A Molecular Assay to Quantify Male and Female Plasmodium falciparum Gametocytes: Results From 2 Randomized Controlled Trials Using Primaquine for Gametocyte Clearance

Stone, Will; Sawa, Patrick; Lanke, Kjerstin; Rijpma, Sanna R.; Oriango, Robin; Nyaurah, Maureen; Osodo, Paul; Osoti, Victor; Mahamar, Almahamoudou; Diawara, Halimatou; Woestenenk, Rob; Graumans, Wouter; Vegte‐Bolmer, Marga van de; Bradley, John; Chen, Ingrid; Brown, Joelle; Siciliano, Giulia; Alano, Pietro; Gosling, Roly; Dicko, Alassane; Drakeley, Chris; Bousema, Teun

doi:10.1093/infdis/jix237

Cited by 51 publications

(119 citation statements)

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“…Of 31 paired experiments, 18 (58.1%) direct skin feeding and 22 (71.0%) membrane feeding experiments resulted in at least one infected mosquito (p=0.289). Total gametocyte density, quantified in venous blood by quantitative reverse transcriptase PCR (qRT-PCR) targeting female-specific Pfs25 mRNA and male-specific Pfmget mRNA (29), was positively associated with the proportion of mosquitoes that became infected following direct skin feeding (Spearman ρ=0.415, p=0.0204) or membrane feeding (Spearman ρ=0.596, p = 0.0004) (Figure 1A). The proportion of infected mosquitoes was higher by direct skin feeding as compared to membrane feeding assays (odds ratio 2.01; 95% CI 1.21 – 3.33, p = 0.007), in line with previous studies (9, 10, 30).…”

Section: Resultsmentioning

confidence: 99%

“…Nucleic acids from these 200μL mosquito pools and from 100μL venous and finger prick whole blood samples in RNAprotect Cell Reagent were isolated using the bead-based MagNAPure LC automatic extractor (Total Nucleic Acid Isolation Kit—High Performance, Roche Applied Science) and eluted in 50μL of water. In these samples, ring-stage asexual parasites, female gametocytes and male gametocytes were quantified by individual quantitative reverse-transcription PCR (qRT-PCR) assays targeting sbp1 (31); Pfs25 (45) and PfMGET (29), respectively. Skin biopsy samples were immediately stored in RNAlater solution after collection.…”

Section: Methodsmentioning

confidence: 99%

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P. falciparum gametocyte density and infectivity in peripheral blood and skin tissue of naturally infected parasite carriers

Meibalan

Barry

et al. 2019

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Transmission of Plasmodium falciparum depends on the presence of mature gametocytes that can be ingested by mosquitoes taking a bloodmeal when feeding on human skin. It has long been hypothesised that skin sequestration contributes to efficient transmission. Although skin sequestration would have major implications for our understanding of transmission biology and the suitability of mosquito feeding methodologies to measure the human infectious reservoir, it has never been formally tested. In two populations of naturally infected gametocyte carriers from Burkina Faso, we assessed transmission potential to mosquitoes and directly quantified male and female gametocytes and asexual parasites in: i) finger prick blood, ii) venous blood, iii) skin biopsies, and in pools of mosquitoes that fed iv) on venous blood or, v) directly on the skin. Whilst more mosquitoes became infected when feeding directly on the skin compared to venous blood, concentrations of gametocytes in the subdermal skin vasculature were identical to that in other blood compartments. Asexual parasite densities, gametocyte densities and sex ratios were identical in the mosquito blood meals taken directly from the skin of parasite carriers and their venous blood.We also observed sparse gametocytes in skin biopsies from legs and arms of gametocyte carriers by microscopy. Taken together, we provide conclusive evidence for the absence of significant skin sequestration of P. falciparum gametocytes. Gametocyte densities in peripheral blood are thus informative for predicting onward transmission potential to mosquitoes. Quantifying this human malaria transmission potential is of pivotal importance for the deployment and monitoring of malaria elimination initiatives.IMPORTANCEOur observations settle a long-standing question in the malaria field and close a major knowledge gap in the parasite cycle. By deploying mosquito feeding experiments and stage-specific molecular and immunofluorescence parasite detection methodologies in two populations of naturally infected parasite carriers, we conclusively reject the hypothesis of gametocyte skin sequestration. Our findings provide novel insights in parasite stage composition in human blood compartments, mosquito bloodmeals and their implications for transmission potential. We demonstrate that gametocyte levels in venous or finger prick blood can be used to predict onward transmission potential to mosquitoes. Our findings thus pave the way for methodologies to quantify the human infectious reservoir based on conventional blood sampling approaches to support the deployment and monitoring of malaria elimination efforts for maximum public health impact.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

P. falciparum gametocyte density and infectivity in peripheral blood and skin tissue of naturally infected parasite carriers

Meibalan

Barry

et al. 2019

Preprint

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“…With a very conservative threshold of 1,000-fold enrichment in three of the four strains tested (Fig 6A), eight of the 15 tested transcripts were highly specific to gametocytes. Transcript abundance in ring-stage parasites was assessed and compared to Pfs25 mRNA, an established and highly abundant yet intron-less female gametocyte specific transcript 11,38 . Five out of eight gametocyte specific transcripts were undetectable in asexual ring stages at ≤10 5 parasites/mL, similar in specificity to Pfs25 (Fig 6B); the five most sensitive gametocyte markers detected gametocytes across the range of 10 2 – 10 6 gametocytes/mL (Fig 6C).…”

Section: Resultsmentioning

confidence: 99%

“…Five targets were tested for their sensitivity and can recognize 100 gametocytes/mL, while the signal is undetectable when fewer than 10 5 -10 6 ring stage parasites/mL are present. In practical terms, these markers may be used to reliably detect gametocytes at densities well below the microscopic threshold of detection in samples without high-densities of asexual parasites, similar to the gametocyte marker that is currently most widely used, the female gametocyte-specific Pfs25 [35].…”

Section: Discussionmentioning

confidence: 99%

Probabilistic data integration identifies reliable gametocyte-specific proteins and transcripts in malaria parasites

Meerstein-Kessel

Lee

Lanke

et al. 2017

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“…Successful transmission of Plasmodium infection from humans to mosquitoes requires that a female Anopheles mosquito imbibes at least one mature male and one mature female gametocyte in a blood meal. The density and the ratio of female to male gametocytes in peripheral blood are therefore key determinants of the dynamics of transmission, transmission epidemiology in endemic settings, and for evaluating the efficacy of transmission blocking interventions, such as drugs and vaccines (1)(2)(3)(4)(5)(6).…”

Section: Introductionmentioning

confidence: 99%

An assay for quantification of male and female gametocytes in human blood by qRT-PCR in the absence of pure gametocyte standards

Wang¹,

Ballard²,

Llewellyn³

et al. 2020

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Background Malaria transmission from humans to mosquitoes requires the presence of gametocytes in human peripheral circulation, and the dynamics of transmission are determined largely by the density and sex ratio of the gametocytes. Molecular methods are thus employed to measure gametocyte densities, particularly when assessing transmission epidemiology and the efficacy of transmission-blocking interventions. However accurate quantification of gametocytes with molecular methods requires pure male and female gametocytes as reference standards, which are not widely available. Methods qRT-PCR assays were used to quantify levels of sex-specific mRNA transcripts in Plasmodium falciparum female and male gametocytes ( pfs25 and pfMGET respectively) using synthetic cRNA standards and in-vitro cultured gametocytes. Assay were validated and assay performance was investigated using blood samples of clinical trial participants (ClinicalTrials.gov reference number NCT02431637 and NCT02431650) using these standards and compared to absolute quantification by droplet digital PCR (ddPCR). Results The number of transcript copies per gametocyte were determined to be 279.3 (95% CI 253.5 - 307.6) for the female-specific transcript pfs25, and 12.5 (95% CI 10.6 - 14.9) for the male-specific transcript pfMGET . These numbers can be used to convert from transcript copies/mL to gametocyte/mL. The reportable range was determined to be 5.71x10 6 to 5.71 gametocytes/mL for pfs25, and 1.73x10 7 to 5.59 for pfMGET. The limit of detection was 3.9 (95% CI 2.5-8.2) gametocytes/mL for pfs25, and 26.9 (95% CI 19.3 - 51.7) gametocytes/mL for PfMGET . Both assays showed minimal intra-assay and inter-assay variability with coefficient of variation < 3%. No cross-reactivity was observed in both assays in uninfected human blood samples. Comparison of results from ddPCR to qRT-PCR assays on clinical blood samples indicated high level agreement (ICC=0.998 for pfs25 and 0.995 for pfMGET ). Conclusions We developed and validated qRT-PCR assays that are able to accurately quantify levels of female and male P. falciparum gametocytes at submicroscopic densities. The assays showed excellent reproducibility, sensitivity, precision, specificity and accuracy. The methodology will enable the estimation of gametocyte density in the absence of pure female and male gametocyte standards, and will facilitate clinical trials and epidemiological studies.

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A Molecular Assay to Quantify Male and Female Plasmodium falciparum Gametocytes: Results From 2 Randomized Controlled Trials Using Primaquine for Gametocyte Clearance

Abstract: SummaryA sensitive molecular assay was developed to quantify male and female Plasmodium falciparum gametocytes. Its application in 2 clinical trials demonstrates that the early effects of primaquine may be due to gametocyte fitness rather than sex ratio.

Cited by 51 publications

References 38 publications

P. falciparum gametocyte density and infectivity in peripheral blood and skin tissue of naturally infected parasite carriers

P. falciparum gametocyte density and infectivity in peripheral blood and skin tissue of naturally infected parasite carriers

Probabilistic data integration identifies reliable gametocyte-specific proteins and transcripts in malaria parasites

An assay for quantification of male and female gametocytes in human blood by qRT-PCR in the absence of pure gametocyte standards

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