# An improved multiplex RT–quantitative PCR assay can reveal sex-specific activity of transmission-blocking drugs on ex vivo gametocytes from Plasmodium falciparum asymptomatic infections

## TL;DR

A new RT–qPCR method helps assess how drugs affect male and female malaria parasites, aiding in the development of transmission-blocking treatments.

## Contribution

An improved multiplex RT–qPCR assay for quantifying sex-specific gametocyte transcripts in malaria parasites.

## Key findings

- The assay revealed that methylene blue and P218 reduced gametocyte transcript levels, with P218 showing the most significant effect.
- Some drugs required a 48-hour post-treatment incubation to observe transcript level declines.
- Differential effects on male and female gametocyte transcripts were observed for two drugs.

## Abstract

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.