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

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

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

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

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

Anti-malarial seroprevalence assessment during an elimination programme in Chabahar District, south-eastern Iran.

BACKGROUND: Iran has achieved a substantial decline in malaria incidence over the past decades. A common feature of malaria-endemic settings is the requirement for more sensitive techniques to describe levels of low transmission. In this study, serological and parasitological methods were used to measure transmission levels of Plasmodium falciparum and Plasmodium vivax during an elimination programme (2012) in Chabahar District, Sistan and Baluchistan Province, south-eastern Iran. METHODS: Participants were randomly selected from 64 different geographical clusters in Chabahar city and surrounding villages. Antibody responses to P. falciparum and P. vivax blood-stage antigens were assessed by ELISA, while microscopy and molecular testing were used to determine parasite carriage by species. Age-adjusted antibody responses were analysed using a reversible catalytic model to calculate seroconversion rates (SCR). RESULTS: There was no evidence of recent transmission in the study areas, indicated by an absence of parasite infections in all ages and low or absent serological responses to either species in young children. The best model for age P. falciparum seroconversion was one with a change in exposure 21 years before sampling was done in Chabahar city (P = 0.018) and 4 years in the villages (P = 0.039). There was a higher level of recent P. vivax transmission compared to P. falciparum, based on the SCRs, in both the city and village settings. CONCLUSION: Serological analysis identified a decline in P. falciparum transmission in the urban areas of Chabahar, consistent with a previously described decrease in malaria in the early 1990s, demonstrating the utility of this approach to reconstruct exposure history. At present, it remains unclear whether the P. vivax antibody responses reflect active transmission due to new infections or relapse infections. The absence of parasitological and serological evidence of recent malaria transmission in Chabahar District is viable evidence for certification of elimination

Single nucleotide polymorphisms in Plasmodium falciparum V type H+ pyrophosphatase gene (pfvp2) and their associations with pfcrt and pfmdr1 polymorphisms

"Uncorrected proof"BACKGROUND: Chloroquine resistance in Plasmodium falciparum malaria has been associated with pfcrt 76T (chloroquine resistance transporter gene) and pfmdr1 86Y (multidrug resistance gene 1) alleles. Pfcrt 76T enables transport of protonated chloroquine out of the parasites digestive vacuole resulting in a loss of hydrogen ions (H(+)). V type H(+) pyrophosphatase (PfVP2) is thought to pump H(+) into the digestive vacuole. This study aimed to describe the geographic distribution of single nucleotide polymorphisms in pfvp2 and their possible associations with pfcrt and pfmdr1 polymorphisms. METHODS: Blood samples from 384 patients collected (1981-2009) in Honduras (n=35), Colombia (n=50), Liberia (n=50), Guinea Bissau (n=50), Tanzania (n=50), Iran (n=50), Thailand (n=49) and Vanuatu (n=50) were analysed. The pfcrt 72-76 haplotype, pfmdr1 copy numbers, pfmdr1 N86Y and pfvp2 V405I, K582R and P711S alleles were identified using PCR based methods. RESULTS: Pfvp2 was amplified in 344 samples. The pfvp2 allele proportions were V405 (97%), 405I (3%), K582 (99%), 582R (1%), P711 (97%) and 711S (3%). The number of patients with any of pfvp2 405I, 582R and/or 711S were as follows: Honduras (2/30), Colombia (0/46), Liberia (7/48), Guinea-Bissau (4/50), Tanzania (3/48), Iran (3/50), Thailand (1/49) and Vanuatu (0/31). The alleles were most common in Liberia (P=0.01) and Liberia+Guinea-Bissau (P=0.01). The VKP haplotype was found in 189/194 (97%) and 131/145 (90%) samples harbouring pfcrt 76T and pfcrt K76 respectively (P=0.007). CONCLUSIONS: The VKP haplotype was dominant. Most pfvp2 405I, 582R and 711S SNPs were seen where CQ resistance was not highly prevalent at the time of blood sampling possibly due to greater genetic variation prior to the bottle neck event of spreading CQ resistance. The association between the pfvp2 VKP haplotype and pfcrt 76T, which may indicate that pfvp2 is involved in CQ resistance, should therefore be interpreted with caution.This work was supported by Swedish International Development Cooperation Agency, Department for research Cooperation (Sida-SAREC Contribution no 75007082/03) and Sigurd och Elsa Goljes Minne Fund (project No. LA2010-0537). MIV is recipient of Post Doctoral fellowship from Fundacao para a Ciencia e Tecnologia (FCT)/Ministerio da Ciencia e Ensino Superior, Portugal - MCES (ref. SFRH/BPD/76614/2011). JU has a postdoctoral position funded by Stockholms lans landsting