: All the caught rodents (47 Tatera indica, 44 Meriones hurriana, 5 Gerbilus nanus and 4 Meriones libycus) were studied for endoparasites emphasizing to their zoonotic aspects. Endoparasites including Spirurida, Hymenolepis diminuta, Hymenolepis nana feraterna, Trichuris trichiura, Skerjabino taenia, Trichostrongylus spp, Entamoeba muris, Chilomastix mesnili and Leishmania spp were parasitologically identified. CONCLUSION: Among 9 genera or species of the identified parasites at least 5 of them have zoonotic and public health importance.

DNA Sequence Polymorphism of the Lactate Dehydrogenase Genefrom Iranian Plasmodium vivax and Plasmodium falciparum Isolates.

BACKGROUND: Parasite lactate dehydrogenase (pLDH) is extensively employed as malaria rapid diagnostic tests (RDTs). Moreover, it is a well-known drug target candidate. However, the genetic diversity of this gene might influence performance of RDT kits and its drug target candidacy. This study aimed to determine polymorphism of pLDH gene from Iranian isolates of P. vivax and P. falciparum. METHODS: Genomic DNA was extracted from whole blood of microscopically confirmed P. vivax and P. falciparum infected patients. pLDH gene of P. falciparum and P. vivax was amplified using conventional PCR from 43 symptomatic malaria patients from Sistan and Baluchistan Province, Southeast Iran from 2012 to 2013. RESULTS: Sequence analysis of 15 P. vivax LDH showed fourteen had 100% identity with P. vivax Sal-1 and Belem strains. Two nucleotide substitutions were detected with only one resulted in amino acid change. Analysis of P. falciparum LDH sequences showed six of the seven sequences had 100% homology with P. falciparum 3D7 and Mzr-1. Moreover, PfLDH displayed three nucleotide changes that resulted in changing only one amino acid. PvLDH and PfLDH showed 75%-76% nucleotide and 90.4%-90.76% amino acid homology. CONCLUSION: pLDH gene from Iranian P. falciparum and P. vivax isolates displayed 98.8-100% homology with 1-3 nucleotide substitutions. This indicated this gene was relatively conserved. Additional studies can be done weather this genetic variation can influence the performance of pLDH based RDTs or not.

Diagnosing Malaria Cases Referred to the Malaria Reference Laboratory in Tehran University of Medical Science, Iran.

BACKGROUND: The number of malaria cases is declining worldwide; however, it remains as a serious health problem. Diagnosing unusual cases is the most important issue to manage the problem. This study designed to describe the number of falciparum and vivax malaria infected patients referred to Malaria Reference Laboratory in Tehran University of Medical Science from 2000 to 2012. METHODS: A retrospective study was conducted based on the collected questionnaires from each patient referred to the laboratory. Diagnosing results and demographic information for positive cases were analyzed using SPSS software. Problematic cases were evaluated for any difficulties in diagnosis or in clinical signs. Scanning and molecular methods were performed whenever there was an atypical case referred to the laboratory. Some of the samples had various difficulties for diagnosing such as presence of fussed gametocytes and schizonts of Plasmodium falciparum in peripheral blood and CCHF like hemoragic disorders. RESULTS: Plasmodium vivax caused a large proportion of the cases (76.1%) in contrast with P. falciparum that included smaller proportion (22.8%) and the rest (1.1) belonged to mixed infection. Most of the positive cases (69.6%) were belonged to Afghani people. Men (94.6%) showed more infection than women (5.4%), moreover the most infection (44.5%) was seen at a range of 21-30 yr. CONCLUSION: In the case of existing atypical issues to diagnose, it is needed to perform more precise microscopical examination beyond the current standard conditions. Sometimes molecular method is required to verify the exact agent of the disease.