Molecular identification of Palearctic members of Anopheles maculipennis in northern Iran

BACKGROUND: Members of Anopheles maculipennis complex are effective malaria vectors in Europe and the Caspian Sea region in northern Iran, where malaria has been re-introduced since 1994. The current study has been designed in order to provide further evidence on the status of species composition and to identify more accurately the members of the maculipennis complex in northern Iran. METHODS: The second internal transcribed spacer of ribosomal DNA (rDNA-ITS2) was sequenced in 28 out of 235 specimens that were collected in the five provinces of East Azerbayjan, Ardebil, Guilan, Mazandaran and Khorassan in Iran. RESULTS: The length of the ITS2 ranged from 283 to 302 bp with a GC content of 49.33 – 54.76%. No intra-specific variations were observed. Construction of phylogenetic tree based on the ITS2 sequence revealed that the six Iranian members of the maculipennis complex could be easily clustered into three groups: the An. atroparvus – Anopheles labranchiae group; the paraphyletic group of An. maculipennis, An. messeae, An. persiensis; and An. sacharovi as the third group. CONCLUSION: Detection of three species of the An. maculipennis complex including An. atroparvus, An. messae and An. labranchiae, as shown as new records in northern Iran, is somehow alarming. A better understanding of the epidemiology of malaria on both sides of the Caspian Sea may be provided by applying the molecular techniques to the correct identification of species complexes, to the detection of Plasmodium composition in Anopheles vectors and to the status of insecticide resistance by looking to related genes

Merozoite surface protein-3α is a reliable marker for population genetic analysis of Plasmodium vivax

BACKGROUND: The knowledge on population structure of the parasite isolates has contributed greatly to understanding the dynamics of the disease transmission for designing and evaluating malaria vaccines as well as for drug applications. msp-1 and msp-3α genes have been used as a genetic marker in population studies of Plasmodium vivax isolates. In this study, msp-3α was compared and assessed with msp-1 marker in order to find whether msp-3α is a reliable genetic marker for P. vivax population studies. METHODS: This comparative study was designed and carried out as the first assessment of diversity in Pvmsp-3α gene by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in the 50 northern and 94 southern P. vivax isolates from Iran, which had been analysed before for msp-1 gene. RESULTS: Three allele size as, Type A (1.8 kb), Type B (1.5 kb) and Type C (1.2 kb) have been detected among both northern and southern isolates based on PCR results. Type C (70%) and Type A (68.7%) were the predominant fragments among northern and southern parasites, respectively. 99 distinct Pvmsp-3α fragments defined by the size were detected in the 94 southern samples by PCR analysis. However, no mixed genotype infections have been detected among northern isolates. Based on restriction pattern from digestion with Hha I and Alu I 12 and 49 distinct allelic variants have been detected among 50 northern and 94 southern isolates. However, based on msp-1 gene, 30 distinct variants identified in all 146-sequenced Iranian P. vivax isolate. CONCLUSION: The results suggested that PCR-RFLP on msp-3α gene is an adequate, applicable and easily used technique for molecular epidemiology studies of P. vivax isolates without the need for further sequencing analysis

Non-variant specific antibody responses to the C-terminal region of merozoite surface protein-1 of Plasmodium falciparum (PfMSP-119) in Iranians exposed to unstable malaria transmission

<p>Abstract</p> <p>Background</p> <p>The C-terminal region of <it>Plasmodium falciparum </it>merozoite surface protein-1 (PfMSP-1₁₉) is a leading malaria vaccine candidate antigen. However, the existence of different variants of this antigen can limit efficacy of the vaccine development based on this protein. Therefore, in this study, the main objective was to define the frequency of PfMSP-1₁₉haplotypes in malaria hypoendemic region of Iran and also to analyse cross-reactive and/or variant-specific antibody responses to four PfMSP-1₁₉variant forms.</p> <p>Methods</p> <p>The PfMSP-1₁₉was genotyped in 50 infected subjects with <it>P. falciparum </it>collected during 2006-2008. Four GST-PfMSP-1₁₉variants (E/TSR/L, E/TSG/L, E/KNG/F and Q/KNG/L) were produced in <it>Escherichia coli </it>and naturally occurring IgG antibody to these proteins was evaluated in malaria patients' sera (n = 50) using ELISA. To determine the cross-reactivity of antibodies against each PfMSP-1₁₉variant in <it>P. falciparum-</it>infected human sera, an antibody depletion assay was performed in eleven corresponding patients' sera.</p> <p>Results</p> <p>Sequence data of the PfMSP-1₁₉revealed five variant forms in which the haplotypes Q/KNG/L and Q/KNG/F were predominant types and the second most frequent haplotype was E/KNG/F. In addition, the prevalence of IgG antibodies to all four PfMSP-1₁₉variant forms was equal and high (84%) among the studied patients' sera. Immunodepletion results showed that in Iranian malaria patients, Q/KNG/L variant could induce not only cross-reactive antibody responses to other PfMSP-1₁₉variants, but also could induce some specific antibodies that are not able to recognize the E/TSG/L or E/TSR/L variant forms.</p> <p>Conclusion</p> <p>The present findings demonstrated the presence of non-variant specific antibodies to PfMSP-1₁₉in Iranian falciparum malaria patients. This data suggests that polymorphism in PfMSP-1₁₉is less important and one variant of this antigen, particularly Q/KNG/L, may be sufficient to be included in PfMSP-1₁₉-based vaccine.</p

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

Molecular assessment of <it>atpase6</it> mutations associated with artemisinin resistance among unexposed and exposed <it>Plasmodium falciparum</it> clinical isolates to artemisinin-based combination therapy

Abstract Background Artemisinin-based combination therapy (ACT) is the mainstay of global efforts for treatment of Plasmodium falciparum malaria, but decline in its efficacy is the most important obstacle towards malaria control and elimination. Therefore, the present molecular analysis provides information on putative mutations associated with artemisinin resistance in P. falciparum clinical population unexposed and exposed to artesunate 4 years after adoption of ACT as the first-line anti-malarial therapy in Iran. Methods In this study, blood samples (n = 226) were collected from uncomplicated P. falciparum-infected patients from different health centers of Chabahar district in Sistan and Baluchistan province in the south-eastern part of Iran, during 2003 to 2010. All collected isolates were analysed for putative candidate mutations (TTA) L263E (GAA), (GAA) E431K (AAA), (GCA) A623E (GAA) and (AGT) S769N (AAT) of pfatpase6 gene using nested PCR/RFLP, followed by sequencing. Furthermore, the gene copy number was assessed by real-time quantitative PCR (RT-qPCR) in the presence of SYBR green. Results Neither the pfatpase6 L263E nor the A623E mutation was detected among all examined isolates. The E431K mutation was found in 23% of the analysed samples unexposed to ACT; however, it was detected in 17.8% (34/191) of P. falciparum isolates exposed to artesunate after 2007. High frequency of this single nucleotide polymorphisms (SNP) (overall 18.6%) among both examined groups (X2 test, P>0.05) indicated that this SNP should be considered as an unrelated mutation to artemisinin resistance. In contrast, S769N mutation was not detected in unexposed isolates; however, it was found in 2.6% (5/191), four years after introduction of ACT in this malaria setting. Also, detected SNPs were not significantly frequent in both unexposed and exposed examined isolates (X2 test, P> 0.05). Investigation in the copy number of pfatpase6 gene revealed a similar number of copy (n = 1) as in an isolate sensitive to artemisinin. Conclusion Taken together, the results suggest, in particular, that pfatpase6 S769N gene needs more consideration for its possible association with artesunate resistance among P. falciparum isolates.</p