A LAMP-SNP Assay Detecting C580Y Mutation in Pfkelch13 Gene from Clinically Dried Blood Spot Samples

Artemisinin resistance (ART) has been confirmed in Greater Mekong Sub-region countries. Currently, C580Y mutation on Pfkelch13 gene is known as the molecular marker for the detection of ART. Rapid and accurate detection of ART in field study is essential to guide malaria containment and elimination interventions. A simple method for collection of malaria-infected blood is to spot the blood on filter paper and is fast and easy for transportation and storage in the field study. This study aims to evaluate LAMP-SNP assay for C580Y mutation detection by introducing an extra mismatched nucleotide at the 3’ end of the FIP primer. The LAMP-SNP assay was performed in a water bath held at a temperature of 56°C for 45 min. LAMP-SNP products were interpreted by both gel-electrophoresis and HNB-visualized changes in color. The method was then tested with 120 P. falciparum DNA from dried blood spot samples. In comparing the LAMP-SNP assay results with those from DNA sequencing of the clinical samples, the 2 results fully agreed to detect C580Y. The sensitivity and specificity of the LAMP-SNP assay showed 100%. There were no cross-reactions with other Plasmodium species and other Pfkelch13 mutations. The LAMP-SNP assay performed in this study was rapid, reliable, and useful in detecting artemisinin resistance in the field study.

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Assessment of dried blood spot samples as a simple method for detection of hepatitis B virus markers

Journal of Medical Virology ◽

10.1002/jmv.22138 ◽

2011 ◽

Vol 83 (9) ◽

pp. 1522-1529 ◽

Cited By ~ 45

Author(s):

Livia Melo Villar ◽

Jaqueline Correia de Oliveira ◽

Helena Medina Cruz ◽

Clara Fumiko Tachibana Yoshida ◽

Elisabeth Lampe ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Dried Blood Spot ◽

Simple Method ◽

B Virus ◽

Hepatitis B Virus Markers ◽

Blood Spot

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Screening for antifolate and artemisinin resistance in Plasmodium falciparum clinical isolates from three hospitals of Eritrea

F1000Research ◽

10.12688/f1000research.54195.1 ◽

2021 ◽

Vol 10 ◽

pp. 628

Author(s):

Harriet Natabona Mukhongo ◽

Johnson Kang'ethe Kinyua ◽

Yishak Gebrekidan Weldemichael ◽

Remmy Wekesa Kasili

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Point Mutations ◽

Artemisinin Resistance ◽

Pcr Amplification ◽

Clinical Isolates ◽

Genetic Mechanism ◽

Dried Blood Spot ◽

Nested Polymerase Chain Reaction ◽

Blood Spot

Background: Antimalarial drug resistance is a major challenge hampering malaria control and elimination. Plasmodium falciparum, the leading causative parasite species, has developed resistance to basically all antimalarials. Continued surveillance of drug resistance using genetic markers provides important molecular data for treatment policies. This study sought to verify the genetic mechanism of resistance to sulfadoxine-pyrimethamine and assess the occurrence of point mutations associated with artemisinin resistance in P. falciparum clinical isolates from Eritrea. Methods: Nineteen dried blood spot samples were collected from patients visiting Adi Quala, Keren and Gash Barka Hospitals, Eritrea. The patients were followed up after receiving treatment with first line artesunate-amodiaquine. Nested polymerase chain reaction and Sanger sequencing techniques were employed to genotype point mutations in the P. falciparum bifunctional dihydrofolate reductase-thymidylate synthase (Pfdhfr, PF3D7_0417200), dihydropteorate synthase (Pfdhps, PF3D7_0810800) and kelch 13 (PfK13, PF3D7_1343700) genes. Results: Eight of nineteen (42%) of the dried blood spot samples were successful for PCR-amplification. Data analyses of the PCR-positive isolates revealed the following point mutations: Pfdhfr N51I in four isolates, C59R in one isolate, S108N in four isolates, a rare non-synonymous substitution V45A in four isolates and Pfdhps K540E in four isolates. No PfK13 point mutations were reported. Conclusions: Pfdhfr C59R and Pfdhps K540E point mutations are reliable markers for the sulfadoxine-pyrimethamine quintuple mutant haplotype combination. These findings highlight first reports in Eritrea, which verify the underlying genetic mechanism of antifolate resistance. Continuous monitoring of the PfK13 marker is recommended.

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Detection of mutations associated with artemisinin resistance in K13 gene of Plasmodium falciparum parasites

Ministry of Science and Technology, Vietnam ◽

10.31276/vjst.63(2).01-04 ◽

2021 ◽

Vol 63 (2) ◽

pp. 1-4

Author(s):

Thi Thu Huyen Tran ◽

◽

Thi Lan Dung Nguyen ◽

Thuy Trang Nguyen ◽

Van Ninh Nguyen ◽

...

Keyword(s):

Mutation Frequency ◽

Nucleotide Substitution ◽

Gene Segment ◽

Artemisinin Resistance ◽

Reference Sequence ◽

Clinical Samples ◽

Blood Samples ◽

Detection Of Mutations ◽

Sanger Method ◽

C580y Mutation

Objective: this study aims to develop a methodology for detecting mutations in the K13 gene and determine mutation frequencies in clinical samples. Method: total DNAs were extracted blood samples collected from 50 patients in failure with artemisinin treatmentin the Central Highlands region. A fragment of the K13 gene was amplified, purified, and sequenced by the Sanger method. The K13 sequences were analysed by using Bioedit and aligned with the reference sequence to determine K13 mutations. Results: successfully amplified the K13 gene segment with size 799 bp in P. falciparum. After sequencing, there was a nucleotide substitution of A>G at position 1740, leading to changes in amino acids C>Y at the respective position 580. In the 50 patient samples, 41/50 (82%) samples showed the C580Y mutation, 9/50 (18%) of the samples had no mutation, and there was no other mutation. Conclusion: the authors have successfully developed and optimised a procedure for detecting the mutation C580Y in K13 in P. falciparum and determined the mutation frequency in the K13 gene.

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Validation and Clinical Evaluation of a Novel Method To Measure Miltefosine in Leishmaniasis Patients Using Dried Blood Spot Sample Collection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02976-15 ◽

2016 ◽

Vol 60 (4) ◽

pp. 2081-2089 ◽

Cited By ~ 11

Author(s):

A. E. Kip ◽

H. Rosing ◽

M. J. X. Hillebrand ◽

S. Blesson ◽

B. Mengesha ◽

...

Keyword(s):

Venous Blood ◽

Dried Blood Spot ◽

Patient Specific ◽

Pharmacokinetic Studies ◽

Sample Collection ◽

Limit Of Quantification ◽

Simple Method ◽

Linear Effect ◽

Combination Treatments ◽

Blood Spot

ABSTRACTTo facilitate future pharmacokinetic studies of combination treatments against leishmaniasis in remote regions in which the disease is endemic, a simple cheap sampling method is required for miltefosine quantification. The aims of this study were to validate a liquid chromatography-tandem mass spectrometry method to quantify miltefosine in dried blood spot (DBS) samples and to validate its use with Ethiopian patients with visceral leishmaniasis (VL). Since hematocrit (Ht) levels are typically severely decreased in VL patients, returning to normal during treatment, the method was evaluated over a range of clinically relevant Ht values. Miltefosine was extracted from DBS samples using a simple method of pretreatment with methanol, resulting in >97% recovery. The method was validated over a calibration range of 10 to 2,000 ng/ml, and accuracy and precision were within ±11.2% and ≤7.0% (≤19.1% at the lower limit of quantification), respectively. The method was accurate and precise for blood spot volumes between 10 and 30 μl and for Ht levels of 20 to 35%, although a linear effect of Ht levels on miltefosine quantification was observed in the bioanalytical validation. DBS samples were stable for at least 162 days at 37°C. Clinical validation of the method using paired DBS and plasma samples from 16 VL patients showed a median observed DBS/plasma miltefosine concentration ratio of 0.99, with good correlation (Pearson'sr= 0.946). Correcting for patient-specific Ht levels did not further improve the concordance between the sampling methods. This successfully validated method to quantify miltefosine in DBS samples was demonstrated to be a valid and practical alternative to venous blood sampling that can be applied in future miltefosine pharmacokinetic studies with leishmaniasis patients, without Ht correction.

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