Molecular characterization of antifolates resistance-associated genes, (dhfr and dhps) in Plasmodium vivax isolates from the Middle East

<p>Abstract</p> <p>Background</p> <p>In Iran, co-infections of <it>Plasmodium vivax </it>and <it>Plasmodium falciparum </it>are common and <it>P. vivax </it>infections are often exposed to sulphadoxine-pyrimethamine (SP). In the present study, the frequency distribution of mutations associated to SP resistance was investigated in <it>pvdhfr </it>and <it>pvdhps </it>genes from field isolates.</p> <p>Methods</p> <p>Clinical isolates of <it>P. vivax </it>were collected in two different malaria endemic regions in northern and south-eastern Iran, between 2001 and 2006. All 189 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 <it>pvdhps </it>genes using nested PCR-RFLP methods</p> <p>Results</p> <p>All 189 examined isolates were found to carry wild-type amino acids at positions 13, 33, 61 and 173, while 57L and 58R and 117N mutations in pure form was detected among 1.1%, 17.5% and 26% examined samples, respectively, with no polymorphisms in different loci of <it>dhps </it>genes. Based on size polymorphism of <it>pvdhfr </it>genes at repeat region, among northern isolates, the frequency distribution for type A and B were 2.2% and 97.8% respectively. However, in southern samples the prevalence of type A, B and C were 7%, 89.5% and 7.7%, respectively. Mixed genotype infections (type B and C) were detected in only 4.2% (6/143) of southern, but in none of the northern isolates. The combination of <it>pvdhfr and pvdhps </it>haplotypes among all 189 samples demonstrated six distinct haplotypes. The two most prevalent haplotypes among all examined samples were I₁₃P₃₃F₅₇S₅₈T₆₁S₁₁₇I₁₇₃/A₃₈₃A₅₅₃(65.6%) and I₁₃P₃₃F₅₇S₅₈T₆₁<b>N</b>₁₁₇I₁₇₃(16.4%). Two other alleles with one point mutation I₁₃P₃₃F₅₇<b>R</b>₅₈T₆₁S₁₁₇I₁₇₃/A₃₈₃A₅₅₃and two mutations I₁₃P₃₃F₅₇<b>R</b>₅₈T₆₁<b>N</b>₁₁₇I₁₇₃/A₃₈₃A₅₅₃accounted for 7.4% and 9.5% of the total isolates.</p> <p>Conclusion</p> <p>The present molecular data provide important information for making decisions on population based drug use in Iran. In addition, since October 2005, with more availability of SP as first-line treatment, <it>P. vivax </it>isolates are more exposed to SP and the selection or spread of resistant <it>pvdhfr </it>and <it>pvdhps </it>alleles might increase in the near future in this region.</p

Molecular characterization of matrix metalloproteinase-1 (MMP-1) in Lucilia sericata larvae for potential therapeutic applications

Background: The salivary glands of Lucilia sericata are the first organs to express specific endopeptidase enzymes. These enzymes play a central role in wound healing, and they have potential to be used therapeutically. Methods: Rapid amplification of cDNA ends and rapid amplification of genomic ends were used to identify the coding sequence of MMP-1 from L. sericata. Different segments of MMP1 gene, namely the middle part, 3\u2032 end, and 5\u2032 end, were cloned, sequenced, and analyzed using bioinformatics tools to determine the distinct features of MMP-1 protein. Results: Assembling the different segments revealed that the complete mRNA sequence of MMP-1 is 1932 bp long. CDS is 1212 bp long and is responsible for the production of MMP-1 of 404 amino acid residues with a predicted molecular weight of 45.1 kDa. The middle part, 3\u2032 end, and 5\u2032 end sequences were 933, 503, and 496 bp. In addition, it was revealed that the MMP-1 genomic sequence includes three exons and two introns. Furthermore, the three-dimensional structure of L. sericata MMP-1 protein was evaluated, and its alignment defined that it has high similarity to chain A of human MMP-2 with 100% confidence, 72% coverage, and 38% identity according to the SWISS-MODEL modeling analysis. Conclusions:MMP-1 of L. sericata has a close relationshipwith its homologs in invertebrates and other insects. The present study significantly contributes to understanding the function, classification, and evolution of the characterized MMP-1 from L. sericata and provides basic required information for the development of an effective medical bioproduct

Analysis of von Willebrand factor A domain-related protein (WARP) polymorphism in temperate and tropical Plasmodium vivax field isolates

<p>Abstract</p> <p>Background</p> <p>The identification of key molecules is crucial for designing transmission-blocking vaccines (TBVs), among those ookinete micronemal proteins are candidate as a general class of malaria transmission-blocking targets. Here, the sequence analysis of an extra-cellular malaria protein expressed in ookinetes, named von Willebrand factor A domain-related protein (WARP), is reported in 91 <it>Plasmodium vivax </it>isolates circulating in different regions of Iran.</p> <p>Methods</p> <p>Clinical isolates were collected from north temperate and southern tropical regions in Iran. Primers have been designed based on <it>P. vivax </it>sequence (ctg_6991) which amplified a fragment of about 1044 bp with no size variation. Direct sequencing of PCR products was used to determine polymorphism and further bioinformatics analysis in <it>P. vivax </it>sexual stage antigen, <it>pvwarp</it>.</p> <p>Results</p> <p>Amplified <it>pvwarp </it>gene showed 886 bp in size, with no intron. BLAST analysis showed a similarity of 98–100% to <it>P. vivax </it>Sal-I strain; however, Iranian isolates had 2 bp mismatches in 247 and 531 positions that were non-synonymous substitution [T (ACT) to A (GCT) and R (AGA) to S (AGT)] in comparison with the Sal-I sequence.</p> <p>Conclusion</p> <p>This study presents the first large-scale survey on <it>pvwarp </it>polymorphism in the world, which provides baseline data for developing WARP-based TBV against both temperate and tropical <it>P. vivax </it>isolates.</p

Detection of malaria parasites by nested PCR in south-eastern, Iran: Evidence of highly mixed infections in Chahbahar district

BACKGROUND: Rapid diagnosis and correct treatment of cases are the main objectives of control programs in malaria-endemic areas. METHODS AND RESULTS: To evaluate these criteria and in a comparative study, blood specimens were collected from 120 volunteers seeking care at the Malaria Health Center in Chahbahar district. One hundred and seven out of 120 Giemsa-stained slides were positive for malaria parasites by microscopy. Eighty-four (70%) and 20 (16.7%) were identified as having only Plasmodium vivax and Plasmodium falciparum infections, respectively, while only 3 (2.5%) were interpreted as having mixed P. vivax-P. falciparum infections. The target DNA sequence of the 18S small sub-unit ribosomal RNA (ssrRNA) gene was amplified by Polymerase Chain Reaction (PCR) and used for the diagnosis of malaria in south-eastern Iran. One hundred twenty blood samples were submitted and the results were compared to those of routine microscopy. The sensitivity of PCR for detection of P. vivax and P. falciparum malaria was higher than that of microscopy: nested PCR detected 31 more mixed infections than microscopy and parasite positive reactions in 9 out of the 13 microscopically negative samples. The results also confirmed the presence of P. vivax and P. falciparum. CONCLUSIONS: These results suggest that, in places where transmission of both P. vivax and P. falciparum occurs, nested PCR detection of malaria parasites can be a very useful complement to microscopical diagnosis

Cloning, expression and transmission-blocking activity of anti-PvWARP, malaria vaccine candidate, in Anopheles stephensi mysorensis

<p>Abstract</p> <p>Background</p> <p>Notwithstanding progress in recent years, a safe, an effective and affordable malaria vaccine is not available yet. Ookinete-secreted protein, <it>Plasmodium vivax </it>von Willebrand factor A domain-related protein (PvWARP), is a candidate for malaria transmission-blocking vaccines (TBVs).</p> <p>Methods</p> <p>The PvWARP was expressed in <it>Escherichia coli </it>BL21 using the pET-23a vector and was purified using Ni-NTA affinity chromatography from a soluble fraction. Polyclonal antibody was raised against rPvWARP and transmission blocking activity was carried out in an <it>Anopheles stephensi</it>-<it>P. vivax </it>model.</p> <p>Results</p> <p>Expression of full length of PvWARP (minus signal peptide) expression showed a 35-kDa protein. The purified protein was recognized by mouse polyclonal antibody directed against rPvWARP. Sera from the animals displayed significantly a blocking activity in the membrane feeding assay of <it>An. stephensi </it>mysorensis.</p> <p>Conclusions</p> <p>This is the first report on <it>P. vivax </it>WARP expression in <it>E. coli </it>that provides an essential base for development of the malaria TBV against <it>P. vivax</it>. This may greatly assist in malaria elimination, especially in the oriental corner of WHO Eastern Mediterranean Regional Office (WHO/EMRO) including Afghanistan, Iran and Pakistan.</p

Identification of the Midgut Microbiota of An. stephensi and An. maculipennis for Their Application as a Paratransgenic Tool against Malaria

The midgut microbiota associated with Anopheles stephensi and Anopheles maculipennis (Diptera: Culicidae) was investigated for development of a paratransgenesis-based approach to control malaria transmission in Eastern Mediterranean Region (EMR). Here, we present the results of a polymerase chain reaction (PCR) and biochemical-based approaches to identify the female adult and larvae mosquitoe microbiota of these two major malaria vectors, originated from South Eastern and North of Iran. Plating the mosquito midgut contents from lab-reared and field-collected Anopheles spp. was used for microbiota isolation. The Gram-negative and Gram-positive bacterial colonies were identified by Gram staining and specific mediums. Selected colonies were identified by differential biochemical tests and 16S rRNA gene sequence analysis. A number of 10 An. stephensi and 32 An. maculipennis adult mosquitoes and 15 An. stephensi and 7 An. maculipennis larvae were analyzed and 13 sequences of 16S rRNA gene bacterial species were retrieved, that were categorized in 3 classes and 8 families. The majority of the identified bacteria were belonged to the γ-proteobacteria class, including Pseudomonas sp. and Aeromonas sp. and the others were some closely related to those found in other vector mosquitoes, including Pantoea, Acinetobacter, Brevundimonas, Bacillus, Sphingomonas, Lysinibacillus and Rahnella. The 16S rRNA sequences in the current study aligned with the reference strains available in GenBank were used for construction of the phylogenetic tree that revealed the relatedness among the bacteria identified. The presented data strongly encourage further investigations, to verify the potential role of the detected bacteria for the malaria control in Iran and neighboring countries

Speculation on the possibility for introducing Anopheles stephensi as a species complex: preliminary evidence based on odorant binding protein 1 intron I sequence

Abstract Background Anopheles stephensi is considered an important malaria vector in Iran, Asia, and recently in the Horn of Africa. Recently, Ansteobp1 intron I sequence has been introduced a new molecular marker for identification of its biological forms including, mysorensis, intermediate and type, using insectary colony specimens. Methods In the current study, new marker ability in molecular identification of biological forms has been evaluated with An. stephensi specimens collected from Iran and Afghanistan malarious provinces. Following DNA extraction and PCR amplification, sequence analysis and constructed phylogenetic tree revealed that type and intermediate forms are distributed in Iran. Results The specimens collected from Afghanistan identified as intermediate and mysorensis forms. Therefore, intermediate form is sympatric species in both countries. Based on the results of Ansteobp1 intron I sequences, An. stephensi could be suggested as new Anopheles complex species including An. stephensi sibling A (type form), An. stephensi sibling B (intermediate form) and An. stephensi sibling C (mysorensis form). This is the first report on the presence of An. stephensi biological forms in Afghanistan. Conclusions Iran is going to eliminate malaria transmission from the country, precise species identification, especially in complex species will be helpful in the prevention of malaria resurgence in the country, mainly because of common fauna of Anopheles species and through border malaria and population movement within Afghanistan, Pakistan, and Iran

Correction: Molecular detection and genetic diversity of avian haemosporidian parasites in Iran.

[This corrects the article DOI: 10.1371/journal.pone.0206638.]