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Research in progress: Evaluating Artemisinin-resistant Plasmodium falciparum malaria using the K13 propeller domain molecular biomarker

January 9, 2016 - 09:03 -- Olajoju Soniran

Hi,
I want to share my research observations (on artemisinin resistance in Plasmodium falciparum) with malaria Professionals in this scientific community and welcome your candid comments.

I am a Ph.D student working on surveillance for artemisinin-resistant Plasmodium falciparum in dried blood spots sampled from Nigeria. I sequenced the K13 propeller domain at Macrogen using the PCR primers published in Nature Ariey 2014 titled 'A molecular marker of artemisinin resistant Plasmodium falciparum malaria'.

25 gDNA samples previously confirmed positive with regular PCR were selected for sequencing in the first phase. Surprisingly, the kelch protein was amplified in only one (1) sample which proved to be sensitive to artemisinin. The kelch protein was not amplified in the remaining 24 samples. A retrial experiment was run by Macrogen and it produced the same result.

I don’t have an explanation for this observation, please I need your candid comment on this. I will post some images of the sequence results(chromatogram).

I look forward to your response.

Thank you

Comments

Submitted by Marc Vanacker (not verified) on

In light of the clinical trial results in Maniema described a few days ago on www.malariaworld.org, do we really need artemisin in Africa. The plant Artemisia afra which does not contain artemisinin is as efficient as Artemisia annua. It is growing wild from South Africa to Ethiopia. Just harvest it and use it instead of buying expensive ACT pills from the chemical industry in the North

Submitted by Aria (not verified) on

Hi,
As you have not explained about other details. I would like to clarify some doubts before jumping to other conclusions.
Are you sure your samples (dried blood spots) have P. falciparum or the DNA quantity and quality is sufficient? Did you try to amplify any other gene from the same DNA? Did you check the species using PCR species diagnostic assay.

Hope this helps.

Rachel

Submitted by Olajoju Soniran (not verified) on

Hi,
I appreciate your comment. In response to your comment, the samples have been confirmed earlier to contain P. falciparum from the field using Rapid Diagnostic kit specific for Pf. In addition to that, PfCrt gene and dhfr gene were successfully amplified from these samples using the regular PCR, and some were sequenced for chloroquine resistance. So, all these preliminary experiments confirmed that the samples contain P. falciparum DNA.
However, after DNA extraction from the filter papers, i couldn't verify the quantity of P. falciparum DNA present in the samples because it's not purely P. falciparum DNA.

Submitted by Kyaw (not verified) on

Hello, have you sequenced the whole kelch 13 gene or only after amino acid position 440 (propeller region). You can also check on the following links for details:
http://www.nejm.org/doi/full/10.1056/NEJMoa1314981
Best wishes, Kyaw

Submitted by Olajoju Soniran (not verified) on

I appreciate your response Kyaw. The whole kelch 13 gene was sequenced following the protocol of the reference article mentioned in the comment posted.

Olajoju Soniran's picture
Submitted by Olajoju Soniran on

I appreciate your comments, the samples contain P. falciparum because Pfcrt and dhfr gene had been amplified successfully before the kelch experiment. I couldn’t quantify the exact DNA quantity of the parasite because the dried blood spot equally contains some human DNA.
In addition, the whole kelch gene was sequenced.

Soniran O.T.(PhD)

Submitted by Ricardo Ataide on

Hi Olajoju,

I don't have an immediate answer for your problem, but here are a couple of things you may want to think about:
- Is there a difference in quality/quantity of DNA obtained in the sample that was amplified as compared to the other samples?
- Do you have a positive control (usually a parasite isolate with known K13 sequence)?
- How have you determined artemisinin-sensitivity? Ring-stage survival assays, or parasite-clearance half-life?

Also, ultimately, you can directly contact the authors of the paper you mentioned (and other papers that have used similar protocols) and ask if they were faced with similar problems.

Cheers,
Ric

Ricardo Ataíde

Olajoju Soniran's picture
Submitted by Olajoju Soniran on

Thanks Ricardo for those line of thought. I will surely apply the suggestions.
Thanks.

Olajoju Soniran

Soniran O.T.(PhD)