Indian Council of Medical Research-National Institute of Malaria Research, Sector 8, Dwarka, New Delhi, 110077, India.
Department of Chemistry, University of Delhi, Delhi, 110007, India.
Malar J. 2018 Jan 8;17(1):11. doi: 10.1186/s12936-017-2153-9.
Malaria remains a global health problem despite availability of effective tools. For malaria elimination, drugs targeting sexual stages of Plasmodium falciparum need to be incorporated in treatment regimen along with schizonticidal drugs to interrupt transmission. Primaquine is recommended as a transmission blocking drug for its effect on mature gametocytes but is not extensively utilized because of associated safety concerns among glucose-6-phosphate dehydrogenase (G6PD) deficient patients. In present work, methylene blue, which is proposed as an alternative to primaquine is investigated for its gametocytocidal activity amongst Indian field isolates. An effort has been made to establish Indian field isolates of P. falciparum as in vitro model for gametocytocidal drugs screening.
Plasmodium falciparum isolates were adapted to in vitro culture and induced to gametocyte production by hypoxanthine and culture was enriched for gametocyte stages using N-acetyl-glucosamine. Gametocytes were incubated with methylene blue for 48 h and stage specific gametocytocidal activity was evaluated by microscopic examination.
Plasmodium falciparum field isolates RKL-9 and JDP-8 were able to reproducibly produce gametocytes in high yield and were used to screen gametocytocidal drugs. Methylene blue was found to target gametocytes in a concentration dependent manner by either completely eliminating gametocytes or rendering them morphologically deformed with mean IC (early stages) as 424.1 nM and mean IC (late stages) as 106.4 nM. These morphologically altered gametocytes appeared highly degenerated having shrinkage, distortions and membrane deformations.
Field isolates that produce gametocytes in high yield in vitro can be identified and used to screen gametocytocidal drugs. These isolates should be used for validation of gametocytocidal hits obtained previously by using lab adapted reference strains. Methylene blue was found to target gametocytes produced from Indian field isolates and is proposed to be used as a gametocytocidal adjunct with artemisinin-based combination therapy. Further exploration of methylene blue in clinical studies amongst Indian population, including G6PD deficient patients, is recommended.
尽管有有效的工具，疟疾仍然是一个全球性的健康问题。为了消除疟疾，需要在治疗方案中加入针对疟原虫有性阶段的药物，与裂殖体杀灭药物一起阻断传播。由于葡萄糖-6-磷酸脱氢酶（G6PD）缺乏症患者存在相关安全问题，因此推荐使用伯氨喹作为传播阻断药物。然而，由于存在安全问题，伯氨喹并未得到广泛应用。在目前的工作中，研究了亚甲蓝作为替代伯氨喹的药物，以评估其对印度田间分离株的配子体杀伤活性。努力建立了疟原虫印度田间分离株的体外模型，用于配子体杀伤药物的筛选。
适应疟原虫体外培养，通过次黄嘌呤诱导配子体产生，并使用 N-乙酰葡萄糖胺富集配子体阶段。将配子体与亚甲蓝孵育 48 小时，通过显微镜检查评估阶段特异性配子体杀伤活性。
疟原虫田间分离株 RKL-9 和 JDP-8 能够稳定地产生高产量的配子体，并用于筛选配子体杀伤药物。亚甲蓝以浓度依赖的方式靶向配子体，要么完全消除配子体，要么使它们形态变形，早期阶段的平均 IC（早期阶段）为 424.1 nM，晚期阶段的平均 IC（晚期阶段）为 106.4 nM。这些形态改变的配子体显得高度退化，出现收缩、扭曲和膜变形。
可以鉴定出能够在体外产生高产量配子体的田间分离株，并用于筛选配子体杀伤药物。这些分离株应该用于验证以前使用实验室适应的参考株获得的配子体杀伤命中。亚甲蓝被发现能够靶向印度田间分离株产生的配子体，并被提议作为与基于青蒿素的联合疗法一起使用的配子体杀伤辅助药物。建议在包括 G6PD 缺乏症患者在内的印度人群中进一步探索亚甲蓝的临床研究。
