Genetic diversity of merozoite surface protein-5 (MSP-5) of Plasmodium vivax isolates from Malaria patients in Iran.

Malaria has not yet been eradicated in Iran, and Plasmodium vivax (P. vivax) is the main cause of malaria in the country. This study aimed to investigate and analyze the amount of genetic diversity of Plasmodium vivax merozoite surface protein-5 (PvMSP-5) exon 1 gene in the southeast of Iran.Thirty-five patients with clinical symptoms of P. vivax malaria participated. The exon 1 of PvMSP-5 was amplified by PCR, and the PCR product of all isolates was sequenced, and genetic polymorphisms were determined using various genetic software.The analysis showed that studied isolates are different from one another in the DnaSP software version. Out of the 612 sites, 477 were monomorphic and 135 were segregated. The total number of mutations was 143. The singleton variable and the parsimony informative sites were 23 and 112, respectively. There were 17 specific haplotypes with haplotype diversity equal to 0.943. Nucleotide diversity was equal to 0.06766 in the isolates. The ratio of nonsynonymous (0.06446) to synonymous (0.07909) mutations was 0.815020. Tajima's D, which expressed coding, and non-coding regions, was 0.72403, which was not deemed significant (P > 0.10).The analysis of intrapopulation diversity revealed nucleotide and haplotype diversity in the msp-5 gene of Iranian P. vivax isolates. In addition to balancing or purifying selection, intragenic recombination also contributed to the variation observed in exon 1 of PvMSP-5, according to the findings.

Molecular Identification of Cutaneous Leishmaniasis Species in the Northcentral Iranian Province of Alborz: Is There a New Focus on Cutaneous Leishmaniasis in the Province?

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.

Associations between Toxoplasma gondii Infection and Multiple Sclerosis: A Case-Control Seroprevalence Study.

Background: Currently, there are conflicting reports on the associations between Toxoplasma gondii infection and multiple sclerosis (MS) in humans. In the present study, a case-control study was carried out to assess associations between seropositivity to T. gondii infection and MS. Methods: This case-control study was carried out on 200 MS patients (cases) attended in Sina Hospital affiliated to Tehran University of Medical Sciences, Tehran, Iran, and 200 healthy subjects from the general population of the same city, March to July 2017. Blood samples were collected from individuals and were examined using Enzyme-linked immunosorbent assay (ELISA) for the presence of T. gondii IgG antibodies and the IgG-positive samples were further analyzed for specific anti-T. gondii IgM. Results: The overall seroprevalence of anti-T. gondii IgG was 44.2% (177/400) in 121 (60.5%) sera of the 200 MS patients (cases) and 56 (28.0%) sera of the 200 controls (OR = 3.94; 95% CI: 2.59-5.99; P < 0.001). Seroprevalence of T. gondii infection in MS patients increased significantly with increasing of age (P < 0.001). In the control group, no statistically significant differences were seen between the seroprevalence of T. gondii infection in various age groups (P = 0.858). Moreover, no statistically significant relationships were reported between the seropositivity to T. gondii and the sex for the cases and controls (P>0.05). Anti-T. gondii IgM antibodies were not detected in anti-T. gondii IgG positive patients. Conclusion: T. gondii infection might be a probability risk factor for MS. However, further studies are necessary to describe clearly the roles of T. gondii infection in MS.

Unraveling the role of natural killer cells in leishmaniasis.

NK cells are known as frontline responders that are efficient in combating several maladies as well as leishmaniasis caused by Leishmania spp. As such they are being investigated to be used for adoptive transfer therapy and vaccine. In spite of the lack of antigen-specific receptors at their surface, NK cells can selectively recognize pathogens, accomplished by the activation of the receptors on the NK cell surface and also as the result of their effector functions. Activation of NK cells can occur through interaction between TLR-2 expressed on NK cells and. LPG of Leishmania parasites. In addition, NK cell activation can occur by cytokines (e.g., IFN-γ and IL-12) that also lead to producing cytokines and chemokines and lysis of target cells. This review summarizes several evidences that support NK cells activation for controlling leishmaniasis and the potentially lucrative roles of NK cells during leishmaniasis. Furthermore, we discuss strategies of Leishmania parasites in inhibiting NK cell functions. Leishmania LPG can utilizes TLR2 to evade host-immune responses. Also, Leishmania GP63 can directly binds to NK cells and modulates NK cell phenotype. Finally, this review analyzes the potentialities to harness NK cells effectiveness in therapy regimens and vaccinations.

Key plasma microRNAs variations in patients with Plasmodium vivax malaria in Iran.

Introduction: As the cause of RBC infection and splenomegaly, malaria remains a major parasitic disease in the world. New specific biomarkers such as MicroRNAs (miRNAs) are developed to accurately diagnose malaria and clarify its pathologic changes. This study aimed at evaluating changes in the plasma miRNAs markers of Plasmodium vivax in patients with malaria in Chabahar, Iran. Materials and methods: For the present descriptive-analytical study conducted in 2018, we collected blood samples from 20 individuals. Real-time quantitative Polymerase Chain Reaction (RT-qPCR) was used to measure the plasma levels of miR-145, miR-155, miR-191 and miR-223-3p. Results: The 2-ΔΔCT method of Real-time PCR showed the plasma levels of miR-223, miR-145 and miR-155 to respectively be 5.6, 16.9 and 1.7 times higher in patients with P. vivax compared to those in healthy individuals. The expressions of all the three miRNAs significantly increased in patients with malaria compared to in the controls (P < 0.05). The expression of miR-191 was 1.405 times higher in patients with malaria compared to that in the controls, although the difference was statistically insignificant. Conclusion: The present study found P. vivax to change host miRNAs such as miR-223, miR-145 and miR-155. These small molecules thus appeared to constitute biomarkers for P. vivax malaria assessment.

Prevalence of Cryptosporidium spp. in children and the elderly in southwestern Iran.

Aim: The current study investigated the prevalence of Cryptosporidium spp. among children under 6 and adults over 60 years of age with diarrhea in the southwest of Iran. Background: Cryptosporidiosis is an opportunistic parasitic infection caused by the species Cryptosporidium that causes gastrointestinal complications and diarrhea. Methods: This cross-sectional study was conducted in Khuzestan province between January 2020 to December 2020. Out of 350 patients referring to medical centers with clinical signs of diarrhea, 57.4% were under six years of age and 42.6% were more than 60 years old. Fecal samples were examined using Modified Ziehl-Neelsen (MZN) staining and nested-PCR techniques. Results: The overall prevalence of Cryptosporidium spp. infection in the study population was 0.9% as determined by microscopic and molecular methods (3/47). Conclusion: The study results confirm the prevalence of parasitic infections as reported in previous studies in other regions of Iran. Preventive health measures are necessary.

Anti-Leishmanial Activity of Artemisia persica, A. spicigera, and A. fragrance against Leishmania major.

BACKGROUND: Neglected tropical diseases (NTDs) like zoonotic cutaneous leishmaniasis (ZCL), is a widespread infectious disease with high mortality and morbidity. Various medications are used for treating the disease, but several side effects and drug resistance have been reported. Herbal medicines are unlimited sources for discovering new medications to treat infectious diseases. We aimed to determine the leishmanicidal activity of three species of Iranian Artemisia herbal plant extracts in in-vitro. METHODS: In-vitro anti-leishmanial activity of ethanolic extracts on both promastigotes and amastigotes was determined by using MTT method. IC50, CC50, EC50 and SI were calculated. The study was done in 2019-2020 in Iran University of Medical Sciences, Tehran, Iran. RESULTS: All of the three Artemisia species significantly reduced the number of parasite promastigotes. Among them, A. persica had the highest leishmanicidal activity against parasite promastigotes. Cytotoxicity assay elucidated that the Artemisia had no toxicity to the host cells, and killed the L. major amastigotes very efficiently. By increasing the dose of extracts, the parasite number in both phases (promastigotes and amastigotes) was reduced significantly. CONCLUSION: These results indicated satisfactory anti-leishmanial activity of Artemisia extracts against ZCL in-vitro. Accordingly, Artemisia ethanolic extracts might be considered as a strong, effective and safe herbal compound for clearing the L. major with less toxicity to the host macrophages cells. Hence, it may be recognized as an excellent herbal therapy for treating the ZCL.

The Drug Resistance of Plasmodium falciparum and P. vivax in Iran: A Review Article.

BACKGROUND: One of the main obstacles to malaria control in the world has been the emergence of resistance in Plasmodium falciparum to chloroquine and other anti-malarial drugs. This study aimed to review studies in Iran on resistance in P. falciparum and P. vivax to drugs, and to reveal the mechanisms and molecular markers of resistance of these two species. METHODS: The databases of PubMed, Scopus, Google Scholar, Magiran, and reputable Iranian journals were searched to find published studies on the resistance in P. falciparum and P. vivax to antimalarial drugs in Iran. RESULTS: There is a significant relationship between resistance to chloroquine in P. falciparum and the emergence of K76T mutation in the P. falciparum chloroquine-resistance transporter gene in Iran. Resistance to sulfadoxine-pyrimethamine (SP) in P. falciparum is also significantly associated with the development of mutations in the dihydrofolate reductase and dihydropteroate synthase genes. Resistance to chloroquine in P. vivax has not been reported in Iran and it is used as a first-line treatment for P. vivax malaria. CONCLUSION: P. falciparum has become resistant to chloroquine in different regions of Iran and is not currently used to treat malaria. Besides, cases have emerged of P. falciparum resistance to SP in different parts of southern Iran, and SP is not administered alone for treating P. falciparum.

Molecular and microscopic prevalence of intestinal microsporidia among HIV+/AIDS patients in the Alborz province, Iran.

BACKGROUND: Microsporidia are a large family of obligate intracellular protozoa; these medically important species are recognized as opportunistic agents in intestinal complications in HIV+/AIDS patients. METHODS: The current cross-sectional study was designed and conducted from October 2018 to June 2019 to determine intestinal microsporidia in HIV+/AIDS patients by trichrome/Zeihl-Neelsen staining and SYBR Green-based real-time PCR. RESULTS: Out of 80 HIV+/AIDS patients, 23.75% (n=19) and 12.5% (n=10) were identified by molecular and microscopic methods, respectively. The predominant species in patients was Encephalitozoon (94%), which was found by quantitative real-time PCR and its high resolution melting tool. CONCLUSION: As far as we know, this is the first report from the Alborz region. The prevalence of intestinal microsporidiosis in this area in HIV+/AIDS patients was higher than both the global and national average. In addition to the need for further studies to prove protozoan pathogenicity in the aforementioned group, preventive measures should be considered.

Seroepidemiological study of toxoplasmosis in women referred to a pre-marriage counseling center in Alborz Province, Iran.

OBJECTIVES: The aim of the current study was to assess prevalence of Toxoplasma infection and its associated risk factors in women of childbearing-age in central Iran. RESULTS: Of 400 serum samples assessed for anti-T. gondii antibodies, 81 (20.25%) samples were positive for anti-T. gondii antibodies, including 74 positive samples (91.3%) for anti-T. gondii IgG and seven positive samples (8.7%) for IgG and IgM. Of seven IgG and IgM positive samples, five and two samples were high and low in IgG avidity, respectively. Based on PCR analysis, Toxoplasma infection was detected in one sample with anti-T. gondii IgM and low IgG avidity. The Chi-square test showed significant correlations of T. gondii seropositivity with history of undercooked meat consumption and contacts with cats (p < 0.05). In the present study, 79.75% of the participants were negative for IgG against T. gondii infection. Furthermore, recently acquired Toxoplasma infection was found using IgG avidity and PCR assays among women of childbearing-age in the study area, which would increase the risk of their fetus becoming infected. Educational program and antenatal screening of childbearing-age women for T. gondii infection may be important primary prevention strategies and help reduce the risk of congenital toxoplasmosis in this population.

Molecular and Seroepidemiological Survey of Visceral Leishmaniasis in Owned Dogs (Canis familiaris) in New Foci of Rural Areas of Alborz Province, Central Part of Iran: A Cross-Sectional Study in 2017.

BACKGROUND: Mediterranean form of visceral leishmaniasis (VL) is endemic among some provinces of Iran. The present study was designed to determine the prevalence of canine visceral leishmaniasis (CVL) in the owned dogs of the rural areas of Alborz Province near Tehran as the capital of Iran. METHODS: This study conducted on 303 owned dogs that selected using a stratified random sampling method. The direct agglutination test (DAT) was used to determine the frequency of Vl. The spleen biopsy was taken from the serology-positive dogs for the confirmation of CVL in the suspected dogs. Nested PCR and sequencing methods were used to determine the type of Leishmania species in the dogs which were parasitological positive. RESULTS: Overall, the DAT results of 9 dogs (2.97%, CI: 1.57-5.55) showed anti Leishmania antibodies at titers ≥ 1:320 indicating VL infection. One dog (0.33%, CI 95%: 0.06-1.85) showed clinical signs and symptoms of VL. There was a significant correlation between the positive cases of CVL and rural area (p< 0.001). The Leishmania was observed in the impression smears that were prepared from spleen biopsy of five the studied dogs. Leishmania infantum were confirmed in all them using nested-PCR assay. The sequence analysis of all five isolates was 95% similar to L. infantum. CONCLUSION: This study shows that domestic cycle of L. infantum has been established in rural areas of Alborz province where located near Tehran as capital city of Iran. It is necessary to increase the awareness and monitoring of the disease periodically.

Evaluating the preventive and curative effects of Toxocara canis larva in Freund's complete adjuvant-induced arthritis.

Helminthic infection and the parallel host immune reactions are the results of a protracted dynamic co-interaction between the host and worms. An assessment of the effect of Toxocara canis infection on arthritis in rats stimulated by Freund's complete adjuvant (FCA) was the main purpose of the investigation. An arthritis model was established by the administration of 0.1 mL FCA in the palmar surface. Cytokine assessment, evaluating oedema and the use of a rheumatoid arthritis (RA) score provided evidence of the protective effects of T canis against adjuvant-induced arthritis (AIA). The cytokines TGF-ß, IFN-É£, IL-10 and IL-17 were measured to assess the anti-inflammatory effect of T canis infection. Besides, arthritis swelling findings were evaluated in rat paws. The data showed that T canis infection significantly modulated the immune response by alleviating inflammatory cytokines and increasing TGF-ß as an anti-inflammatory cytokine. Evaluations of arthritis swelling showed low severity and faster recuperation. These findings suggest that the products derived from T canis eggs might be a potential therapeutic candidate to treat autoimmune diseases like the arthritis.

Paediatric visceral leishmaniasis: a retrospective study on clinical manifestations, demographic features and laboratory findings of hospitalised cases in Iran between 2006 and 2016.

Visceral leishmaniasis (VL) is a neglected disease. Our retrospective study describes 38 clinical and epidemiological characteristics of VL in patients admitted to a paediatric hospital in Tehran, Iran, who came from different geographical regions, indicating that the disease has spread to most parts of the country. Some 76.3% of the children documented suffered with symptoms of the disease for two months before admission.

Genetic Diversity in C-terminal of SERA5 Gene in the Blood Stage of Human Isolates of Plasmodium vivax in Sistan and Baluchistan, Iran.

BACKGROUND: Vivax malaria is more prevalent in the malarious areas of Iran, which makes vaccine research a high priority. Serine Repeat Antigens (SERA) have essential role in the parasite life cycle and high expression profiles of PvSERA5 make it suitable vaccine candidates. This study aimed to evaluate the genetic diversity of C-terminal region of PvSERA5 in Iranian isolates of Plasmodium vivax in Sistan and Baluchistan. METHODS: Totally, 49 blood samples were taken from symptomatic malaria patients in Sistan and Baluchistan Province in 2016. Mono-infection to P. vivax was confirmed by 18srRNA-Nested-PCR. Genomic DNA was extracted and C-terminal region of PvSERA5 was amplified by specific primers. PCR-products have been sequenced and analysis was done by using bioinformatics software, mainly DnaSP & MEGA5. RESULTS: Genetic diversity was calculated 14.8% in C-terminal region of PvSERA5 in Iranian isolates, 19 different sequences and 4 haplotypes existed. The amount of Tajima's D (0.3805) and ratio of non-synonymous to synonymous mutation (1.82) showed that C-terminal region of PvSERA5 is under positive natural selection; also intragenic recombination could interfere. CONCLUSION: Results could be helpful in any research, regarding this antigen as vaccine candidate in Iran or worldwide.

The effect of chloroquine dose and primaquine on Plasmodium vivax recurrence: a WorldWide Antimalarial Resistance Network systematic review and individual patient pooled meta-analysis.

BACKGROUND: Chloroquine remains the mainstay of treatment for Plasmodium vivax malaria despite increasing reports of treatment failure. We did a systematic review and meta-analysis to investigate the effect of chloroquine dose and the addition of primaquine on the risk of recurrent vivax malaria across different settings. METHODS: A systematic review done in MEDLINE, Web of Science, Embase, and Cochrane Database of Systematic Reviews identified P vivax clinical trials published between Jan 1, 2000, and March 22, 2017. Principal investigators were invited to share individual patient data, which were pooled using standardised methods. Cox regression analyses with random effects for study site were used to investigate the roles of chloroquine dose and primaquine use on rate of recurrence between day 7 and day 42 (primary outcome). The review protocol is registered in PROSPERO, number CRD42016053310. FINDINGS: Of 134 identified chloroquine studies, 37 studies (from 17 countries) and 5240 patients were included. 2990 patients were treated with chloroquine alone, of whom 1041 (34·8%) received a dose below the target 25 mg/kg. The risk of recurrence was 32·4% (95% CI 29·8-35·1) by day 42. After controlling for confounders, a 5 mg/kg higher chloroquine dose reduced the rate of recurrence overall (adjusted hazard ratio [AHR] 0·82, 95% CI 0·69-0·97; p=0·021) and in children younger than 5 years (0·59, 0·41-0·86; p=0·0058). Adding primaquine reduced the risk of recurrence to 4·9% (95% CI 3·1-7·7) by day 42, which is lower than with chloroquine alone (AHR 0·10, 0·05-0·17; p<0·0001). INTERPRETATION: Chloroquine is commonly under-dosed in the treatment of vivax malaria. Increasing the recommended dose to 30 mg/kg in children younger than 5 years could reduce substantially the risk of early recurrence when primaquine is not given. Radical cure with primaquine was highly effective in preventing early recurrence and may also improve blood schizontocidal efficacy against chloroquine-resistant P vivax. FUNDING: Wellcome Trust, Australian National Health and Medical Research Council, and Bill & Melinda Gates Foundation.

Visceral Leishmaniasis in Rural Areas of Alborz Province of Iran and Implication to Health Policy.

Visceral leishmaniasis (VL) or kala-azar mainly affects children in endemic areas. This study was conducted to determine the seroprevalence of VL using direct agglutination test (DAT) in children living in rural districts of Alborz Province located 30 km from Tehran capital city of Iran. Multi-stage cluster random sampling was applied. Blood samples were randomly collected from 1,007 children under 10 years of age in the clusters. A total of 37 (3.7%) of the studied population showed anti-Leishmania infantum antibodies with titers of ≥1:800. There was a significant association between positive sera and various parts of the rural areas of Alborz Province (P<0.002). Two children with anti-Leishmania infantum antibodies titers of ≥1:3,200 indicated kala-azar clinical features and treated with anti-leishmaniasis drugs in pediatric hospital. The findings of this study indicated that Leishmania infection is prevalent in rural areas of Alborz Province. Therefore, it is necessary to increase the awareness and alertness among physicians and public health managers, particularly in high-risk rural areas of the province in Iran.

Detection of mixed-species infections of Plasmodium falciparum and Plasmodium vivax by nested PCR and rapid diagnostic tests in southeastern Iran.

Coexistence of two species of Plasmodium in a single host has disrupted the diagnosis and treatment of malaria. This study was designed to evaluate the ability of rapid diagnostic test (RDT) kits for the diagnosis of mixed-species malaria infections in southeastern Iran. A total of 100 malaria patients were included in the study out of 164 randomly suspected symptomatic malaria patients from May to November 2012. Nested polymerase chain reaction (PCR) was also used to judge the ability of microscopy versus RDT kits for detecting mixed species. The sensitivity of light microscopy for the detection of mixed-species malaria infections was 16.6% (95% confidence interval [CI] = 3-49.1). Nested PCR revealed 12 patients with mixed-species infection. The CareStart Pv/Pf Combo kit detected 58% of the mixed-species infections, which were determined by nested PCR (sensitivity = 58.3%; 95% CI = 28.5-83.5). For identifying P. falciparum, P. vivax, and mixed-species infections, the concordance rates (kappa statistics) of microscopy and CareStart Pv/Pf Combo kit with nested PCR were 0.76 and 0.79, respectively (P = 0.001). This study underlines the effectiveness of RDT kits to improve the differentiation of mixed-species malaria infections in endemic areas where the prevalence of chloroquine resistance is high.

Genetic Variation and Selection of Domain I of the Plasmodium vivax Apical Membrane Antigen-1(AMA-1) Gene in Clinical Isolates from Iran.

BACKGROUND: Apical Membrane antigen 1 (AMA-1) is positioned on the surface of merozoite and it may play a role in attack to red blood cells. The main aim of present study was to determine the genetic variation, as well as, to detect of selection at domain I of AMA-1 gene Plasmodium vivax isolates in Iran. METHODS: Blood samples were collected from 58 patients positive for P. vivax, mono infection and the domain I of AMA-1 gene was amplified by nested PCR and then sequenced. RESULTS: A total 33 different haplotypes were identified among 58 Iranian sequences. The 23 new haplotypes were determined in this study that was not reported previously in other regions of the world. There were totally observed 36 point mutations at the nucleotide level in the analyzed sequences. Sequences analyses indicated 25 amino acid changes at 20 positions in which 5 sites demonstrated thrimorphic polymorphism and the others were dimorphic in the domain I of the Iranian PvAMA-1 isolates. CONCLUSION: Our findings indicated relatively high level of allelic diversity at the domain I of PvAMA-1 among P.vivax isolates of Iran. Since, PvAMA-1 is considering as vaccine candidate antigen, these data provide valuable information for the development of a PvAMA-1 based malaria vaccine.