Assays for quantification of male and female gametocytes in human blood by qRT-PCR in the absence of pure sex-specific gametocyte standards

摘要

Successful transmission has been reported to occur at sub-microscopic gametocyte levels both in endemic areas during asymptomatic infections, and in volunteer infection studies (VIS), also referred to as controlled human malaria infection (CHMI)-transmission studies [6, 8, 9]. Understanding the contribution of sub-microscopic infections to the human infectious reservoir would greatly assist elimination efforts. Gametocyte densities have traditionally been measured by thick or thin film microscopy; however, both methods have limited sensitivity and cannot accurately measure gametocyte densities below 10,000 gametocytes per mL [10]. To overcome the limited sensitivity of microscopy, quantitative reverse transcription PCR (qRT-PCR) and quantitative nucleic acid sequence based amplification (NASBA) assays targeting gametocyte specific mRNA transcripts have been developed [6, 11–15]. The highly conserved pfs25 gene transcript is present in abundance in female gametocytes and is, therefore, commonly targeted for quantification by qRT-PCR [7, 14]. More recently, male-specific gametocyte mRNA transcripts have been described [5, 16, 17], and among them pfMGET (Pf3D7_1469900) shows an abundant transcription profile [5] making it an appropriate candidate for quantitation of male gametocytes. The use of both female- and male-specific assays allows evaluation of the gametocyte sex ratio, a parameter of interest in quantifying infectivity to mosquitoes, particularly at lower gametocyte densities [5, 18–21]. The Pfs25-PfMGET combination for sex ratio determination was recently validated by immunofluorescence assays on field samples from gametocyte donors [22]. This ratio may be particularly important for assessment of gametocytocidal drug activity, as the less abundant male gametocytes may be more readily sterilized or killed by some anti-malarial drugs [14, 23, 24].