Molecular quantification of Plasmodium parasite density from the blood retained in used RDTs

Robinson, Ailie; Busula, Annette O.; Muwanguzi, Julian; Powers, Stephen J.; Masiga, Daniel K.; Bousema, Teun; Takken, Willem; Boer, Jetske G. de; Logan, James G.; Beshir, Khalid B.; Sutherland, Colin J.

doi:10.1038/s41598-019-41438-0

Cited by 20 publications

(21 citation statements)

References 35 publications

(66 reference statements)

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“…Large-scale RDTs in endemic countries are now used as sources of parasite DNA [10,11] and have helped develop molecular tools to improve malaria surveillance [12][13][14]. Recently, numerous studies have detected asexual forms of Plasmodium falciparum in RDT samples by PCR [15].…”

Section: Introductionmentioning

confidence: 99%

RETRACTED ARTICLE: Molecular detection and quantification of Plasmodium falciparum gametocytes carriage in used RDTs in malaria elimination settings in northern Senegal

Guiguemdé

Dièye²,

Lô

et al. 2020

Malar J

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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.

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Section: Introductionmentioning

confidence: 99%

RETRACTED ARTICLE: Molecular detection and quantification of Plasmodium falciparum gametocytes carriage in used RDTs in malaria elimination settings in northern Senegal

Guiguemdé

Dièye²,

Lô

et al. 2020

Malar J

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“…P. falciparum International standard (Pf INT), a reagent comprising lyophilised whole blood from a single hyperparasitaemic individual was obtained from NIBSC UK. Undiluted, this reagent represents a parasitaemia of 9.8% which is equivalent to 4.9 x 10 5 parasites per µl (14,16).…”

Section: Plasmodium Falciparum Dna Strainsmentioning

confidence: 99%

“…DNA was extracted from DBS from Eritrea and from whole blood from the MRL and from cultured laboratory isolates using a robotic DNA extraction system (Qiasymphony, QIAGEN, Germany), as previously described (16). The clinical samples from Kenya and Tanzania were extracted using the QIAamp Blood Mini Kit (Qiagen, UK) as per the manufacturer's instructions.…”

Section: Dna Extractionmentioning

confidence: 99%

A Novel Multiplex qPCR Assay for Detection of Plasmodium falciparum with Histidine-rich Protein 2 and 3 (pfhrp2 and pfhrp3) Deletions in Polyclonal Infections

Grignard

Nolder

Sepúlveda

et al. 2020

Preprint

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AbstractBackgroundRapid diagnostic tests (RDTs) that detect the malaria antigen histidine-rich protein 2 (HRP2) are widely used in endemic areas globally to confirm Plasmodium falciparum infection in febrile patients. The emergence of parasites lacking the gene encoding HRP2 and escaping RDT detection threatens progress in malaria control and elimination. Many health facilities in malaria endemic countries are dependent on RDTs for diagnosis and some National Health Service hospitals without expert microscopists rely on them for diagnosis out of hours. It is vital to study the emergence and the extent of such parasites globally to guide diagnostic policy. Currently, verification of the presence of such parasites in a blood sample requires a series of PCR assays to confirm the presence of P. falciparum and in the absence of amplicons from pfhrp2 and/or pfhrp3, which encodes a cross-reactive protein isoform. These tests have different limits of detection and many laboratories have reported difficulty in confirming the absence of pfhrp2 and pfhrp3 with certainty.MethodsWe developed and validated a novel and rapid multiplex real time quantitative (qPCR) assay to detect pfhrp2, pfhrp3, confirmatory parasite and human reference genes simultaneously. We also applied the assay to detect pfhrp2 and pfhrp3 deletion in 462 field samples from different endemic countries and UK travellers.ResultsThe qPCR assay showed limit of detection and quantification of 0.76-1.5 parasites per μl. The amplification efficiency, coefficient of determination (R2) and slope for the genes were 96-1.07%, 0.96-0.98 and −3.375 2 to −3.416 respectively. The assay demonstrated diagnostic sensitivity of 100% (n=19, 95% CI= (82.3%; 100%)) and diagnostic specificity of 100% (n=31; 95% CI= (88.8%; 100%)) in detecting pfhrp2 and pfhrp3 in. In addition, the qPCR assay estimates P. falciparum parasite density and can detect pfhrp2 and pfhrp3 deletions masked in polyclonal infections. We report pfhrp2 and pfhrp3 deletions in parasite isolates from Kenya, Tanzania and in UK travellers.ConclusionThe new qPCR assay is simple to use and offers significant advantages in speed and ease of interpretation. It is easily scalable to routine surveillance studies in countries where P. falciparum parasites lacking pfhrp2 and pfhrp3 are a threat to malaria control.

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“…Large-scale RDTs in endemic countries are now used as sources of parasite DNA [11,12] and have helped develop molecular tools to improve malaria surveillance [13,14,15]. Recently, numerous studies have detected asexual forms of Plasmodium falciparum in RDT samples by PCR [16].…”

Section: Introductionmentioning

confidence: 99%

Molecular detection and quantification of Plasmodium falciparum gametocytes carriage in used RDTs in malaria elimination settings in northern Senegal

Guiguemdé

Dièye²,

Lô³

et al. 2020

Preprint

View full text Add to dashboard Cite

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 for the parasite's 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 P. falciparum on these 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 technique in order to use this new tool 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: We 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, described for the first time, is a simple and effective tool to better understand the level of malaria transmission and reach elimination. Keywords: Malaria, RDT, Gametocytes, DNA extraction, Quantification, Plasmodium falciparum , qPCR.

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Molecular quantification of Plasmodium parasite density from the blood retained in used RDTs

Cited by 20 publications

References 35 publications

RETRACTED ARTICLE: Molecular detection and quantification of Plasmodium falciparum gametocytes carriage in used RDTs in malaria elimination settings in northern Senegal

RETRACTED ARTICLE: Molecular detection and quantification of Plasmodium falciparum gametocytes carriage in used RDTs in malaria elimination settings in northern Senegal

A Novel Multiplex qPCR Assay for Detection of Plasmodium falciparum with Histidine-rich Protein 2 and 3 (pfhrp2 and pfhrp3) Deletions in Polyclonal Infections

Molecular detection and quantification of Plasmodium falciparum gametocytes carriage in used RDTs in malaria elimination settings in northern Senegal

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