Molecular surveillance of Plasmodium vivax dhfr and dhps mutations in isolates from Afghanistan

<p>Abstract</p> <p>Background</p> <p>Analysis of dihydrofolate reductase (<it>dhfr</it>) and dihydropteroate synthase (<it>dhps</it>) mutations in <it>Plasmodium vivax </it>wild isolates has been considered to be a valuable molecular approach for mapping resistance to sulphadoxine-pyrimethamine (SP). The present study investigates the frequency of SNPs-haplotypes in the <it>dhfr </it>and <it>dhps </it>genes in <it>P. vivax </it>clinical isolates circulating in two malaria endemic areas in Afghanistan.</p> <p>Methods</p> <p><it>P. vivax </it>clinical isolates (n = 171) were collected in two different malaria endemic regions in north-west (Herat) and east (Nangarhar) Afghanistan in 2008. All collected isolates were analysed for SNP-haplotypes at positions 13, 33, 57, 58, 61, 117 and 173 of the <it>pvdhfr </it>and 383 and 553 of the <it>pvdhps </it>genes using PCR-RFLP methods.</p> <p>Results</p> <p>All 171 examined isolates were found to carry wild-type amino acids at positions 13, 33, 57, 61 and 173, while 58R and 117N mutations were detected among 4.1% and 12.3% of Afghan isolates, respectively. Based on the size polymorphism of <it>pvdhfr </it>genes at repeat region, type B was the most prevalent variant among Herat (86%) and Nangarhar (88.4%) isolates. Mixed genotype infections (type A/B and A/B/C) were detected in only 2.3% (2/86) of Herat and 1.2% (1/86) of Nangarhar isolates, respectively. The combination of <it>pvdhfr </it>and <it>pvdhps </it>haplotypes among all 171 samples demonstrated six distinct haplotypes. The two most prevalent haplotypes among all examined samples were wild-type (86%) and single mutant haplotype I₁₃P₃₃F₅₇S₅₈T₆₁<b>N </b>₁₁₇I_173/A₃₈₃A₅₅₃(6.4%).</p> <p>Double (I₁₃P₃₃S₅₇<b>R</b>₅₈T₆₁<b>N</b>₁₁₇I₁₇₃/A₃₈₃A₅₅₃) and triple mutant haplotypes (I₁₃P₃₃S₅₇<b>R </b>₅₈T₆₁<b>N</b>₁₁₇I₁₇₃/<b>G</b>₃₈₃A₅₅₃) were found in 1.7% and 1.2% of Afghan isolates, respectively. This triple mutant haplotype was only detected in isolates from Herat, but in none of the Nangarhar isolates.</p> <p>Conclusion</p> <p>The present study shows a limited polymorphism in <it>pvdhfr </it>from Afghan isolates and provides important basic information to establish an epidemiological map of drug-resistant vivax malaria, and updating guidelines for anti-malarial policy in Afghanistan. The continuous usage of SP as first-line anti-malarial drug in Afghanistan might increase the risk of mutations in the <it>dhfr </it>and <it>dhps </it>genes in both <it>P. vivax </it>and <it>Plasmodium falciparum </it>isolates, which may lead to a complete SP resistance in the near future in this region. Therefore, continuous surveillance of <it>P. vivax </it>and <it>P. falciparum </it>molecular markers are needed to monitor the development of resistance to SP in the region.</p

Comparative Gene Expression Profiling of P. falciparum Malaria Parasites Exposed to Three Different Histone Deacetylase Inhibitors

Histone deacetylase (HDAC) inhibitors are being intensively pursued as potential new drugs for a range of diseases, including malaria. HDAC inhibitors are also important tools for the study of epigenetic mechanisms, transcriptional control, and other important cellular processes. In this study the effects of three structurally related antimalarial HDAC inhibitors on P. falciparum malaria parasite gene expression were compared. The three hydroxamate-based compounds, trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA; Vorinostat®) and a 2-aminosuberic acid derivative (2-ASA-9), all caused profound transcriptional effects, with ∼2–21% of genes having >2-fold altered expression following 2 h exposure to the compounds. Only two genes, alpha tubulin II and a hydrolase, were up-regulated by all three compounds after 2 h exposure in all biological replicates examined. The transcriptional changes observed after 2 h exposure to HDAC inhibitors were found to be largely transitory, with only 1–5% of genes being regulated after removing the compounds and culturing for a further 2 h. Despite some structural similarity, the three inhibitors caused quite diverse transcriptional effects, possibly reflecting subtle differences in mode of action or cellular distribution. This dataset represents an important contribution to our understanding of how HDAC inhibitors act on malaria parasites and identifies alpha tubulin II as a potential transcriptional marker of HDAC inhibition in malaria parasites that may be able to be exploited for future development of HDAC inhibitors as new antimalarial agents

STATUS OF INSECTICIDE RESISTANCE IN ANOPHELES CULICIFACIES (DIPTERA: CULICIDAE) IN GHASREGHAND DISTRICT, SISTAN AND BALUCHISTAN PROVINCE, IRAN, (1997)

Anopheles culicifacies s.l. plays an important role In transmission of malaria in Sistan and Baluchistan province, southeastern Iran. Adult susceptibility test on fieltt-collccled mosquitoes was conducted in Ohasreghand district. WHO diagnostic test procedures revealed that adult females were resistant to 0.4% dieUirin (mortality 64.5 &amp;plusmn; 3.13), tolerant to 0.1% propoxur (mortality 88.5 &amp;plusmn; 2.24) and susceptible to 4% DDT (mortality 98.75 &amp;plusmn; 0.8). 5% malathion (mortality 100%), 0.1% bendiocarb (mortality 98.86 &amp;plusmn; 0.7), 0.25% pcrmcthrin (mortality 98.4 &amp;plusmn; 0.1), ami 0.1% lamhdacyhalothrin (mortality 100%). Malathion and lamhdacyhalothrin had the highest efficacy against this species when they were exposed at the diagnostic dose for 1 hour followed by a 24 hour recovery period. DieUirin, DDT a nil malathion had been used for malaria control as an indoor residual spraying. Tlic implication of these findings in the control programme is discussed

Polymorphism of Merozoite Surface Protein-3α Gene of Plasmo-dium vivax in Isolates of Iran

Background: The worldwide distribution of P. vivax has expanded significantly and the number of reported cases has been on the rise. Approximately 88% of malaria cases in Iran are caused by Plasmodium vivax, and in order to management of the disease, understanding the population genetic structure of the parasite is necessary for designing and applying drugs and vaccines. Among many potential candidates, merozoite surface protein-3α gene (PvMSP-3α) is promising target to develop an effective vaccine. This study was carried out to determine the variation of this gene, as a genetic marker, in Plasmodium vivax isolates in malarious areas of Iran. Methods: Diversity in PvMSP-3α gene was assessed in 85 Plasmodium vivax isolated from four southern and east-southern provinces of the country by PCR/RFLP method. Amplification was performed with two primer pair sets in a nested PCR format and the products were digested by the enzyme HhaI in RFLP method. Results: Based on the size of the PCR products, we observed three biotypes A (about 1900bp), B (about 1400bp) and C (about 1100bp) of PvMSP-3α gene. Biotype A was predominant. According to RFLP patterns, 10 allelic groups of the gene were observed, that, 7, 2 and 1 groups correspond to the biotype A, B and C, respectively. Mixed genotype and multiple infections were not seen. Conclusion: RFLP method with HhaI enzyme is a useful method for determining the polymorphism of biotype A of PvMSP-3agene

Ophthalmomyiasis Caused by Flesh Fly (Dip¬tera: Sarcophagidae) in a Patient with Eye Malignancy in Iran

Here we describe a case of ophthalmomyiasis in a male patient with basal cell carcinoma. During the operation several live and motile maggots were removed from the lesion. Preliminary examination on the larvae confirmed their affiliation to the genus Sarcophaga (Diptera: Sarcophagidae).This genus is widely distributed throughout the world and species are very difficult to identify. The authors made at¬tempt to approach species identification by rearing larvae to the adult flesh flies, but due to shortage of adult male specimen, reliable diagnosis in the level of species was not obtained. Possible interaction between ocular myiasis and malignancy concerning the case has not been addressed in this paper

Variation of the Chloroquine Resistance Transporter (Crt) Gene in Chloroquine-Resistant and Chloroquine-Sensitive Plasmodium berghei

Background: The emergence and spread of chloroquine resistant Plasmodium falciparum in the world stimulated some investigators to consider different aspects of chloroquine resistance in human and rodent Plasmodia. Using animal Plasmo­dia, particularly primate and rodent Plasmodia can be useful model for human Plasmodia studies. In this study we have tried to consider and compare the sequence of chloroquine resistance transporter (crt) gene among chloroquine-resistant and chloroquine-sensitive strains of Plasmodium berghei. Methods: This experimental study was performed at the Malaria Laboratory of School of public health. DNA was ex­tracted from two strains of P. berghei which their resistance and sensitivity had been demonstrated in mice with treatment by chloroquine. By using specific primer for crt gene some parts of this gene were amplified by PCR, and obtained frag­ments were then sequenced and compared. Results: There were considerable differences in crt gene between two strains. Sequenced 1212 bp of crt gene fragment in the two strains showed 43 differences at nucleotides level and 16 differences in presumed coding amino acids. Conclusion: crt can be addressed as a considerable gene which involves in induction of resistance to chloroquine in P. berghei, as P. falciparum. The results increased such a promise that considering crt gene in chloroquine-sensitive and chlo­roquine-resistant P. berghei can prepare suitable and helpful fields for more understanding the molecular aspects of chloro­quine-resistance in Plasmodia and reversing the effectiveness of 4-aminoquinolines particularly chloroquine for treatment of drug resistant Plasmodia

Genetic diversity in the circumspordzoite protein gene of Plasmodium falciparum from major endemic regions of Iran

Circumsporozoite protein (CSP) is one of the stage specific antigens, which is used for the development of vaccines and to investigate the genetic diversity in Plasmodium falciparum malaria parasite. Polymerase chain reaction was used for typing of CSP genes on 67 positive falciparum malaria patients from Sistan and Baluchistan Province of Iran. Three fragments were detected for CSP gene. Twenty, 38 and 4 samples showed 700, 750 and 800 by fragments, respectively. Sequences of some samples were aligned and compared with P.falcipamm csp gene in gene bank. While the falciparum malaria endemic region of Iran is classified in low to moderate group but, extensive polymorphism was observed in the samples that could be taken into account in designing malaria vaccine