Transmission-blocking Effects of Primaquine and Methylene Blue Suggest Plasmodium falciparum Gametocyte Sterilization Rather Than Effects on Sex Ratio

Bradley, John; Soumaré, Harouna M; Mahamar, Almahamoudou; Diawara, Halimatou; Roh, Michelle E.; Delves, Michael; Drakeley, Chris; Churcher, Thomas S; Dicko, Alassane; Gosling, Roly; Bousema, Teun

doi:10.1093/cid/ciz134

Cited by 23 publications

(27 citation statements)

References 11 publications

(23 reference statements)

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“…While the transmission-blocking effect of PQ may precede the gametocyte-clearing effect and gametocytes persisting after PQ may not result in onward transmission to mosquitoes (1, 6, 23), the results of the present study suggest that the efficacy of low-dose PQ may be affected by CYP2D6 metabolizer status. We previously demonstrated that PQ pharmacokinetics are influenced by genetically inferred CYP2D6 metabolizer status (22), suggesting that lower concentrations of the PQ active metabolites may occur in CYP2D6 PM/IM individuals.…”

Section: Discussionmentioning

confidence: 55%

CYP2D6 Polymorphisms and the Safety and Gametocytocidal Activity of Single-Dose Primaquine for Plasmodium falciparum

Pett

Bradley

Okebe

et al. 2019

Antimicrob Agents Chemother

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Single-dose primaquine (PQ) clears mature gametocytes and reduces the transmission of Plasmodium falciparum after artemisinin combination therapy. Genetic variation in CYP2D6, the gene producing the drug-metabolizing enzyme cytochrome P450 2D6 (CYP2D6), influences plasma concentrations of PQ and its metabolites and is associated with PQ treatment failure in Plasmodium vivax malaria. Using blood and saliva samples of varying quantity and quality from 8 clinical trials across Africa (n = 1,076), we were able to genotype CYP2D6 for 774 samples (72%). We determined whether genetic variation in CYP2D6 has implications for PQ efficacy in individuals with gametocytes at the time of PQ administration (n = 554) and for safety in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals treated with PQ (n = 110). Individuals with a genetically inferred CYP2D6 poor/intermediate metabolizer status had a higher gametocyte prevalence on day 7 or 10 after PQ than those with an extensive/ultrarapid CYP2D6 metabolizer status (odds ratio [OR] = 1.79 [95% confidence interval {CI}, 1.10, 2.90]; P = 0.018). The mean minimum hemoglobin concentrations during follow-up for G6PD-deficient individuals were 11.8 g/dl for CYP2D6 extensive/ultrarapid metabolizers and 12.1 g/dl for CYP2D6 poor/intermediate metabolizers (P = 0. 803). CYP2D6 genetically inferred metabolizer status was also not associated with anemia following PQ treatment (P = 0.331). We conclude that CYP2D6 poor/intermediate metabolizer status may be associated with prolonged gametocyte carriage after treatment with single-low-dose PQ but not with treatment safety.

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Section: Discussionmentioning

confidence: 55%

CYP2D6 Polymorphisms and the Safety and Gametocytocidal Activity of Single-Dose Primaquine for Plasmodium falciparum

Pett

Bradley

Okebe

et al. 2019

Antimicrob Agents Chemother

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“…The oocyst grows over time and, upon rupture, releases sporozoites that travel via haemolymph to the mosquito’s salivary glands and render the mosquito infectious to humans. Whilst the density of gametocytes is strongly associated with the likelihood of mosquitoes becoming infected [12, 13], transmission efficiency may differ between populations [12, 14] and is influenced by factors such as human immune responses [15] and treatment [13]. Malaria transmission thus needs to be quantified directly by allowing mosquitoes to feed on the skin of malaria-infected individuals or on venous blood drawn from infected individuals in direct membrane feeding assays (DMFA) [16, 17].…”

Section: Introductionmentioning

confidence: 99%

Is that a real oocyst? Insectary establishment and identification of Plasmodium falciparum oocysts in midguts of Anopheles mosquitoes fed on infected human blood in Tororo, Uganda

Musiime

Okoth

Conrad

et al. 2019

Malar J

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Background The human infectious reservoir for malaria consists of individuals capable of infecting mosquitoes. Oocyst prevalence and density are typical indicators of human infectivity to mosquitoes. However, identification of oocysts is challenging, particularly in areas of low malaria transmission intensity where few individuals may infect mosquitoes, and infected mosquitoes tend to have few oocysts. Here, features that differentiate oocysts from other oocyst-like in mosquito midguts are explained and illustrated. In addition, the establishment and maintenance of infrastructure to perform malaria transmission experiments is described. This work may support other initiatives to set up membrane feeding infrastructure and guide oocyst detection in low transmission settings. Methods In 2014, an insectary was developed and equipped in Tororo district, Uganda. A colony of Anopheles gambiae s.s. mosquitoes (Kisumu strain) was initiated to support infectivity experiments from participants enrolled in a large cohort study. Venous blood drawn from participants who were naturally infected with malaria parasites was used for membrane feeding assays, using 60–80 mosquitoes per experiment. Approximately 9–10 days after feeding, mosquitoes were dissected, and midguts were stained in mercurochrome and examined by light microscopy for Plasmodium falciparum oocysts and similar structures. In supportive experiments, different staining procedures were compared using in vitro cultured parasites. Results A stable colony of the Kisumu strain of An. gambiae s.s. was achieved, producing 5000–10,000 adult mosquitoes on a weekly basis. Challenges due to temperature fluctuations, mosquito pathogens and pests were successfully overcome. Oocysts were characterized by: presence of malaria pigment, clearly defined edge, round shape within the mosquito midgut or on the peripheral tissue and always attached to the epithelium. The main distinguishing feature between artifacts and mature oocysts was the presence of defined pigment within the oocysts. Conclusions Oocysts may be mistaken for other structures in mosquito midguts. Distinguishing real oocysts from oocyst-like structures may be challenging for inexperienced microscopists due to overlapping features. The characteristics and guidelines outlined here support identification of oocysts and reliable detection at low oocyst densities. Practical advice on sustaining a healthy mosquito colony for feeding experiments is provided. Following the reported optimization, the established infrastructure in Tororo allows assessments of infectivity of naturally infected parasite carriers.

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“…This emphasized the importance of quantifying female and male gametocytes, particularly in individuals with low-density Plasmodium infections. Gametocyte sex ratio is also important in characterizing the transmission-blocking effects of antimalarial drugs [ 20 , 22 ].…”

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confidence: 99%

qRT-PCR versus IFA-based Quantification of Male and Female Gametocytes in Low-Density Plasmodium falciparum Infections and Their Relevance for Transmission

Gruenberg

Hofmann

Nate

et al. 2019

The Journal of Infectious Diseases

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Background Accurate quantification of female and male gametocytes and sex ratios in asymptomatic low-density malaria infections are important for assessing their transmission potential. Gametocytes often escape detection even by molecular methods, therefore ultralow gametocyte densities were quantified in large blood volumes. Methods Female and male gametocytes were quantified in 161 PCR-positive Plasmodium falciparum infections from a cross-sectional survey in Papua New Guinea. Ten-fold concentrated RNA from 800 µL blood was analyzed using female-specific pfs25 and male-specific pfmget or mssp qRT-PCR. Gametocyte sex ratios from qRT-PCR were compared with those from immunofluorescence assays (IFA). Results Gametocytes were identified in 58% (93/161) P. falciparum-positive individuals. Mean gametocyte densities were frequently below 1 female and 1 male gametocyte/µL by qRT-PCR. The mean proportion of males was 0.39 (95% confidence interval, 0.33–0.44) by pfs25/pfmget qRT-PCR; this correlated well with IFA results (Pearsons r2 = 0.91; P < .001). A Poisson model fitted to our data predicted 16% P. falciparum-positive individuals that are likely to transmit, assuming at least 1 female and 1 male gametocyte per 2.5 µL mosquito bloodmeal. Conclusions Based on model estimates of female and male gametocytes per 2.5 µL blood, P. falciparum-positive individuals detected exclusively by ultrasensitive diagnostics are negligible for human-to-mosquito transmission. Estimating the transmission potential of ultralow-density malaria infections informs interventions. Almost all infections with ≥1 female and male gametocyte per 2.5 µL mosquito bloodmeal, and thus with highest likelihood of contributing to human-to-mosquito transmission, were detectable by standard molecular diagnostics.

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Transmission-blocking Effects of Primaquine and Methylene Blue Suggest Plasmodium falciparum Gametocyte Sterilization Rather Than Effects on Sex Ratio

Cited by 23 publications

References 11 publications

CYP2D6 Polymorphisms and the Safety and Gametocytocidal Activity of Single-Dose Primaquine for Plasmodium falciparum

CYP2D6 Polymorphisms and the Safety and Gametocytocidal Activity of Single-Dose Primaquine for Plasmodium falciparum

Is that a real oocyst? Insectary establishment and identification of Plasmodium falciparum oocysts in midguts of Anopheles mosquitoes fed on infected human blood in Tororo, Uganda

qRT-PCR versus IFA-based Quantification of Male and Female Gametocytes in Low-Density Plasmodium falciparum Infections and Their Relevance for Transmission

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