Ciardo Mariagrazia, Henry Noëlie B, Soulama Issiaka, Sermé Samuel S, Rotili Dante, Mai Antonello, Lombardo Fabrizio, Alano Pietro, Siciliano Giulia
Dipartimento Malattie Infettive, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome 00161, Italy.
Centre National de Recherche et de Formation sur le Paludisme, Ouagadougou, Burkina Faso.
J Antimicrob Chemother. 2025 Jul 1;80(7):1907-1914. doi: 10.1093/jac/dkaf146.
To develop a multiplex RT-quantitative PCR (RT-qPCR) assay to quantify sex-specific Plasmodium falciparum gametocyte transcripts (pfCCp4, pfMGET), for evaluating the impact of drug treatments on gametocyte viability for malaria transmission-blocking drug development.
We optimized an RT-qPCR assay incorporating a normalization transcript to use the ΔΔCt method (differences in Cycle threshold) to quantify gametocyte transcript levels. The assay was used on ex vivo gametocytes from P. falciparum natural infections exposed for 24 h to six drugs impairing mosquito transmission, as measured by the direct membrane feeding assay. Follow-up in vitro experiments showed that an additional 48 h incubation, following drug wash-out, was required to monitor decline in transcript levels and potential sex-specific effects.
The optimized assay revealed efficacy of drug treatment as a reduction in transcript levels for two of the six drugs tested: 30% for pfMGET and 80% for pfCCp4 in methylene blue (5 µM)-treated samples, and 75% for both sex-specific transcripts in samples treated with P218 (0.25 µM). In the remaining drugs, a 48 h incubation period post drug wash-out was required to measure a decline in transcript levels. Furthermore, a differential reduction in the levels of male versus female gametocyte transcripts suggested sex-specific effects for two of the drugs.
The multiplex RT-qPCR assay provides a reliable method to assess the inhibitory effects of drug treatments on P. falciparum gametocytes, with the potential to evaluate both overall and sex-specific impacts on gametocyte viability. This assay represents a valuable tool in the development and evaluation of transmission-blocking drugs, particularly in distinguishing effects on male and female gametocytes.
开发一种多重逆转录定量聚合酶链反应（RT-qPCR）检测方法，用于定量疟原虫配子体特异性转录本（pfCCp4、pfMGET），以评估药物治疗对疟原虫配子体活力的影响，助力疟疾传播阻断药物的研发。
我们优化了一种RT-qPCR检测方法，该方法纳入了一个标准化转录本，以使用ΔΔCt法（循环阈值差异）来定量配子体转录本水平。通过直接膜饲法检测，将该检测方法应用于来自恶性疟原虫自然感染的离体配子体，这些配子体暴露于六种损害蚊虫传播的药物中24小时。后续的体外实验表明，药物洗脱后需要额外孵育48小时，以监测转录本水平的下降以及潜在的性别特异性影响。
优化后的检测方法显示，在测试的六种药物中有两种药物治疗有效，即转录本水平降低：在亚甲蓝（5μM）处理的样本中，pfMGET降低30%，pfCCp4降低80%；在P218（0.25μM）处理的样本中，两种性别特异性转录本均降低75%。在其余药物中，药物洗脱后需要48小时的孵育期来测量转录本水平的下降。此外，雄配子体和雌配子体转录本水平的差异降低表明其中两种药物存在性别特异性影响。
多重RT-qPCR检测方法提供了一种可靠的方法来评估药物治疗对恶性疟原虫配子体的抑制作用，有潜力评估对配子体活力的总体和性别特异性影响。该检测方法是开发和评估传播阻断药物的宝贵工具，特别是在区分对雄配子体和雌配子体的影响方面。
