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 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

Map of Iran indicating the location of the study area in Chabahar and Sarbaz districts situated in the south-eastern corner of Baluchistan Province, from where the isolates were collected

<p><b>Copyright information:</b></p><p>Taken from "Prevalence of mutations associated with antimalarial drugs in isolates prior to the introduction of sulphadoxine-pyrimethamine as first-line treatment in Iran"</p><p>http://www.malariajournal.com/content/6/1/148</p><p>Malaria Journal 2007;6():148-148.</p><p>Published online 13 Nov 2007</p><p>PMCID:PMC2214731.</p><p></p> : Sistan and Baluchistan province, : Chabahar, : Pishi

Frequency distribution of the combination haplotypes obtained from 206 isolates collected in Sistan and Baluchistan of Iran

<p><b>Copyright information:</b></p><p>Taken from "Prevalence of mutations associated with antimalarial drugs in isolates prior to the introduction of sulphadoxine-pyrimethamine as first-line treatment in Iran"</p><p>http://www.malariajournal.com/content/6/1/148</p><p>Malaria Journal 2007;6():148-148.</p><p>Published online 13 Nov 2007</p><p>PMCID:PMC2214731.</p><p></p> The haplotype NNAwas the most prevalent among Chabahar (48%) and Sarbaz (43%) isolates. Mutated amino acids are boldfaced

Prevalence of mutations associated with antimalarial drugs in <it>Plasmodium falciparum </it>isolates prior to the introduction of sulphadoxine-pyrimethamine as first-line treatment in Iran

<p>Abstract</p> <p>Background</p> <p>This work was carried out to assess the patterns and prevalence of resistance to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) in Iran.</p> <p>Methods</p> <p>The prevalence of <it>pfcrt </it>K76T, <it>pfmdr1 </it>N86Y, <it>pfdhfr </it>N51I, C59R, S108N/T and I164L and codons S436F/A, A437G, K540E, A581E, and A613S/T in <it>pfdhps </it>genes were genotyped by PCR/RFLP methods in 206 <it>Plasmodium falciparum </it>isolates from Chabahar and Sarbaz districts in Sistan and Baluchistan province, Iran, during 2003–2005.</p> <p>Results</p> <p>All <it>P. falciparum </it>isolates carried the 108N, while 98.5% parasite isolates carried the 59R mutation. 98.5% of patients carried both 108N and 59R. The prevalence of <it>pfdhps </it>437G mutation was 17% (Chabahar) and 33% (Sarbaz) isolates. 20.4% of samples presented the <it>pfdhfr </it>108N, 59R with <it>pfdhps </it>437G mutations. The frequency of allele <it>pfcrt </it>76T was 98%, while 41.4% (Chabahar) and 27.7% (Sarbaz) isolates carried <it>pfmdr1 </it>86Y allele. Eight distinct haplotypes were identified in all 206 samples, while the most prevalent haplotype was <b>T</b>_76/N_86/N₅₁<b>R</b>₅₉<b>N</b>_108/A₄₃₇among both study areas.</p> <p>Conclusion</p> <p>Finding the fixed level of CQ resistance polymorphisms (<it>pfcrt </it>76T) suggests that CQ must be withdrawn from the current treatment strategy in Iran, while SP may remain the treatment of choice for uncomplicated malaria.</p

COVID-19 prognostic modeling using CT radiomic features and machine learning algorithms: Analysis of a multi-institutional dataset of 14,339 patients

Background: We aimed to analyze the prognostic power of CT-based radiomics models using data of 14,339 COVID-19 patients. Methods: Whole lung segmentations were performed automatically using a deep learning-based model to extract 107 intensity and texture radiomics features. We used four feature selection algorithms and seven classifiers. We evaluated the models using ten different splitting and cross-validation strategies, including non-harmonized and ComBat-harmonized datasets. The sensitivity, specificity, and area under the receiver operating characteristic curve (AUC) were reported. Results: In the test dataset (4,301) consisting of CT and/or RT-PCR positive cases, AUC, sensitivity, and specificity of 0.83 ± 0.01 (CI95%: 0.81-0.85), 0.81, and 0.72, respectively, were obtained by ANOVA feature selector + Random Forest (RF) classifier. Similar results were achieved in RT-PCR-only positive test sets (3,644). In ComBat harmonized dataset, Relief feature selector + RF classifier resulted in the highest performance of AUC, reaching 0.83 ± 0.01 (CI95%: 0.81-0.85), with a sensitivity and specificity of 0.77 and 0.74, respectively. ComBat harmonization did not depict statistically significant improvement compared to a non-harmonized dataset. In leave-one-center-out, the combination of ANOVA feature selector and RF classifier resulted in the highest performance. Conclusion: Lung CT radiomics features can be used for robust prognostic modeling of COVID-19. The predictive power of the proposed CT radiomics model is more reliable when using a large multicentric heterogeneous dataset, and may be used prospectively in clinical setting to manage COVID-19 patients.</p