Molecular detection and quantification of Plasmodium falciparum gametocytes carriage in used RDTs in malaria elimination settings in northern Senegal
Authors: Tamba Souané
Babacar Faye
Aminata Lam
Marie Pièrre Diouf
Magatte Ndiaye
Kiswendsida Thierry Guiguemde
Aminata Collé Lô
Roger Clément Kouly Tine
Moussa Diop
Yakou Dieye
Isaac Akhénaton Manga
Gnagna Dieng Sow
Journal: Malaria journal (2020)
Discipline: CLINICAL MEDICINE
Keywords: DNA extraction; Gametocytes; Malaria; Plasmodium falciparum; Quantification
Affiliations: Department of Medical Parasitology, Medical Faculty, Cheikh Anta Diop University, Dakar, Senegal. ., PATH, Malaria Control and Evaluation Partnership (MACEPA), Dakar, Senegal., Department of Medical Parasitology, Medical Faculty, Cheikh Anta Diop University, Dakar, Senegal.

Abstract:  Background: Malaria surveillance requires powerful tools and strategies to achieve malaria elimination. Rapid diagnostic tests for malaria (RDTs) are easily deployed on a large scale and are helpful sources of parasite DNA. The application of sensitive molecular techniques to these RDTs is a modern tool for improving malaria case detection and drug resistance surveillance. Several studies have made it possible to extract the DNA of Plasmodium falciparum from RDTs. The knowledge of gametocyte carriage in the population is important to better assess the level of parasite transmission in elimination settings. The aim of this study was to detect P. falciparum gametocytes from used RDTs by quantitative PCR for molecular monitoring of malaria transmission. Methods: DNA was extracted from 303 RDT devices (SD Bioline Malaria Pf) using the Chelex-100 protocol. qPCR was performed in a 20 μL reaction to detect and quantify transcripts of the pfs25 gene. The cycle threshold (Ct) was determined by the emission fluorescence corresponding to the initial amount of amplified DNA. Results: The study found an overall prevalence of 53.47% with an average Ct of 32.12 ± 4.28 cycles. In 2018, the prevalence of gametocytes was higher in the Ranérou district (76.24%) than in the Saint-Louis district (67.33%) where an increase in the number of gametocyte carriers in 2018 was noted, in comparison with 2017. Conclusions: RDTs are a good source of DNA for molecular monitoring of gametocyte carriage. This method is a simple and effective tool to better understand the level of malaria transmission with a view to elimination. 

Retracted: No

PubPeer comments:
 The authors report detecting gametocyte carriage by amplificating Plasmodium DNA retrieved from used RDTs. By definition, gametocyte genes are present in all plasmodium stages. However, they are only expressed at certain stages. Only by amplificating RNA transcripts can gametocyte presence be detected. The authors are therefore detecting parasites, but not specifically those EXPRESSING gametocyte specific transcripts. They DO NOT detect gametocytes. It seems impossible that editors and reviewers would miss this key point? I wonder what happened in the review process. , 
 ** The authors report having detected the carriage of gametocytes by amplifying the Plasmodium DNA recovered from the RDTs used. ** The editors and reviewers made the same comment so did not miss the key point you are describing. The pertinence of the comment should not be on the review process nor the competence of the reviewers but must remain scientific. In the manuscript it is specified that the authors used a sequence of Pfs25 primers wich are specific to mature gametocytes and can be considered to quantify only female gametocytes. This has been demonstrated by Schneider et al. (2015) who has shown that the DNA sequences of the target regions (DNA produced in vitro: ivDNA) and the cDNA were identical and no significant nucleotide polymorphism were detected in the primer regions among almost 2000 samples of P. falciparum collected worldwide. The use of specific DNA to detect female gametocytes becomes easily applicable for large-scale studies in low-income countries. Your concern is well-founded and your suggestions are welcome to improve gametocyte detection techniques. , 
 #2 The specific marker of gametocyte stage is the detection of Pfs25 messengerRNA, not Pfs25 DNA. All Plasmodium parasite stages (asexual blood stages, sporozoites, gametocytes...) contain Pfs25 DNA, since this gene is part of their nucleic genome. However, only gametocytes EXPRESS this gene as mRNA and then as a protein (this is why it s called stage-specific). In order to detect gametocytes, Schneider et al conducted the following protocol : 1) perform a specific RNA extraction 2) perform reverse-transcription to obtain cDNA 3) amplify this cDNA by PCR Steps 2 + 3 are were summarized as  RT-qPCR (see Schneider et al, 2015) i.e.  Reverse-Transcriptase quantitative PCR . This should not be confused with  real-time PCR , often shortened as RT-PCR, in which the amplification of DNA is monitored cycle after cycle using a fluorescent dye. If RNA is not isolated specifically from DNA (step 1), and if no reverse transcription is performed before PCR amplification (step 2), then during step 3, PCR will amplify nucleic Pfs25 DNA which is present in any Plasmodium stage. Guiguemde et al report that DNA was extracted from used RDT and amplified by real-time PCR (methods sections of the abstract and of the article). These methods do not report steps 1 and 2: they do not appear designed to provide specific evidence of the presence of Pfs25 mRNA. So, it does nt seem possible to conclude to the presence of gametocytes. In addition, RNA is much more unstable than DNA. RNA has to be preserved in specific conditions after blood collection. RDTs are not designed to stabilize RNA. It is therefore very uncertain to rely on used RDTs to collect RNA. ,