Molecular identification and anti-malarial drug resistance profile of Plasmodium falciparum from patients attending Kisoro Hospital, southwestern Uganda

Abstract Background The evolution of malaria infection has necessitated the development of highly sensitive diagnostic assays, as well as the use of dried blood spots (DBS) as a potential source of deoxyribonucleic acid (DNA) yield for polymerase chain reaction (PCR) assays. This study identified the different Plasmodium species in malaria-positive patients, and the anti-malarial drug resistance profile for Plasmodium falciparum using DBS samples collected from patients attending Kisoro Hospital in Kisoro district, Southwestern Uganda. Methods The blood samples were prospectively collected from patients diagnosed with malaria to make DBS, which were then used to extract DNA for real-time PCR and high-resolution melting (HRM) analysis. Plasmodium species were identified by comparing the control and test samples using HRM-PCR derivative curves. Plasmodium falciparum chloroquine (CQ) resistance transporter (pfcrt) and kelch13 to screen the samples for anti-malarial resistance markers. The HRM-PCR derivative curve was used to present a summary distribution of the different Plasmodium species as well as the anti-malarial drug profile. Results Of the 152 participants sampled, 98 (64.5%) were females. The average age of the participants was 34.9 years (range: 2 months–81 years). There were 134 samples that showed PCR amplification, confirming the species as Plasmodium. Plasmodium falciparum (N = 122), Plasmodium malariae (N = 6), Plasmodium ovale (N = 4), and Plasmodium vivax (N = 2) were the various Plasmodium species and their proportions. The results showed that 87 (71.3%) of the samples were sensitive strains/wild type (CVMNK), 4 (3.3%) were resistant haplotypes (SVMNT), and 31 (25.4%) were resistant haplotypes (CVIET). Kelch13 C580Y mutation was not detected. Conclusion The community served by Kisoro hospital has a high Plasmodium species burden, according to this study. Plasmodium falciparum was the dominant species, and it has shown that resistance to chloroquine is decreasing in the region. Based on this, molecular identification of Plasmodium species is critical for better clinical management. Besides, DBS is an appropriate medium for DNA preservation and storage for future epidemiological studies.

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Molecular identification and characterization of rat Abcc1 cDNA: existence of two splicing variants and species difference in drug-resistance profile

Journal of Experimental Therapeutics and Oncology ◽

10.1046/j.1359-4117.2003.01089.x ◽

2003 ◽

Vol 3 (3) ◽

pp. 136-146 ◽

Cited By ~ 2

Author(s):

Shin-ichiro Takayanagi ◽

Toshihisa Ishikawa

Keyword(s):

Drug Resistance ◽

Molecular Identification ◽

Species Difference ◽

Resistance Profile ◽

Splicing Variants ◽

Drug Resistance Profile ◽

Identification And Characterization

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Prevalence of pfk13 and pfmdr1 polymorphisms in Bounkiling, Southern Senegal

PLoS ONE ◽

10.1371/journal.pone.0249357 ◽

2021 ◽

Vol 16 (3) ◽

pp. e0249357

Author(s):

Ambroise Ahouidi ◽

Rafael Oliveira ◽

Lis Lobo ◽

Cyrille Diedhiou ◽

Souleymane Mboup ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Pcr Amplification ◽

Parasite Clearance ◽

Dried Blood Spots ◽

Multi Drug Resistance ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Multidrug Resistance Gene ◽

Filter Papers

Background Delayed Plasmodium falciparum parasite clearance has been associated with Single Nucleotide Polymorphisms (SNPs) in the kelch protein propeller domain (coded by pfk13 gene). SNPs in the Plasmodium falciparum multidrug resistance gene 1 (pfmdr1) are associated with multi-drug resistance including the combination artemether-lumefantrine. To our knowledge, this is the first work providing information on the prevalence of k13-propeller and pfmdr1 mutations from Sédhiou, a region in the south of Senegal. Methods 147 dried blood spots on filter papers were collected from symptomatic patients attending a hospital located in Bounkiling City, Sédhiou Region, Southern Senegal. All samples were collected between 2015–2017 during the malaria transmission season. Specific regions of the gene pfk13 and pfmdr1 were analyzed using PCR amplification and Sanger sequencing. Results The majority of parasites (92.9%) harboured the pfk13 wild type sequence and 6 samples harboured synonymous changes. Regarding pfmdr1, wild-type alleles represented the majority except at codon 184. Overall, prevalence of 86Y was 11.9%, 184F was 56.3% and 1246Y was 1.5%. The mutant allele 184F decreased from 73.7% in 2015 to 40.7% in 2017. The prevalence of haplotype NFD decreased from 71.4% in 2015 to 20.8% in 2017. Conclusions This study provides the first description of pfk13 and pfmdr1 genes variations in Bounkiling, a city in the Sédhiou Region of Senegal, contributing to closing the gap of information on anti-malaria drug resistance molecular markers in southern Senegal.

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Differential ex vivo drug resistance profile in first and subsequent relapsed childhood acute myeloid leukemia in comparison to initial diagnosis

Medical and Biological Sciences ◽

10.2478/v10251-012-0027-5 ◽

2012 ◽

Vol 26 (2) ◽

Author(s):

Beata Kuryło-Rafińska ◽

Beata Kołodziej ◽

Małgorzata Kubicka ◽

Mariusz Wysocki ◽

Jan Styczyński

Keyword(s):

Drug Resistance ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Ex Vivo ◽

Initial Diagnosis ◽

Resistance Profile ◽

Drug Resistance Profile ◽

Childhood Acute Myeloid Leukemia ◽

Acute Myeloid

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The Drug Resistance Profile of Mycobacterium abscessus Group Strains from Korea

Annals of Laboratory Medicine ◽

10.3343/alm.2014.34.1.31 ◽

2014 ◽

Vol 34 (1) ◽

pp. 31-37 ◽

Cited By ~ 38

Author(s):

Seung Heon Lee ◽

Hee Kyung Yoo ◽

Seol Hee Kim ◽

Won-Jung Koh ◽

Chang Ki Kim ◽

...

Keyword(s):

Drug Resistance ◽

Mycobacterium Abscessus ◽

Resistance Profile ◽

Drug Resistance Profile

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Deletions of pfhrp2 and pfhrp3 genes in rapid diagnostic test-negative Plasmodium falciparum isolates from Uganda

10.21203/rs.3.rs-42855/v1 ◽

2020 ◽

Author(s):

Sam L Nsobya ◽

Andrew Walakira ◽

Elizabeth Namirembe ◽

Moses Kiggundu ◽

Joaniter I Nankabirwa ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Protein Expression ◽

False Negative ◽

Plasmodium Species ◽

Pcr Amplification ◽

Rapid Diagnostic Tests ◽

False Negatives ◽

Cross Sectional ◽

True Prevalence ◽

Blood Smears

Abstract Background: Rapid diagnostic tests (RDTs) play a key role in malaria case management. The most widely used RDT identifies Plasmodium falciparum based on immunochromatographic recognition of P. falciparum histidine-rich protein 2 (PfHRP2). Deletion of the homologous pfhrp2 and pfhrp3 genes leads to false-negative PfHRP2-based RDTs, and has been reported in P. falciparum from South America and Africa. However, identification of pfhrp2/pfhrp3 deletions has usually been based only on failure to amplify these genes using PCR, without confirmation based on protein expression, and our understanding of the true prevalence of deletions is incomplete. Methods: We investigated pfhrp2 / pfhrp3 deletions in blood samples from cross-sectional surveys in 2012-13 in three regions of varied malaria transmission intensity in Uganda. We evaluated samples with positive Giemsa-stained thick blood smears and negative PfHRP2-based RDTs by PCR amplification of conserved subunit ribosomal DNA for Plasmodium species, PCR amplification of pfhrp2 and pfhrp3 genes to identify deletions, and bead-based immunoassays for expression ofPfHRP2. Results: Of 3516 samples collected in cross-sectional surveys, 1493 (42.5%) had positive blood smears, of which 96 (6.4%) were RDT-negative. Of these 96 RDT-negative samples, P. falciparum was identified in 56 (58%) and only non-falciparum plasmodial DNA in 40 (42%). In all 56 P. falciparum -positive samples there was a failure to amplify pfhrp2 or pfhrp3 :in 25 (45%) pfhrp2 was not amplified, in 39 (70%) pfhrp3 was not amplified, and in 19 (34%) neither gene was amplified. For the 39 P. falciparum -positive, RDT-negative samples available for analysis of protein expression, PfHRP2 was not identified by immunoassay in only four samples (10.3%); these four samples all had failure to amplify both pfhrp2 and pfhrp3 by PCR. Thus, only four of 96 (4.2%) smear-positive, RDT-negative samples had P. falciparum infections with deletion of pfhrp2 and pfhrp3 confirmed by failure to amplify the genes by PCR and lack of expression of PfHRP2 demonstrated by immunoassay. Conclusion: False negative RDTs were uncommon, and deletions in pfhrp2 and pfhrp3 explained some of these findings, although most false negatives were not due to deletion of the pfhrp2 and pfhrp3 genes.

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