Dataset Information

Molecular assessment of kelch13 non-synonymous mutations in Plasmodium falciparum isolates from Central African Republic (2017-2019).

ABSTRACT:

Background

Over the last decade, artemisinin-based combination therapy (ACT) has contributed substantially to the decrease in malaria-related morbidity and mortality. The emergence of Plasmodium falciparum parasites resistant to artemisinin derivatives in Southeast Asia and the risk of their spread or of local emergence in sub-Saharan Africa are a major threat to public health. This study thus set out to estimate the proportion of P. falciparum isolates, with Pfkelch13 gene mutations associated with artemisinin resistance previously detected in Southeast Asia.

Methods

Blood samples were collected in two sites of Bangui, the capital of the Central African Republic (CAR) from 2017 to 2019. DNA was extracted and nested PCR were carried out to detect Plasmodium species and mutations in the propeller domain of the Pfkelch13 gene for P. falciparum samples.

Results

A total of 255 P. falciparum samples were analysed. Plasmodium ovale DNA was found in four samples (1.57%, 4/255). Among the 187 samples with interpretable Pfkelch13 sequences, four samples presented a mutation (2.1%, 4/187), including one non-synonymous mutation (Y653N) (0.5%, 1/187). This mutation has never been described as associated with artemisinin resistance in Southeast Asia and its in vitro phenotype is unknown.

Conclusion

This preliminary study indicates the absence of Pfkelch13 mutant associated with artemisinin resistance in Bangui. However, this limited study needs to be extended by collecting samples across the whole country along with the evaluation of in vitro and in vivo phenotype profiles of Pfkelch13 mutant parasites to estimate the risk of artemisinin resistance in the CAR.

SUBMITTER: Nzoumbou-Boko R

PROVIDER: S-EPMC7247190 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Molecular assessment of kelch13 non-synonymous mutations in Plasmodium falciparum isolates from Central African Republic (2017-2019).

Nzoumbou-Boko Romaric R Panté-Wockama Chris-Boris Gildas CG Ngoagoni Carine C Petiot Nathalie N Legrand Eric E Vickos Ulrich U Gody Jean-Chrysostome JC Manirakiza Alexandre A Ndoua Christophe C Lombart Jean-Pierre JP Ménard Didier D

Malaria journal 20200524 1

<h4>Background</h4>Over the last decade, artemisinin-based combination therapy (ACT) has contributed substantially to the decrease in malaria-related morbidity and mortality. The emergence of Plasmodium falciparum parasites resistant to artemisinin derivatives in Southeast Asia and the risk of their spread or of local emergence in sub-Saharan Africa are a major threat to public health. This study thus set out to estimate the proportion of P. falciparum isolates, with Pfkelch13 gene mutations ass ...[more]

PMID: 32448203

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Project description:Since several malaria parasite species are usually present in a particular area, co-infections with more than one species of Plasmodium are more likely to occur in humans infected in these areas. In many mixed infections, parasite densities of the cryptic species may be low and often not recognized in clinical practice.Two children (3 and 6 years old) adopted recently from Central African Republic were admitted to hospital because of intermittent fever. Thin blood smears stained with Giemsa showed Plasmodium falciparum and Plasmodium malariae co-infection for both children at admission. They were both treated with atovaquone-proguanil combination for 3 days. At day 7, both thin blood smears examination remained negative but at day 28, thin blood smear was positive for P. malariae trophozoites and for Plasmodium ovale for the girl and her brother, respectively. Samples collected at day 1 and day 28 were submitted to real-time PCR showing the presence of the three parasite species (P. falciparum, P malariae and P. ovale) in admission blood samples from the two children and only P. ovale at day 28.Twenty-eight days follow-up after treatment led to detection of a third parasite species in the blood of these two patients suggesting covert co-infection and a delayed appearance of one cryptic species following treatment. Concurrently infecting malaria species could be mutually suppressive, with P. falciparum tending to dominate other species. These observations provide more evidence that recommendations for treatment of imported malaria should take into account the risk of concurrent or cryptic infection with Plasmodium species. Clinicians and biologists should be aware of the underestimated frequency of mixed infections with cryptic species and of the importance of patient follow-up at day 28. Future guidelines should shed more light on the treatment of mixed infection and on the interest of using artemisinin-based combinations for falciparum and non-falciparum species.

Project description:BackgroundBecause of the loss of chloroquine (CQ) effectiveness, the Democratic Republic of Congo (DRC)'s malaria treatment policy replaced CQ by sulfadoxine-pyrimethamine (SP) as first-line treatment of uncomplicated malaria in 2003, which in turn was replaced by artemisinin-based combination therapies (ACT) in 2005. The World Health Organization (WHO) recommends monitoring of anti-malarial drug resistance every 2 years. The study aimed to provide baseline data for biennial molecular surveillance of anti-malarial drug resistance by comparing data from a study conducted in 2019 to previously published data from a similar study conducted in 2017 in the DRC.MethodsFrom July to November 2019, a cross-sectional study was conducted in ten sites which were previously selected for a similar study conducted in 2017 across the DRC. P. falciparum malaria was diagnosed by a rapid diagnostic test (RDT) or by microscopy and dried blood samples (DBS) were taken from patients who had a positive test. Segments of interest in pfcrt and pfk13 genes were amplified by conventional PCR before sequencing.ResultsOut of 1087 enrolled patients, 906 (83.3%) were PCR-confirmed for P. falciparum. Like in the 2017-study, none of the mutations known to be associated with Artemisinine (ART) resistance in Southeast Asia was detected. However, non-synonymous (NS) mutations with unknown functions were observed among which, A578S was detected in both 2017 and 2019-studies. The overall prevalence of pfcrt-K76T mutation that confers CQ-resistance was 22.7% in 2019-study compared to 28.5% in 2017-study (p-value = 0.069), but there was high variability between sites in the two studies. Like in 2017-study, the pfcrt 72-76 SVMNT haplotype associated with resistance to amodiaquine was not detected.ConclusionThe study reported, within 2 years, the non-presence of molecular markers currently known to be associated with resistance to ART and to AQ in P. falciparum isolated in the DRC. However, the presence of polymorphisms with as-yet unknown functions was observed, requiring further characterization. Moreover, an overall decrease in the prevalence of CQ-resistance marker was observed in the DRC, but this prevalence remained highly variable from region to region.

Project description:IntroductionDrug resistance remains a major challenge in malaria treatment, especially after the emergence of resistance to artemisinin-based combined therapies. Plasmodium falciparum Kelch13 gene mutations are implicated in conferring artemisinin resistance. Thus, this study was aimed at determining the occurrence of Kelch13 (K13) propeller resistance gene polymorphism mutations in Bushenyi district, Uganda.MethodsParticipants suspected to have malaria were recruited. P. falciparum was confirmed using antigen histidine-rich protein 2 (HRP2) (Pf) (Access Bio, Inc, USA) and microscopy. Malaria-positive patients were treated with artemeter-lumefantrine (AL). Blood was withdrawn from participants who tested positive for parasites after day 3 and kept in blood filter papers (ET31CHR; Whatman Limited, Kent, UK). DNA was extracted using chelex-suspension method. Nested polymerase chain reaction (PCR) was conducted and the second-round products sequenced using Sanger's method. Sequenced products were analyzed using DNAsp 5.10.01 software and then blasted on to the NCBI for K13-propeller gene sequence identity using the Basic Local Alignment Search Tool (BLAST).ResultsOut of 283 enrolled participants, 194 completed the follow-up schedule. A total of 134 (69%) had no parasites on day 3, while 60 (31%) had parasites on that day. Out of the 60 samples, 40 (62%) were positively amplified as P. falciparum, with polymorphisms in the K13-propeller gene detected in 3 (7.5%) out of the 40 amplicons. Polymorphisms at codon 1929, 1788 and 1801 were detected separately in one sample each. Sequences have been deposited in NCBI with accession numbers PRJNA720348 and PRJNA720800.ConclusionPolymorphisms in the K13-propeller gene previously reported to be associated with artemisinin resistance were not detected in the P. falciparum isolates from Bushenyi district, Uganda. More studies need to be conducted on the new mutations detected so as to understand their association, if any, with ACT resistance.

Project description:BackgroundCurrent malaria control and elimination strategies rely mainly on efficacious antimalarial drugs. However, drug resistance is a major threat facing malaria control programs. Determination of drug resistance molecular markers is useful in the monitoring and surveillance of malaria drug efficacy. This study aimed to determine the mutations and haplotypes frequencies of different genes linked with antimalarial drug resistance in certain areas in Sudan.MethodsA total of 226 dried blood spots (DBS) of microscopically diagnosed P. falciparum isolates were collected from Khartoum and three other areas in Sudan during 2015-2017. Plasmodium falciparum confirmation and multiplicity of infection was assessed using the Sanger's 101 SNPs-barcode and speciation was confirmed using regions of the parasite mitochondria. Molecular genotyping of drug resistance genes (Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, exonuclease, Pfk13, parasite genetic background (PGB) (Pfarps10, ferredoxin, Pfcrt, Pfmdr2)) was also performed. All genotypes were generated by selective regions amplicon sequencing of the parasite genome using the Illumina MiSeq platform at the Wellcome Sanger Institute, UK then genotypes were translated into drug resistance haplotypes and species determination.FindingsIn total 225 samples were confirmed to be P. falciparum. A higher proportion of multiplicity of infection was observed in Gezira (P<0.001) based on the Sanger 101 SNPs -barcode. The overall frequency of mutant haplotype Pfcrt 72-76 CVIET was 71.8%. For Pfmdr1, N86Y was detected in 53.6%, Y184F was observed in 88.1% and D1246Y was detected in 1.5% of the samples. The most frequently observed haplotype was YFD 47.4%. For Pfdhfr (codons 51, 59,108,164), the ICNI haplotype was the most frequent (80.7%) while for Pfdhps (codons 436, 437, 540, 581, 613) the (SGEAA) was most frequent haplotype (41%). The Quadruple mutation (dhfr N51I, S108N + dhps A437G, K540E) was the highest frequent combined mutation (33.9%). In Pfkelch13 gene, 18 non-synonymous mutations were detected, 7 of them were detected in other African countries. The most frequent Pfk13 mutation was E433D detected in four samples. All of the Pfk13 mutant alleles have not been reported to belong to mutations associated with delayed parasite clearance in Southeast Asia. PGB mutations were detected only in Pfcrt N326S\I (46.3%) and Pfcrt I356T (8.2%). The exonuclease mutation was not detected. There was no significant variation in mutant haplotypes between study areas.ConclusionsThere was high frequency of mutations in Pfcrt, Pfdhfr and Pfdhps in this study. These mutations are associated with chloroquine and sulfadoxine-pyrimethamine (SP) resistance. Many SNPs in Pfk13 not linked with delayed parasite clearance were observed. The exonuclease E415G mutation which is linked with piperaquine resistance was not reported.

Dataset Information

Molecular assessment of kelch13 non-synonymous mutations in Plasmodium falciparum isolates from Central African Republic (2017-2019).

Background

Methods

Results

Conclusion

Publications

Molecular assessment of kelch13 non-synonymous mutations in Plasmodium falciparum isolates from Central African Republic (2017-2019).

Similar Datasets

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