Dataset Information

Targeted deep amplicon sequencing of kelch 13 and cytochrome b in Plasmodium falciparum isolates from an endemic African country using the Malaria Resistance Surveillance (MaRS) protocol.

ABSTRACT: BACKGROUND:Routine molecular surveillance for imported drug-resistant malaria parasites to the USA and European Union is an important public health activity. The obtained molecular data are used to help keep chemoprophylaxis and treatment guidelines up to date for persons traveling to malaria endemic countries. Recent advances in next-generation sequencing (NGS) technologies provide a new and effective way of tracking malaria drug-resistant parasites. METHODS:As part of a technology transfer arrangement between the CDC Malaria Branch and the Istituto Superiore di Sanità (ISS), Rome, Italy, the recently described Malaria Resistance Surveillance (MaRS) protocol was used to genotype 148 Plasmodium falciparum isolates from Eritrea for kelch 13 (k13) and cytochrome b (cytb) genes, molecular markers associated with resistance to artemisinin (ART) and atovaquone/proguanil (AP), respectively. RESULTS:Spanning the full-length k13 gene, seven non-synonymous single nucleotide polymorphisms (SNPs) were found (K189N, K189T, E208K, D281V, E401Q, R622I and T535M), of which none have been associated with artemisinin resistance. No mutations were found in cytochrome b. CONCLUSION:All patients successfully genotyped carried parasites susceptible to ART and AP treatment. Future studies between CDC Malaria Branch and ISS are planned to expand the MaRS system, including data sharing, in an effort to maintain up to date treatment guidelines for travelers to malaria endemic countries.

SUBMITTER: L'Episcopia M

PROVIDER: S-EPMC7071742 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Targeted deep amplicon sequencing of kelch 13 and cytochrome b in Plasmodium falciparum isolates from an endemic African country using the Malaria Resistance Surveillance (MaRS) protocol.

L'Episcopia Mariangela M Kelley Julia J Patel Dhruviben D Schmedes Sarah S Ravishankar Shashidahar S Menegon Michela M Perrotti Edvige E Nurahmed Abduselam M AM Talha Albadawi A AA Nour Bakri Y BY Lucchi Naomi N Severini Carlo C Talundzic Eldin E

Parasites & vectors 20200314 1

<h4>Background</h4>Routine molecular surveillance for imported drug-resistant malaria parasites to the USA and European Union is an important public health activity. The obtained molecular data are used to help keep chemoprophylaxis and treatment guidelines up to date for persons traveling to malaria endemic countries. Recent advances in next-generation sequencing (NGS) technologies provide a new and effective way of tracking malaria drug-resistant parasites.<h4>Methods</h4>As part of a technolo ...[more]

PMID: 32171330

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Project description:BackgroundTimely molecular surveillance of Plasmodium falciparum kelch 13 (k13) gene mutations is essential for monitoring the emergence and stemming the spread of artemisinin resistance. Widespread artemisinin resistance, as observed in Southeast Asia, would reverse significant gains that have been made against the malaria burden in Africa. The purpose of this study was to assess the prevalence of k13 polymorphisms in western Kenya and Ethiopia at sites representing varying transmission intensities between 2018 and 2022.MethodsDried blood spot samples collected through ongoing passive surveillance and malaria epidemiological studies, respectively, were investigated. The k13 gene was genotyped in P. falciparum isolates with high parasitaemia: 775 isolates from four sites in western Kenya (Homa Bay, Kakamega, Kisii, and Kombewa) and 319 isolates from five sites across Ethiopia (Arjo, Awash, Gambella, Dire Dawa, and Semera). DNA sequence variation and neutrality were analysed within each study site where mutant alleles were detected.ResultsSixteen Kelch13 haplotypes were detected in this study. Prevalence of nonsynonymous k13 mutations was low in both western Kenya (25/783, 3.19%) and Ethiopia (5/319, 1.57%) across the study period. Two WHO-validated mutations were detected: A675V in three isolates from Kenya and R622I in four isolates from Ethiopia. Seventeen samples from Kenya carried synonymous mutations (2.17%). No synonymous mutations were detected in Ethiopia. Genetic variation analyses and tests of neutrality further suggest an excess of low frequency polymorphisms in each study site. Fu and Li's F test statistic in Semera was 0.48 (P > 0.05), suggesting potential population selection of R622I, which appeared at a relatively high frequency (3/22, 13.04%).ConclusionsThis study presents an updated report on the low frequency of k13 mutations in western Kenya and Ethiopia. The WHO-validated R622I mutation, which has previously only been reported along the north-west border of Ethiopia, appeared in four isolates collected from eastern Ethiopia. The rapid expansion of R622I across Ethiopia signals the need for enhanced monitoring of the spread of drug-resistant P. falciparum parasites in East Africa. Although ACT remains currently efficacious in the study areas, continued surveillance is necessary to detect early indicators of artemisinin partial resistance.

Project description:BACKGROUND:Artemisinin-based combination therapy (ACT) is recommended at the initial phase for treatment of Plasmodium falciparum, to reduce morbidity and mortality in all countries where malaria is endemic. Polymorphism in portions of P. falciparum gene encoding kelch (K13)-propeller domains is associated with delayed parasite clearance after ACT. Of about 124 different non-synonymous mutations, 46 have been identified in Southeast Asia (SEA), 62 in sub-Saharan Africa (SSA) and 16 in both the regions. This is the first study designed to analyse the prevalence of polymorphism in the P. falciparum k13-propeller domain in the Jazan region of southwest Saudi Arabia, where malaria is endemic. METHODS:One-hundred and forty P. falciparum samples were collected from Jazan region of southwest Saudi Arabia at three different times: 20 samples in 2011, 40 samples in 2016 and 80 samples in 2020 after the implementation of ACT. Plasmodium falciparum kelch13 (k13) gene DNA was extracted, amplified, sequenced, and analysed using a basic local alignment search tool (BLAST). RESULTS:This study obtained 51 non-synonymous (NS) mutations in three time groups, divided as follows: 6 single nucleotide polymorphisms (SNPs) '11.8%' in samples collected in 2011 only, 3 (5.9%) in 2011and 2016, 5 (9.8%) in 2011 and 2020, 5 (9.8%) in 2016 only, 8 (15.7%) in 2016 and 2020, 14 (27.5%) in 2020 and 10 (19.6%) in all the groups. The BLAST revealed that the 2011 isolates were genetically closer to African isolates (53.3%) than Asian ones (46.7%). Interestingly, this proportion changed completely in 2020, to become closer to Asian isolates (81.6%) than to African ones (18.4%). CONCLUSIONS:Despite the diversity of the identified mutations in the k13-propeller gene, these data did not report widespread artemisinin-resistant polymorphisms in the Jazan region where these samples were collected. Such a process would be expected to increase frequencies of mutations associated with the resistance of ACT.

Project description:The emergence of artemisinin-resistant Plasmodium falciparum parasites in Southeast Asia threatens malaria control and elimination. The interconnectedness of parasite populations may be essential to monitor the spread of resistance. Combining a published barcoding system of geographically restricted single-nucleotide polymorphisms (SNPs), mainly mitochondria of P. falciparum with SNPs in the K13 artemisinin resistance marker, could elucidate the parasite population structure and provide insight regarding the spread of drug resistance. We explored the diversity of mitochondrial SNPs (bp position 611-2825) and identified K13 SNPs from malaria patients in the districts of India (Ranchi), Tanzania (Korogwe), and Senegal (Podor, Richard Toll, Kaolack, and Ndoffane). DNA was amplified using a nested PCR and Sanger-sequenced. Overall, 199 K13 sequences (India: N = 92; Tanzania: N = 48; Senegal: N = 59) and 237 mitochondrial sequences (India: N = 93; Tanzania: N = 48; Senegal: N = 96) were generated. SNPs were identified by comparisons with reference genomes. We detected previously reported geographically restricted mitochondrial SNPs (T2175C and G1367A) as markers for parasites originating from the Indian subcontinent and several geographically unrestricted mitochondrial SNPs. Combining haplotypes with published P. falciparum mitochondrial genome data suggested possible regional differences within India. All three countries had G1692A, but Tanzanian and Senegalese SNPs were well-differentiated. Some mitochondrial SNPs are reported here for the first time. Four nonsynonymous K13 SNPs were detected: K189T (India, Tanzania, Senegal); A175T (Tanzania); and A174V and R255K (Senegal). This study supports the use of mitochondrial SNPs to determine the origin of the parasite and suggests that the P. falciparum populations studied were susceptible to artemisinin during sampling because all K13 SNPs observed were outside the propeller domain for artemisinin resistance.

Project description:BackgroundUnderstanding the dynamics of gametocyte production in polyclonal Plasmodium falciparum infections requires a genotyping method that detects distinct gametocyte clones and estimates their relative frequencies. Here, a marker was identified and evaluated to genotype P. falciparum mature gametocytes using amplicon deep sequencing.MethodsA data set of polymorphic regions of the P. falciparum genome was mined to identify a gametocyte genotyping marker. To assess marker resolution, the number of unique haplotypes in the marker region was estimated from 95 Malawian P. falciparum whole genome sequences. Specificity of the marker for detection of mature gametocytes was evaluated using reverse transcription-polymerase chain reaction of RNA extracted from NF54 mature gametocytes and rings from a non-gametocyte-producing strain of P. falciparum. Amplicon deep sequencing was performed on experimental mixtures of mature gametocytes from two distinct parasite clones, as well as gametocyte-positive P. falciparum field isolates to evaluate the quantitative ability and determine the limit of detection of the genotyping approach.ResultsA 400 bp region of the pfs230 gene was identified as a gametocyte genotyping marker. A larger number of unique haplotypes was observed at the pfs230 marker (34) compared to the sera-2 (18) and ama-1 (14) markers in field isolates from Malawi. RNA and DNA genotyping accurately estimated gametocyte and total parasite clone frequencies when evaluating agreement between expected and observed haplotype frequencies in gametocyte mixtures, with concordance correlation coefficients of 0.97 [95% CI: 0.92-0.99] and 0.92 [95% CI: 0.83-0.97], respectively. The detection limit of the genotyping method for male gametocytes was 0.41 pfmget transcripts/µl [95% CI: 0.28-0.72] and for female gametocytes was 1.98 ccp4 transcripts/µl [95% CI: 1.35-3.68].ConclusionsA region of the pfs230 gene was identified as a marker to genotype P. falciparum gametocytes. Amplicon deep sequencing of this marker can be used to estimate the number and relative frequency of parasite clones among mature gametocytes within P. falciparum infections. This gametocyte genotyping marker will be an important tool for studies aimed at understanding dynamics of gametocyte production in polyclonal P. falciparum infections.

Dataset Information

Targeted deep amplicon sequencing of kelch 13 and cytochrome b in Plasmodium falciparum isolates from an endemic African country using the Malaria Resistance Surveillance (MaRS) protocol.

Publications

Targeted deep amplicon sequencing of kelch 13 and cytochrome b in Plasmodium falciparum isolates from an endemic African country using the Malaria Resistance Surveillance (MaRS) protocol.

Similar Datasets

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