Number and proportion of P. falciparum gametocytes vary from acute infection to chronic parasite carriage despite unaltered sexual commitment rate
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Evaluation Summary:
The work evaluates the production of P. falciparum sexual stage parasites in blood samples from asymptomatic parasite carriers through the dry season and symptomatic malaria patients in the wet season. Sexual stage parasites are required for malaria transmission via mosquito, but historically their low levels in peripheral blood have limited analysis. The work here monitored asexual and sexual stage parasitemia and found that the relative expression of early gametocyte genes, including ap2-g, is similar in samples from asymptomatic and asymptotic individuals. The data was used to model gametocyte availability from the initial symptomatic infection through the chronic phase and advances the understanding of factors that contribute to malaria transmission.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Plasmodium falciparum infections persist through long dry seasons at low parasitaemia without causing malaria symptoms and thus remain untreated. In asymptomatic children, increased circulation of infected erythrocytes without adhering to the vascular endothelium is observed during the dry months, compared to febrile malaria in the wet season. However, alterations of parasite sexual commitment and gametocytogenesis have not been investigated. Here, we compared the expression of genes related to sexual commitment and gametocytogenesis, the proportion and density of P. falciparum gametocytes, and the blood concentration of phospholipids in dry season asymptomatic individuals versus symptomatic subjects in the wet season. Additionally, we adapted a within-host mathematical model considering asexual and sexually-committed parasites and gametocytes to understand the dynamics of gametocyte number and proportion as infections progress. Compared to clinical malaria cases, transcripts of late-stage gametocytes were predominantly upregulated in the dry season, associating with increased proportions of mature gametocytes; while transcription of genes related to parasite sexual commitment was unaltered throughout the year. Our data suggest that gametocyte density and proportion diverge as infections progress from recent transmission to chronic carriage, without alterations in the sexual commitment rate over time.
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Author Response:
Reviewer #2:
The overall approach of this study is to compare gametocyte related parameters of infected blood samples from asymptomatic children, in some cases followed over time, with matched samples from uncomplicated malaria infections during the transmission season. A variety of parameters are analysed to investigate which mechanisms are used by the parasite to ensure that gametocytes are present in adequate numbers at the end of the dry season to be efficiently taken by mosquitoes reappearing at the start of the rainy season.
Authors analyse expression levels of 333 P. falciparum gametocyte specific transcripts from a previously published transcriptome study on infected blood samples from asymptomatic children and from children with uncomplicated malaria. They conduct a Principal Component Analysis on 12 …
Author Response:
Reviewer #2:
The overall approach of this study is to compare gametocyte related parameters of infected blood samples from asymptomatic children, in some cases followed over time, with matched samples from uncomplicated malaria infections during the transmission season. A variety of parameters are analysed to investigate which mechanisms are used by the parasite to ensure that gametocytes are present in adequate numbers at the end of the dry season to be efficiently taken by mosquitoes reappearing at the start of the rainy season.
Authors analyse expression levels of 333 P. falciparum gametocyte specific transcripts from a previously published transcriptome study on infected blood samples from asymptomatic children and from children with uncomplicated malaria. They conduct a Principal Component Analysis on 12 samples from each condition, revealing that PCA1 explains 65% of the seasonality variance, and identify 146 transcripts upregulated and 59 downregulated in the asymptomatic vs symptomatic carriers. This result may be expected as gametocyte physiology is likely different in the two very different conditions.
The following step is investigating male and female gametocyte densities and relative proportions on asexual stages by Realtime analysis of specific transcripts. The main conclusions of this part of the manuscript are 1- that symptomatic malaria cases in the wet season have higher parasite levels and consequently gametocyte densities than asymptomatic cases in the dry season and 2- that the lower parasitaemia levels in the asymptomatic cases show a comparatively higher proportion of sexual stages, precisely of female and, albeit not significantly, of male gametocytes.
Some observations and unclear points on this part of the work are the following:
• More details are needed to clarify how density and proportion of the different parasite stages presented in Figure 1 panels C, D and E have been derived from the Realtime experiments.
• Male and female gametocyte numbers are calculated using published calibration curves for genes pfs25 (female) and pfMGET (males). Incidentally, these are published in https://malariajournal.biomedcentral.com/articles/10.1186/s12936-018-2584-y and not in reference 32.
This has now been corrected.
• The calibration curves for the pfGLY and the pf17 transcripts, used to quantify total parasites and total gametocytes, respectively, and those used to quantify sexual commitment are instead missing and should be shown or referred to. This is relevant both for the determination of parasite numbers and for the use of the DeltaDeltaCt method.
We now provide the standard curves obtained with serial dilutions of synthetic DNA of P25, PfMGET, Pfg17 and Glycine-tRNA ligase and the respective primer efficiencies in the new Fig. S3.
• In panel C, it is intriguing that total parasite densities coincide with gametocyte densities. Comparing total gametocyte density in panel C with those of male and female gametocytes in panel D, it is puzzling that the former, quantified by the gametocyte specific marker pfg17, dramatically differs from those determined using pfs25 and pfMGET.
We believe it is difficult to compare levels of gametocyte densities across different transcripts, because the best approximation to equal primer efficiencies and amplifications often falls short of perfect. Prior reports by Essuman et al. JID 2017 also report the gametocyte specific marker Pfg17 to clearly outperform P25 in detecting gametocyte positive individuals in asymptomatic carriers potentially explaining the observed differences.
• It would be interesting to see if the proportion of male and female gametocytes on total parasites, calculated by the DeltaDeltaCt method in panel E, is comparable with the value that can be calculated from the density data in panels C and D.
• Finally, it would be valuable to derive from these data the gametocyte sex ratio to more synthetically describe the different infections.
We used qPCR and made use of standard curves to quantify the density of total, and male and female gametocytes to allow comparisons between groups of samples over time and different clinical symptoms. We did not compare these attributes between or across the groups, given the difficulty in achieving perfect purity in the standards with sorted gametocytes and the limitations imposed by imperfect match in different primers efficiencies'. We believe, however, that our approach is reasonable and adapted to the questions addressed and proposed interpretations.
A general point on this part of the work is that the transcriptome work was conducted on 12 vs 12 samples from asymptomatic and clinical cases, whereas the subsequent experiments were conducted on 35 samples from the dry season and 27 clinical cases. This is not very clear from the text and it rather looks like that the latter experiments were designed to better characterise the samples used in the transcriptome analysis. Also, the subsequent analysis of phospholipid levels and of the sexual commitment transcript levels appear to be performed on a subset of the samples.
We used different sets of samples for the different analyses of Malian samples and we improved the corresponding explanations within the results section. In figure 1 samples collected in 2012, the same year as the 12 + 12 samples used in the RNAseq analysis reported in Andrade et al. (presented on the PCA and heatmap) were used for both parasite transcript levels and plasma phospholipid analyses. In figures 2 and 4 we show samples collected in 2017/18 to better characterize the phenotype seen in Fig 1 with the inclusion of more time-points and longitudinal analyses of parasite transcript levels and plasma phospholipids. We revised the text to improve the presentation of the correct experimental layout throughout the results section.
In the following section, authors describe a longitudinal study on a smaller cohort of children from the above asymptomatic cases. Analysis of parasite densities, of phospholipid levels and of transcript levels of genes involved in sexual commitment essentially confirms the single point analyses described in the first part of the manuscript. The issues raised above on the determination of male and female gametocyte numbers and relative proportions conducted on these samples apply here as well.
As 2-4fold reduced levels of phospholipids, described to affect rate of gametocytogenesis, were measured in the samples from uncomplicated malaria, sera from symptomatic and asymptomatic cases were compared to measure whether those with reduced phospholipid levels induced sexual commitment of in vitro cultivated parasites. However, as authors themselves state, the observed differences in Lyso-PC levels between the sera were predictably too small to produce an effect in these experiments, as compared with the fold difference described to see effect on sexual commitment.
Having concluded so far that level of early gametocyte and sexual commitment markers do not change in the wet vs the dry season and that the low parasitaemias associated with the latter condition show a higher proportion of sexual stage transcripts/cells, another mRNA expression comparison has been conducted on a set of 163 transcripts selected as some "define early stages" and others "are characteristic of late stages". Assumptions, rationale and conclusions of this analysis are not entirely clear. The predictive value of the gene sets is not clear: available gametocyte stage-specific transcriptomic data fail to identify large numbers of transcripts unambiguously and specifically associated to early vs late gametocytes, so use of the 163 genes requires details on their stage specific diagnostic power. The analysis shows that 43 out of 54 "late gametocyte" transcripts vs 42 out of 109 "early gametocyte" transcripts are upregulated in the samples from asymptomatic cases, which leads authors to propose that this condition has "an effect during the 8-12 days of gametocyte development, in both/either the sexual and/or the asexual parasite compartments". This part requires attention and a clearer formulation of rationale and conclusions.
We now clarify how the subset of Early/Late gametocyte genes was defined and added a new column (F) in Table S1 to help the reader.
In conclusion, this work produced evidences consistent with the hypothesis that the efficient clearance of asexual stages in low parasitaemia asymptomatic infections explains the increase in the proportion of mature circulating gametocytes.
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Evaluation Summary:
The work evaluates the production of P. falciparum sexual stage parasites in blood samples from asymptomatic parasite carriers through the dry season and symptomatic malaria patients in the wet season. Sexual stage parasites are required for malaria transmission via mosquito, but historically their low levels in peripheral blood have limited analysis. The work here monitored asexual and sexual stage parasitemia and found that the relative expression of early gametocyte genes, including ap2-g, is similar in samples from asymptomatic and asymptotic individuals. The data was used to model gametocyte availability from the initial symptomatic infection through the chronic phase and advances the understanding of factors that contribute to malaria transmission.
(This preprint has been reviewed by eLife. We include the …
Evaluation Summary:
The work evaluates the production of P. falciparum sexual stage parasites in blood samples from asymptomatic parasite carriers through the dry season and symptomatic malaria patients in the wet season. Sexual stage parasites are required for malaria transmission via mosquito, but historically their low levels in peripheral blood have limited analysis. The work here monitored asexual and sexual stage parasitemia and found that the relative expression of early gametocyte genes, including ap2-g, is similar in samples from asymptomatic and asymptotic individuals. The data was used to model gametocyte availability from the initial symptomatic infection through the chronic phase and advances the understanding of factors that contribute to malaria transmission.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Demonstrating that the proportion of asexual parasites that convert to gametocytes is similar in asymptomatic and symptomatic infections using the established biomarker ap2-g, suggests that the apparent increase in gametocyte density observed during symptomatic infection is due to an increase in total parasitemia, not a higher conversion rate. Moreover, they show that that mean conversion rate is not changed even though lysophosphatidylcholine (lysoPC) concentrations are significantly lower during a symptomatic infection. LysoPC deficient media has previously been shown to stimulate gametocyte conversion in vitro, but they confirm in vitro that lysoPC levels did not drop low enough in the symptomatic plasma to enhance conversion. Together the results suggest that under these conditions environmental factors …
Reviewer #1 (Public Review):
Demonstrating that the proportion of asexual parasites that convert to gametocytes is similar in asymptomatic and symptomatic infections using the established biomarker ap2-g, suggests that the apparent increase in gametocyte density observed during symptomatic infection is due to an increase in total parasitemia, not a higher conversion rate. Moreover, they show that that mean conversion rate is not changed even though lysophosphatidylcholine (lysoPC) concentrations are significantly lower during a symptomatic infection. LysoPC deficient media has previously been shown to stimulate gametocyte conversion in vitro, but they confirm in vitro that lysoPC levels did not drop low enough in the symptomatic plasma to enhance conversion. Together the results suggest that under these conditions environmental factors are not influencing gametocyte production at the population level, which is a major question in the design of transmission blocking strategies.
Additional information about the quantification of parasite and gametocyte numbers is needed to critically analyze the data comparing asexual and total, male, and female gametocyte proportions in symptomatic infections and overtime in asymptomatic infections. As they state in line 287, these comparisons are complicated by the long development time for P. falciparum gametocytes. The mature gametocytes in a blood samples have been sequestered for ~10 days as they develop prior to release into the blood stream, while the asexual parasitemia continues to expand through 4-5 life cycles. Consequently, it is difficult to compare the asexual/gametocyte ratio in a sample from a chronic ongoing infection and an acute symptomatic infection. This difference also complicates the transcriptome analysis. The symptomatic children would have to be followed overtime to track gametocyte dynamics. Another advance is the development of a model that recapitulates their data and should allow future analysis of factors that would have the most impact on reducing gametocyte production.
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Reviewer #2 (Public Review):
The overall approach of this study is to compare gametocyte related parameters of infected blood samples from asymptomatic children, in some cases followed over time, with matched samples from uncomplicated malaria infections during the transmission season. A variety of parameters are analysed to investigate which mechanisms are used by the parasite to ensure that gametocytes are present in adequate numbers at the end of the dry season to be efficiently taken by mosquitoes reappearing at the start of the rainy season.
Authors analyse expression levels of 333 P. falciparum gametocyte specific transcripts from a previously published transcriptome study on infected blood samples from asymptomatic children and from children with uncomplicated malaria. They conduct a Principal Component Analysis on 12 samples …
Reviewer #2 (Public Review):
The overall approach of this study is to compare gametocyte related parameters of infected blood samples from asymptomatic children, in some cases followed over time, with matched samples from uncomplicated malaria infections during the transmission season. A variety of parameters are analysed to investigate which mechanisms are used by the parasite to ensure that gametocytes are present in adequate numbers at the end of the dry season to be efficiently taken by mosquitoes reappearing at the start of the rainy season.
Authors analyse expression levels of 333 P. falciparum gametocyte specific transcripts from a previously published transcriptome study on infected blood samples from asymptomatic children and from children with uncomplicated malaria. They conduct a Principal Component Analysis on 12 samples from each condition, revealing that PCA1 explains 65% of the seasonality variance, and identify 146 transcripts upregulated and 59 downregulated in the asymptomatic vs symptomatic carriers. This result may be expected as gametocyte physiology is likely different in the two very different conditions.
The following step is investigating male and female gametocyte densities and relative proportions on asexual stages by Realtime analysis of specific transcripts. The main conclusions of this part of the manuscript are 1- that symptomatic malaria cases in the wet season have higher parasite levels and consequently gametocyte densities than asymptomatic cases in the dry season and 2- that the lower parasitaemia levels in the asymptomatic cases show a comparatively higher proportion of sexual stages, precisely of female and, albeit not significantly, of male gametocytes.
Some observations and unclear points on this part of the work are the following:
• More details are needed to clarify how density and proportion of the different parasite stages presented in Figure 1 panels C, D and E have been derived from the Realtime experiments.
• Male and female gametocyte numbers are calculated using published calibration curves for genes pfs25 (female) and pfMGET (males). Incidentally, these are published in https://malariajournal.biomedcentral.com/articles/10.1186/s12936-018-2584-y and not in reference 32.
• The calibration curves for the pfGLY and the pf17 transcripts, used to quantify total parasites and total gametocytes, respectively, and those used to quantify sexual commitment are instead missing and should be shown or referred to. This is relevant both for the determination of parasite numbers and for the use of the DeltaDeltaCt method.
• In panel C, it is intriguing that total parasite densities coincide with gametocyte densities. Comparing total gametocyte density in panel C with those of male and female gametocytes in panel D, it is puzzling that the former, quantified by the gametocyte specific marker pfg17, dramatically differs from those determined using pfs25 and pfMGET.
• It would be interesting to see if the proportion of male and female gametocytes on total parasites, calculated by the DeltaDeltaCt method in panel E, is comparable with the value that can be calculated from the density data in panels C and D.
• Finally, it would be valuable to derive from these data the gametocyte sex ratio to more synthetically describe the different infections.
A general point on this part of the work is that the transcriptome work was conducted on 12 vs 12 samples from asymptomatic and clinical cases, whereas the subsequent experiments were conducted on 35 samples from the dry season and 27 clinical cases. This is not very clear from the text and it rather looks like that the latter experiments were designed to better characterise the samples used in the transcriptome analysis. Also, the subsequent analysis of phospholipid levels and of the sexual commitment transcript levels appear to be performed on a subset of the samples.
In the following section, authors describe a longitudinal study on a smaller cohort of children from the above asymptomatic cases. Analysis of parasite densities, of phospholipid levels and of transcript levels of genes involved in sexual commitment essentially confirms the single point analyses described in the first part of the manuscript. The issues raised above on the determination of male and female gametocyte numbers and relative proportions conducted on these samples apply here as well.As 2-4fold reduced levels of phospholipids, described to affect rate of gametocytogenesis, were measured in the samples from uncomplicated malaria, sera from symptomatic and asymptomatic cases were compared to measure whether those with reduced phospholipid levels induced sexual commitment of in vitro cultivated parasites. However, as authors themselves state, the observed differences in Lyso-PC levels between the sera were predictably too small to produce an effect in these experiments, as compared with the fold difference described to see effect on sexual commitment.
Having concluded so far that level of early gametocyte and sexual commitment markers do not change in the wet vs the dry season and that the low parasitaemias associated with the latter condition show a higher proportion of sexual stage transcripts/cells, another mRNA expression comparison has been conducted on a set of 163 transcripts selected as some "define early stages" and others "are characteristic of late stages". Assumptions, rationale and conclusions of this analysis are not entirely clear. The predictive value of the gene sets is not clear: available gametocyte stage-specific transcriptomic data fail to identify large numbers of transcripts unambiguously and specifically associated to early vs late gametocytes, so use of the 163 genes requires details on their stage specific diagnostic power. The analysis shows that 43 out of 54 "late gametocyte" transcripts vs 42 out of 109 "early gametocyte" transcripts are upregulated in the samples from asymptomatic cases, which leads authors to propose that this condition has "an effect during the 8-12 days of gametocyte development, in both/either the sexual and/or the asexual parasite compartments". This part requires attention and a clearer formulation of rationale and conclusions.
In conclusion, this work produced evidences consistent with the hypothesis that the efficient clearance of asexual stages in low parasitaemia asymptomatic infections explains the increase in the proportion of mature circulating gametocytes.
-