Abstract
Background
Understanding the influence of gametocyte dynamics on transmission of Plasmodium falciparum from humans to mosquitoes may support malaria elimination initiatives.
Methods
Longitudinal parasitemia, gametocytemia, and mosquito transmission data from 91 participants in 9 controlled human malaria infection studies conducted in Brisbane, Australia (n = 8) and Nijmegen, the Netherlands (n = 1) were analyzed to explore the relationship of parasite and participant characteristics on gametocytes and transmission. Transmission potential estimated using Poisson probabilities was compared to transmission to Anopheles stephensi mosquitoes assessed using direct and membrane feeding assays across 5 clinical trials and 30 participants.
Results
Maximum gametocyte density over time per participant was positively correlated with asexual parasitemia at time of first treatment (ρ minimum = 0.44, maximum = 0.89). Median maximum gametocyte densities occurring 19–21 days postinoculation were 1087 gametocytes/mL for female and 219 gametocytes/mL for male gametocytes. Both female and male gametocyte densities were higher among individuals who infected mosquitoes, as were pretreatment parasite growth rates and parasite densities. Infectivity rates varied between 53% and 54% for direct skin, 29% and 33% for direct membrane, 54% and 100% for membrane after serum replacement, and 89% and 100% for enriched membrane feeding assays. Potential for transmission was correctly predicted for 71%–74% of direct feeding assays and 50%–78% of membrane after serum replacement feeding assays.
Conclusions
This study confirms that transmissible gametocyte densities can be consistently achieved in controlled infections. Higher asexual densities and growth rates were associated with higher gametocyte densities and higher transmission. Detailed understanding of how low-level gametocytemia contributes to transmission will guide both experimental infection studies and interventions to interrupt transmission.
Keywords: Plasmodium falciparum, gametocytes, mosquito infectivity, transmission, induced blood stage malaria
Using data from controlled infections, this study assesses how gametocyte development influences the transmission of Plasmodium falciparum from humans to mosquitoes. Factors examined include host, parasite drug treatment, study design, and the type and timing of transmission assay used.
Individuals in malaria-endemic areas with asymptomatic infection commonly have low levels of parasitemia but sufficiently high gametocyte densities to render them infectious to mosquitoes [1]. Scalable techniques to identify and treat all infectious individuals would be desirable in preelimination settings. However, detecting such individuals requires highly sensitive molecular techniques [2]. In controlled human malaria infections (CHMIs), study participants are intentionally infected with Plasmodium parasites to evaluate investigational malaria vaccines and drugs. Recently, CHMIs have been enhanced to evaluate transmission of Plasmodium falciparum [3–9] and Plasmodium vivax [10] from humans to mosquitoes. In studies with P falciparum, infection is initiated either by sporozoites from bites of infected mosquitoes or by induced blood stage malaria (IBSM) infection whereby P falciparum–infected erythrocytes are administered intravenously. CHMI studies that evaluate transmission-blocking interventions require sufficient levels of circulating gametocytes to reliably transmit to mosquitoes. As gametocytogenesis depends on the replication of asexual parasites, the level of gametocytes required for an experimental transmission study needs to be balanced against the potential risks of escalating blood stage malaria infection to achieve higher gametocytemia.
Transmission from humans to mosquitoes is evaluated by direct skin feeding or membrane feeding assays following gametocyte maturation [11]. Critical determinants of successful mosquito transmission include density of gametocytes in the host's blood [4, 12], human genetic factors [13], human clinical and immune responses [14, 15], maturity of gametocytes [16, 17], and gametocyte sex ratio [12, 18, 19]. For low parasitemia infections, as typically observed in CHMI studies, transmission may be hindered by low levels of gametocytes, with male gametocyte density the likely limiting factor [12]. Implementation of polymerase chain reaction (PCR) assays targeting sex-specific messenger RNA (mRNA) transcripts, such as the male markers pfmget and mssp and the female markers pfs25 and ccp4, enables more sensitive sex-specific gametocyte quantitation [20]. Although quantification of sex-specific gametocytes can be imprecise [21], simultaneously detecting and quantifying male and female gametocytes using highly sensitive assays may help predict onward transmission potential [12, 20]. Understanding how individuals with different gametocyte densities contribute to onward transmission is important for guiding elimination strategies, including drug and vaccine approaches to interrupt transmission.
This study combined data from participants previously enrolled in 8 CHMI clinical trials conducted at QIMR Berghofer (n = 79 participants) and 1 CHMI clinical trial from Nijmegen (n = 12 participants) to evaluate factors contributing to gametocyte development and transmission success, by analyzing available longitudinal parasitemia, gametocytemia, and mosquito transmission data. Using Poisson probabilities based on male and female gametocyte densities to predict onward transmission in settings of low levels of parasitemia, we evaluated predicted transmission across 4 different feeding assays [12]. Insights from this work should guide development of future CHMI transmission studies and provide a baseline for studies evaluating malaria transmission at low parasitemia levels in endemic areas by improving understanding of the contribution of low-level gametocytemia on malaria transmission.
METHODS
Induced Blood Stage Malaria Studies
We analyzed longitudinal gametocyte density and parasitemia data from 79 participants in 8 IBSM clinical trials conducted at QIMR Berghofer, Brisbane, Australia, between 2013 and 2019 (Brisbane studies [4, 6, 7, 22–25]), and from 12 participants from a clinical trial conducted at Radboud University Medical Center, Nijmegen, the Netherlands, in 2018 (Nijmegen study [3]). All Brisbane studies were approved by the QIMR Berghofer Medical Research Institute Human Research Ethics Committee, and the Nijmegen study was approved by the Central Committee for Research Involving Human Subjects and Western Institutional Review Board. All participants provided written informed consent. Trial registrations, study details, and references are presented in Supplementary Table 1. The data for the Nijmegen study are available online [26].
Participants were inoculated intravenously on day 0 with human erythrocytes infected with P falciparum 3D7 parasites and treated with antimalarials to clear asexual parasitemia. Antimalarial treatment included piperaquine [3, 4, 25], DSM265 [22, 23], KAE609 [24], MMV048 [6], and artesunate [7], with dosing and timing of administration varying between studies (Supplementary Table 1). In the Nijmegen study, 5 individuals received additional low-dose piperaquine if asexual parasitemia exceeded 1500 parasites/mL after first treatment and before day 21 [3].
Blood samples were collected by venipuncture at protocol-specified time-points, with more frequent sampling during growth and clearance of asexual parasites (typically twice daily from days 4 to treatment, at least twice daily for 4 days after treatment, and daily sampling until day 20), and less frequent sampling (typically every 2–3 days) during the period of gametocyte circulation when pathogenic asexual parasites were not expected. Total parasitemia, encompassing both asexual parasites and gametocytes, was monitored using a quantitative PCR (qPCR) assay targeting the P falciparum 18S ribosomal RNA (rRNA) gene in Brisbane [27] and Nijmegen [28]. The appearance of gametocytes in the peripheral blood was subsequently confirmed by qPCR for male and/or female gametocytes, with male gametocytes tested for 45 of 79 participants in the 5 most recent Brisbane studies [4, 6, 7, 22]. Female gametocytes were assessed using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) for pfs25 mRNA in the Brisbane studies [29] and ccp4 mRNA in the Nijmegen study [30], and male gametocytes using qRT-PCR for pfmget at both sites [29, 30]. The lower limit of reportable range for gametocytes was 6 gametocytes/mL in the Brisbane studies (based on limit for female gametocytes) [29], and the limit of detection was 5 gametocytes/mL in the Nijmegen study [3]. Time to patency for gametocytes was defined as the first time-point above assay thresholds.
Infectivity of gametocytes to Anopheles stephensi mosquitoes (Sind-Kasur Nijmegen strain) was assessed for 18 of 79 participants in 4 Brisbane studies [4, 6, 7] and all 12 Nijmegen participants. All participants were first treated to clear asexual parasitemia on day 8. Symptoms increased with delayed treatment and elevated parasitemia, highlighting the need to carefully time treatment to balance transmission potential with participant safety. In Brisbane studies, participants with higher gametocytemia were selected for mosquito feeding assays; assays were conducted between days 17 and 30 and included in analyses only if conducted before rescue treatment or treatment with artemether-lumefantrine, primaquine, or atovaquone-proguanil (Supplementary Table 1). In the Brisbane studies, infectivity was assessed using direct skin feeding assays (DFA; n = 34), direct membrane feeding assays on venous whole blood (DMFA; n = 66), membrane feeding assays with serum replacement (MFA-SR; n = 26), and enriched membrane feeding assays after Percoll enrichment (eMFA-PE; n = 9) [4]. In the Nijmegen study, transmission was assessed using feeding assays for all 12 participants at day 21; inclusion of feeding assay data at days 24 and 29 was restricted to the 6 participants treated with piperaquine at day 21, as sulfadoxine-pyrimethamine compromised the transmissibility of gametocytes [3]. Therefore, infectivity in the Nijmegen study was assessed using 24 DFA, 24 DMFA, 6 MFA-SR, and 24 MFA after enrichment for gametocytes by magnetic-activated cell sorting (eMFA-MACS) [3].
In the Brisbane studies, mosquitoes were dissected 7–10 days after feeding and the 18S rDNA qPCR assay was used to detect and quantify oocysts in midguts [31]. In the Nijmegen study, mosquitoes were dissected 7–9 days after feeding and microscopy was used to detect and quantify midgut oocysts. Transmission to mosquitoes was defined as at least 1 oocyst-positive mosquito per feeding assay.
Statistical Methods
Analyses are presented separately for the Brisbane and Nijmegen studies. Nonparametric Kruskal–Wallis tests or 1-way analysis of variance were used to compare continuous outcomes between groups. Chi-squared tests or Fisher exact tests were used to compare categorical outcomes between groups. Correlations or associations between independent data were assessed using Spearman correlations or linear regression. Correlations or associations with repeated measurements per individual were assessed using repeated-measures correlation or linear mixed-effects models.
Parasitemia and Gametocyte Dynamics
Parasite growth rates were estimated using a random intercept nonlinear sine wave mixed-effects model [32], with group differences assessed using a time-by-group interaction. Parasitemia prior to recent schizont rupture preceding first antimalarial treatment was estimated from sine wave mixed models as the parasitemia at days 5.9 and 7.5 for participants treated on day 7 or 8, respectively, in the Brisbane studies, and at day 7.5 for Nijmegen (Supplementary Material 1). The proportion of male gametocytes was logit-transformed for analyses and back-transformed for interpretation; only time-points with detected male and female gametocytes were included in these analyses.
Gametocyte densities were either summarized for each individual by considering maximum gametocyte densities, or longitudinal changes were assessed as maximum gametocyte density within specified time-points (<20, 20–22, and >22 days since inoculation), and differences over time analyzed using random intercept linear mixed-effects models. Day 22 is assumed to correspond to the last and highest wave of gametocyte commitment occurring 14–15 days after treatment of asexual parasites, accounting for sequestration of 8–12 days and 2–3 days for circulating gametocytes to reach maturity [33].
Potential Transmission to Mosquitoes
Samples were considered to have potential for transmission based on a Poisson distribution to align with at least 1 female and 1 male gametocyte in an approximate mosquito blood meal volume of 2.5 µL [20]. Transmission potential was defined if the estimated probability of at least 1 gametocyte of each sex in a mosquito meal exceeded 0.4 (calculations in Supplementary Material 2). The first and last day for transmission potential for each individual was recorded.
Observed Transmission to Mosquitoes
Analyses considering transmission as a binary variable were conducted either for the feeding session, or summarized as overall transmission (yes/no) across all experiments for a particular feeding assay conducted for an individual participant. Differences between observed transmission and derived potential transmission were assessed with McNemar test. The relationship between logit-transformed proportion of mosquitoes infected for each feeding assay and log10-transformed female gametocyte density was estimated using a linear regression and back-transformed to the original scale. Linear mixed models assessed trends over time in logit-transformed proportion of mosquitoes infected, with a random effect for participant and an adjustment for log10-transformed female gametocyte density on the day of the feeding experiment.
Statistical analyses were performed using R Studio, version 2024.04.2 with R version 4.4.0.
RESULTS
Study and Participant Characteristics
The study included 79 individuals from 18 cohorts conducted in Brisbane and 12 individuals from 1 cohort in Nijmegen (Table 1). Median parasite densities at time of antimalarial treatment for asexual parasites were 14 256 (min–max, 271–393 678) and 14 299 (min–max, 1519–156 767) parasites/mL in the Brisbane and Nijmegen studies, respectively. All participants had female gametocytes detected; male gametocytes were detected in 38 of 45 (84%) Brisbane participants, and in all Nijmegen participants. Maximum densities of gametocytes were similar between study sites (Table 1), as were trends in gametocyte density over time (Supplementary Figure 1). Of the individuals tested for both gametocyte sexes, the Poisson distribution predicted that 20 (44.4%) Brisbane and 8 (66.7%) Nijmegen participants would have potential to transmit to mosquitoes for at least 1 time-point.
Table 1.
Characteristics of Study Participants and Parasitemia (Asexual and Gametocyte) Dynamics in the Brisbane and Nijmegen Studies
| Characteristic | Brisbane Studies | Nijmegen Study |
|---|---|---|
| Number of study cohorts | 18 | 1 |
| Number of individuals | 79 | 12 |
| Age, y | 24 (18–55) | 24 (19–29) |
| Sex, male, No. (%) | 56 (71%) | 4 (33%) |
| Day of treatment for asexual parasites, No. (%) | ||
| Day 7 | 40 (51%) | 0 (0%) |
| Day 8 | 39 (49%) | 12 (100%) |
| Inoculum size, No. (%) | ||
| 1800 | 33 (42%) | 0 (0%) |
| 2800 | 46 (58%) | 12 (100%) |
| Parasitemia at time of first treatment, parasites/mL | 14 256 (271–393 678) | 14 299 (1519–156 767) |
| Parasitemia prior to pretreatment schizont rupture, parasites/mL | 2534 (10–38 580) | 2060 (188–16 309) |
| Pretreatment parasite growth ratea, log10 parasites/mL/d (95% CI) | 0.71 (.68–.74) | 0.76 (.71–.82) |
| Female gametocytes, No. | 79 | 12 |
| Female gametocytes detected, No. (%) | 79 (100%) | 12 (100%) |
| First day of female gametocyte | 14 (10–22) | 11 (10–13) |
| Maximum female gametocyte density/mL | 830 (11–12 683) | 4736 (933–13 927) |
| Day of maximum female gametocyte density | 20 (14–26) | 21 (19–24) |
| Male gametocytes, No. | 45 | 12 |
| Male gametocytes detected, No. (%) | 38 (84%) | 12 (100%) |
| First day of male gametocyte | 15 (11–22) | 13 (12–15) |
| Maximum male gametocyte density/mL | 245 (7–1577) | 572 (114–1972) |
| Day of maximum male gametocyte density | 20 (14–28) | 19 (15–29) |
| Gametocyte characteristics | ||
| Individuals tested for male and female gametocytes, No. (%) | 45 (57%) | 12 (100%) |
| Potential for transmissionb, No. (%) | 20 (44%) | 8 (67%) |
| First day for transmission potentialb | 17 (15–28) | 17 (15–19) |
| Last recorded day for transmission potentialb | 24 (15–30) | 29 (21–29) |
Data are presented as median (minimum–maximum) unless otherwise specified.
Abbreviation: CI, confidence interval.
aEstimated as the coefficient and 95% CI interval for the time effect from a random intercept nonlinear mixed-effects model with log10 parasites/mL as the response variable.
bPotential for transmission based on Poisson distribution of having at least 1 male and 1 female gametocyte in a 2.5-µL blood sample.
Investigating Potential Factors Associated With Gametocyte Development
Gametocyte levels over the course of the malaria infection were similar in the Brisbane and Nijmegen studies (Supplementary Figure 1). Female and male gametocyte densities were positively correlated, and there was strong positive correlation between parasitemia at time of first treatment and maximum female (Brisbane: ρ = 0.55, P < .001; Nijmegen: ρ = 0.83, P = .002) and male gametocyte densities (Brisbane: ρ = 0.44, P = .006; Nijmegen ρ = 0.89, P < .001) (Figure 1). Similar correlations between parasitemia at time of first treatment and maximum gametocytes were observed for each time-period of <20, 20–22, and >22 days since inoculation (Supplementary Figure 2), with significant increasing trends in gametocyte density at later time-points (Supplementary Table 2).
Figure 1.
Association between (A) maximum female gametocyte density and parasitemia at time of first treatment (n = 79 Brisbane and n = 12 Nijmegen); (B) maximum male gametocyte density and parasitemia at time of first treatment (n = 45 Brisbane and n = 12 Nijmegen); (C) male gametocyte density and female gametocyte density (n = 378 observations in 45 Brisbane participants and n = 128 observations in 12 Nijmegen participants); and (D) maximum male and maximum female gametocyte density (n = 45 Brisbane and n = 12 Nijmegen). Data points in circles indicate samples from the Brisbane studies and triangles represent samples from the Nijmegen study. Dashed lines represent limit of detection for parasitemia and gametocyte assays. Lines of best fit of detected parasitemia and gametocytes are presented with 95% confidence intervals (CIs) for the Brisbane and Nijmegen studies for (A) and (B). Correlation for repeated measures with 95% CIs reported in (C) and Spearman correlation with bootstrapped 95% CIs reported in (A), (B), and (D).
Treating participants with antimalarials at day 8 rather than day 7 for 46 individuals inoculated with 2800 viable parasites in the Brisbane studies was associated with higher parasitemia at time of first treatment (P = .039) though there were no significant differences in maximum male or female gametocyte densities, or potential for transmission (Supplementary Table 3). For each time-period of <20, 20–22, and >22 days postinoculation, there were nonsignificant differences of maximum gametocyte density between those treated on day 7 or day 8 (Supplementary Table 4).
Exploring Potential Factors Associated With Transmission Success in Feeding Sessions
Corresponding gametocyte and parasitemia data were available for 135 feeding assays from 18 participants in the Brisbane studies and 78 feeding assays from 12 participants in the Nijmegen study. All participants were inoculated with approximately 2800 parasites and treated with first antimalarial on day 8. Characteristics of transmission to Anopheles mosquitoes using either DFA, DMFA, MFA-SR, or eMFA for the 2 sites are presented in Table 2. Infectivity rates were similar at both sites, with mean infectivity rates of approximately 53% for DFA and 30% for DMFA. Rates ranged between 54% and 100% for MFA-SR and 89 and 100% for eMFA in the Brisbane and Nijmegen studies, respectively.
Table 2.
Characteristics of Transmission to Anopheles Mosquitoes by 4 Different Feeding Assays, Presented for Each Feeding Assay Type for the Brisbane and Nijmegen Studies
| Feeding Assay | Study | No. of Feeding Sessions | No. of Individuals | Feeding Sessions per Individual, Median (Min–Max) | No. of Feeding Sessions Positivea | Percent Infectivity Ratea, Mean (95% CI) | No. of Mosquitoes Dissectedb, Median (Min–Max) | Percentage of Mosquitoes Infectedc, Median (Min–Max) |
|---|---|---|---|---|---|---|---|---|
| DFA | Brisbane | 34 | 12 | 3 (1–3) | 18 | 52.9 (35.1–70.2) | 30 (29–31) | 3.3 (0–16.7) |
| Nijmegen | 24 | 12 | 2 (1–3) | 13 | 54.2 (32.8–74.4) | 31 (18–36) | 3.0 (0–20.0) | |
| DMFA | Brisbane | 66 | 17 | 4 (1–6) | 22 | 33.3 (22.2–46.0) | 50 (49–92) | 0 (0–10.9) |
| Nijmegen | 24 | 12 | 2 (1–3) | 7 | 29.2 (12.6–51.1) | 25 (25–26) | 0 (0–12.0) | |
| MFA-SR | Brisbane | 26 | 9 | 3 (2–3) | 14 | 53.8 (33.4–73.4) | 50 (41–50) | 2.0 (0–14.0) |
| Nijmegen | 6 | 6 | 1 (1–1) | 6 | 100 (54.1–100) | 25 (23–25) | 22 (4–40.0) | |
| eMFAd | Brisbane | 9 | 6 | 1.5 (1–2) | 8 | 88.9 (51.8–99.7) | 50 (37–50) | 20 (0–52.0) |
| Nijmegen | 24 | 12 | 2 (1–3) | 24 | 100 (85.8–100) | 18 (15–21) | 73 (5–100) |
Abbreviations: CI, confidence interval; DFA, direct skin feeding assay; DMFA, direct membrane feeding assay on venous whole blood; eMFA, enriched membrane feeding assay; MFA-SR, membrane feeding assays with serum replacement.
aA session is considered positive or infective to mosquitoes if at least 1 mosquito is positive with at least 1 oocyst.
bNumber of mosquitoes that were dissected per feeding session and tested for presence of oocyst via 18S quantitative polymerase chain reaction (qPCR).
cPercentage of mosquitoes tested per feeding session via 18S qPCR that have at least 1 oocyst.
deMFA-PE used in Brisbane studies, and eMFA-MACS used in the Nijmegen study.
The relationship between log10-transformed female gametocyte density and proportion of mosquitoes infected was nonlinear, with infectivity increasing more rapidly as female gametocyte density increased before plateauing (Figure 2). Infectivity rates in MRA-SR and eMFAs increased rapidly with increasing female gametocyte density in the original sample (Figure 2C and 2D), with more variability and a less obvious relationship observed for DFA and DMFA (Figure 2A and 2B, Supplementary Figure 3). Gametocytes infected mosquitoes at very low densities for all types of feeding assays; DMFA required higher female gametocyte densities than DFA or experiments where gametocytes were concentrated by eMFA. Simultaneous consideration of the relationship of female and male gametocyte densities with mosquito infection was more complex and stochastic (Supplementary Figure 4), with transmission in DFA and DMFA observed for male gametocyte densities <200 gametocytes/mL (Brisbane, n = 4; Nijmegen, n = 1 feeding sessions). The proportion of mosquitoes infected in feeding assays adjusted for female gametocyte density increased as the follow-up time increased for DMFA (Nijmegen: time effect, P = .050), with a nonsignificant time effect for MFA-SR (Brisbane: P = .089) and eMFA (Nijmegen: P = .11) (Supplementary Material 3).
Figure 2.
Scatterplot of observed mosquito infectivity (proportion of dissected mosquitoes with at least 1 oocyst) and female gametocyte density (gametocytes [gams]/mL) for mosquito feeding assays performed using direct skin feeding assays (DFA, A), direct membrane feeding assays on venous whole blood (DMFA, B), membrane feeding assays with serum replacement (MFA-SR, C), and enriched membrane feeding assays (eMFA, D). Green points indicate samples from the Brisbane studies and purple points represent samples from the Nijmegen study. Solid points represent those with probable transmission, open circles represent those with no probable transmission, and open squares represent those with no male gametocyte data tested to evaluate probable transmission. Lines and 95% confidence intervals for the expected proportion of mosquitoes infected were generated from a linear model using logit probabilities. For the eMFA, the female gametocyte density is prior to enrichment and eMFA-PE used in Brisbane studies and eMFA-MACS used in the Nijmegen study.
When considering transmission as a dichotomous variable, feeding sessions with transmission had higher female gametocyte density, with significant differences in the Brisbane studies for DFA (P < .001) and DMFA (P = .002) and marginal significance for MFA-SR (P = .051) (Table 3). Similarly, feeding sessions with transmission had higher male gametocyte density for DFA (Brisbane, P = .002; Nijmegen, P = .060) and MFA-SR (Brisbane, P = .023). Comparisons for eMFA were not possible due to high transmission rates (8 of 9 sessions in Brisbane studies, and all 24 sessions in Nijmegen). There were no significant associations for proportion of male gametocytes and transmission in DFA or DMFA (Table 3). The accuracy of predicting transmission potential of a sample, estimated using Poisson probabilities based on female and male gametocyte densities, varied by site and feeding assay. The predicted transmission potential correctly classified 74% and 71% of observed transmission outcomes of DFA for the Brisbane and Nijmegen studies, respectively (Supplementary Figure 5). For the Brisbane and Nijmegen studies, respectively, the accuracy was 60% and 54% for DMFA, 78% and 50% for MFA-SR, and 63% and 42% for eMFA. McNemar test suggested significant discordance between estimated transmission potential and observed transmission by DMFA (Table 3 and Supplementary Figure 5). There was a significant difference in transmission for MFA-SR up to, or after, 22 days postinoculation (Brisbane, P = .045), but no significant differences between these time-points for DFA or DMFA (Table 3).
Table 3.
Association of Mosquito Transmission for the 4 Different Feeding Assays (as Defined as at Least 1 Mosquito With at Least 1 Oocyst Present) With Female and Male Gametocyte Density, Proportion Male Gametocytes, Potential for Transmission, and Timing of Feeding Session
| Feeding Assay | Brisbane Studies | Nijmegen Study | ||||
|---|---|---|---|---|---|---|
| Transmission | No Transmission | P Value | Transmission | No Transmission | P Value | |
| DFA | n = 18 | n = 16 | n = 13 | n = 11 | ||
| Female gametocytes/mL | 4612 (1803–12 683) | 1539 (234–7011) | <.001 | 5172 (1036–11 998) | 3948 (569–13 927) | .13 |
| Male gametocytes/mL | 523 (55–1577) | 102 (18–486)a | .002 | 537 (137–1795) | 394 (99–998) | .060 |
| Proportion male, mean (95% CI)b | 0.26 (.22–.30) | 0.25 (.22–.28)a | .74 | 0.12 (.09–.15) | 0.11 (.09–.14) | .82 |
| Potential for transmission, No. (%) | 14 (77.8%) | 4 (30.8%)a | 1.00c | 12 (92.3%) | 6 (54.5%) | .059c |
| Session >22 d since inoculation, No. (%) | 9 (50.0%) | 6 (37.5%) | .46 | 8 (61.5%) | 4 (36.4%) | .41d |
| DMFA | n = 22 | n = 44 | n = 7 | n = 17 | ||
| Female gametocytes/mL | 3765 (1100–12 683) | 1658 (30–9862) | .002 | 5677 (3029–11 128) | 2534 (569–13 927) | .053 |
| Male gametocytes/mL | 330 (36–839) | 192 (6–1577)e | .11 | 537 (402–1795) | 394 (99–1181) | .092 |
| Proportion male, mean (95% CI)b | 0.25 (.22–.29) | 0.26 (.25–.29)e | .47 | 0.10 (.07–.16) | 0.12 (.10–.15) | .49 |
| Potential for transmission, No. (%) | 16 (72.7%) | 18 (47.4%)e | .014c | 7 (100%) | 11 (64.7%) | <.001c |
| Session >22 d since inoculation, No. (%) | 12 (54.5%) | 21 (47.7%) | .60 | 6 (85.7%) | 6 (35.3%) | .069d |
| MFA-SR | n = 14 | n = 12 | n = 6 | n = 0 | ||
| Female gametocytes/mL | 2534 (1369–7972) | 1262 (189–5547) | .051 | 4089 (1087–11 998) | … | |
| Male gametocytes/mL | 397 (55–609) | 129 (12–567)f | .023 | 637 (201–1795) | … | |
| Proportion male, mean (95% CI)b | 0.27 (.23–.32) | 0.24 (.20–.28)f | .20 | 0.14 (.08–.24) | … | |
| Potential for transmission, No. (%) | 12 (85.7%) | 3 (33.3%)f | .65c | 5 (83.3%) | … | |
| Session >22 d since inoculation, No. (%) | 9 (64.3%) | 3 (25.0%) | .045 | 6 (100%) | … | |
| eMFAh | n = 8 | n = 1 | n = 24 | n = 0 | ||
| Female gametocytes/mL | 3558 (1595–9862) | 1531 | 4047 (569–13 927) | … | ||
| Male gametocytes/mL | 283 (102–1577) | g | 468 (99–1795) | … | ||
| Proportion male, mean (95% CI)b | 0.25 (.20–.31) | g | 0.12 (.10–.14) | … | ||
| Potential for transmission, No. (%) | 5 (62.5%) | g | 18 (75%) | … | ||
| Session >22 d since inoculation, No. (%) | 2 (25%) | 0 (0%) | 12 (50%) | … | ||
Data are presented as median (range) unless otherwise specified. Unit of measurement for analyses is the feeding session.
Abbreviations: CI, confidence interval; DFA, direct skin feeding assay; DMFA, direct membrane feeding assay on venous whole blood; eMFA, enriched membrane feeding assay; MFA-SR, membrane feeding assays with serum replacement.
an = 13 due to 3 missing/nondetected male gametocyte data.
bTwo-sample t-test and mean (95% CI) calculated on the logit-transformed proportion of male gametocytes, and back-transformed for interpretation.
cMcNemar test.
dFisher exact test.
en = 38 due to 6 missing/nondetected male gametocyte data.
fn = 9 due to 3 missing/nondetected male gametocyte data.
gn = 0 due to 1 missing/nondetected male gametocyte data.
heMFA-PE used in Brisbane studies and eMFA-MACS used in the Nijmegen study.
Exploring Potential Factors Associated With Transmission at Participant Level
For DFA and DMFA, participants who were infectious to mosquitoes for at least 1 feeding session had higher parasite growth rates and pretreatment parasitemia densities than those participants who were not infectious to mosquitoes (Tables 4 and 5). Eight (66.7%) Brisbane participants and 8 (66.7%) Nijmegen participants were infectious to mosquitoes in at least 1 DFA feeding session. Participants who were infectious to mosquitoes for at least 1 DFA had significantly higher parasitemia at pretreatment schizont rupture and time of first treatment (Brisbane, P = .011; Nijmegen, P = .062), higher parasite growth rates, and higher maximum female density and maximum male gametocyte density, and also reached gametocyte patency earlier (Brisbane, P = .007; Nijmegen, P = .85) (Table 4). A higher median parasitemia at time of first treatment was associated with infectivity to mosquitoes for at least 1 DMFA feeding session (Brisbane, P = .33; Nijmegen, P = .028), as was parasite growth rate (Brisbane, P = .11; Nijmegen, P < .001) and maximum female gametocyte density (Brisbane, P = .064; Nijmegen, P = .012) (Table 5).
Table 4.
Association of Participant, Parasite, and Gametocyte Characteristics With Participants’ Transmission Status
| Characteristica | Brisbane Studies | Nijmegen Study | ||||
|---|---|---|---|---|---|---|
| DFA Transmission (n = 8) | No DFA Transmission (n = 4) | P Value | DFA Transmission (n = 8) | No DFA Transmission (n = 4) | P Value | |
| Age, y | 27 (20–34) | 23.5 (20–32) | .50 | 25.5 (19–29) | 21.5 (19–24) | .20 |
| Sex, male, No. (%) | 5 (62.5%) | 3 (75.0%) | 1 | 3 (37.5%) | 1 (25.0%) | 1 |
| Parasitemia at time of first treatment, parasites/mL | 49 644 (20 588–107 981) | 9219 (2015–63 804) | .042 | 31 655 (4025–156 767) | 3467 (1519–4631) | .011 |
| Parasitemia prior to recent schizont rupture, parasites/mL | 7528 (3205–13 533) | 1748 (613–4101) | .011 | 4530 (188–16 309) | 660 (376–1629) | .062 |
| Pretreatment parasite growth rate, log10 parasites/mL/d (95% CI) | 0.771 (.714–.828) | 0.691 (.628–.754) | .002b | 0.796 (.743–.849) | 0.673 (.610–.737) | <.001b |
| Female gametocytes | ||||||
| Detected female gametocytes, No. (%) | 8 (100%) | 4 (100%) | 1 | 8 (100%) | 4 (100%) | 1 |
| First day of female gametocyte | 12 (11–13) | 13 (13–15) | .007 | 11 (10–13) | 11 (10–13) | .85 |
| Maximum female gametocyte density/mL | 5927 (2560–12 683) | 1691 (397–2985) | .011 | 5250 (1046–13 927) | 1243 (933–3948) | .027 |
| Day of maximum female gametocyte density | 23 (20–25) | 22 (20–24) | .66 | 20 (19–24) | 21 (21–24) | .14 |
| Male gametocytes | ||||||
| Detected male gametocytes, No. (%) | 8 (100%) | 3 (75%) | .33 | 8 (100%) | 4 (100%) | 1 |
| First day of male gametocyte | 14 (12–15) | 15 (11–15)c | 1 | 13 (12–15) | 13.5 (13–15) | .34 |
| Maximum male gametocyte density/mL | 617 (359–1577) | 94 (49–102)c | .014 | 803 (210–1972) | 187 (114–578) | .027 |
| Day of maximum male gametocyte density | 24 (19–28) | 24 (20–24)c | .91 | 19 (17–29) | 20 (19–24) | .41 |
Univariate analyses. Unit of measurement is the participant. A participant is defined as having experienced transmission if the participant was infectious to mosquitoes for at least 1 DFA feeding session.
Abbreviations: CI, confidence interval; DFA, direct skin feeding assay.
aUnless otherwise specified, data represent median (minimum–maximum) and P value estimated using Kruskal–Wallis test for continuous variables, and frequency and percentage and P value from Fisher exact test for categorical variables.
bEstimated as the coefficient and 95% CI for the time effect from a random intercept nonlinear mixed-effects model with log10 parasites/mL as the response variable.
cn = 3 for the number of participants who were tested for male gametocyte density.
Table 5.
Association of Participant, Parasite, and Gametocyte Characteristics With Participants’ Transmission Status
| Characteristica | Brisbane Studies | Nijmegen Study | ||||
|---|---|---|---|---|---|---|
| DMFA Transmission (n = 10) |
No DMFA Transmission (n = 7) | P Value | DMFA Transmission (n = 5) | No DMFA Transmission (n = 7) | P Value | |
| Age, y | 27 (20–48) | 24 (20–34) | .40 | 26 (20–29) | 20 (19–26) | .049 |
| Sex, male, No. (%) | 8 (80.0%) | 5 (71.4%) | 1 | 2 (40.0%) | 2 (28.6%) | 1 |
| Parasitemia at time of first treatment, parasites/mL | 35 858 (6025–79 307) | 14 515 (2015–107 981) | .33 | 38 833 (8837–156 767) | 4025 (1519–32 648) | .028 |
| Parasitemia prior to recent schizont rupture, parasites/mL | 5583 (442–12 099) | 2758 (613–13 533) | .77 | 5509 (1272–16 309) | 770 (188–5251) | .042 |
| Pretreatment parasite growth rate, log10 parasites/mL/d (95% CI) | 0.756 (.697–.815) | 0.716 (.654–mpa#ndash;.778) | .11b | 0.818 (.760–mpa#ndash;.876) | 0.706 (.649–mpa#ndash;.764) | <.001b |
| Female gametocytes | … | |||||
| Detected female gametocytes, No. (%) | 10 (100%) | 7 (100%) | 1 | 5 (100%) | 7 (100%) | 1 |
| First day of female gametocyte | 12 (11–13) | 13 (12–17) | .009 | 12 (10–13) | 10 (10–13) | .60 |
| Maximum female gametocyte density/mL | 5034 (1958–12 683) | 2102 (397–7011) | .064 | 5677 (4301–13 927) | 1398 (933–5199) | .012 |
| Day of maximum female gametocyte density | 20 (17–25) | 23 (19–24) | .20 | 21 (19–24) | 21 (19–24) | 1 |
| Male gametocytes | ||||||
| Detected male gametocytes, No. (%) | 10 (100%) | 6c (100%) | 1 | 5 (100%) | 7 (100%) | 1 |
| First day of male gametocyte | 15 (12–17) | 15 (11–17)c | .73 | 13 (13–15) | 13 (12–15) | .93 |
| Maximum male gametocyte density/mL | 617 (219–1577) | 245 (49–806)c | .065 | 1032 (537–1972) | 210 (114–908) | .062 |
| Day of maximum male gametocyte density | 20 (17–28) | 24 (19–24)c | .16 | 19 (17–29) | 19 (17–24) | .66 |
Univariate analyses. Unit of measurement is the participant. A participant is defined as having experienced transmission if the participant was infectious to mosquitoes for at least 1 DMFA feeding session.
Abbreviations: CI, confidence interval; DMFA, direct membrane feeding assay on venous whole blood.
aUnless otherwise specified, data represent median (minimum–maximum) and P value estimated using Kruskal–Wallis test for continuous variables, and frequency and percentage and P value from Fisher exact test for categorical variables.
bEstimated as the coefficient and 95% CI for the time effect from a random intercept nonlinear mixed-effects model with log10 parasites/mL as the response variable.
cn = 3 for the number of participants who were tested for male gametocyte density.
DISCUSSION
This descriptive study assesses observed and potential transmission of P falciparum infections in 9 IBSM studies conducted in Brisbane, Australia, and Nijmegen, the Netherlands. This study assesses how gametocyte production and onward transmission to mosquitoes vary by preceding asexual parasite density and growth dynamics. Utilizing longitudinal data with sensitive assays to detect low parasite and gametocyte densities, this analysis highlights the complex relationship between gametocyte density, gametocyte maturity, and mosquito infectivity, as well as the impact of different feeding assays on transmission outcomes.
Female gametocyte density showed a strong nonlinear relationship with proportion of infected mosquitoes; however, incorporating male gametocyte density made the relationship more complex due to stochasticity and variable transmission at lower gametocyte densities. Although accounting for both male and female gametocyte densities better predicts human-to-mosquito infection than using total gametocyte or female gametocyte densities alone [12], it remains unclear whether this reflects improved estimation of overall gametocyte biomass by combining 2 markers, versus a specific effect of sex ratio.
Predicted transmission potential from sex-specific gametocyte densities correctly classified 74% and 71% of observed transmission outcomes of DFA sessions in the Brisbane and Nijmegen studies, respectively (Supplementary Figure 5). However accuracy was lower for DMFA and eMFA, and more variable for MFA-SR, likely because these feeding assays depend less on gametocytemia in the original sample. Transmission is inherently stochastic, and discrepancies between predicted transmission potential and observed transmission may reflect differences of gametocyte composition in the blood samples used for DMFA, eMFA, and MFA-SR than of the mosquito blood meal. Despite these challenges, estimating transmission potential may assist in the design of future studies using DFA experiments by identifying participants most likely to transmit to mosquitoes. These predictions could be improved by considering model-based approaches [12].
Transmission to mosquitoes is highly variable and stochastic at low gametocyte densities, and as demonstrated here, differs by feeding assay type and timing. Although DFA replicates natural transmission, it is more challenging to implement in experimental settings than feeding assays using blood collected by venipuncture. This work explored associations between pretreatment parasitemia, gametocytemia, and onward transmission as an option to identify individuals for inclusion in subsequent transmission studies. Future studies with larger sample sizes could support development and validation of predictive models for onward transmission.
Data from 2 laboratories were analyzed without explicitly adjusting for differences in laboratory procedures such as qPCR methods [34], assay precision [12], conversion of gametocyte transcripts into gametocyte counts [20], and gametocyte concentration methods. Nonetheless, trends were generally similar across sites, with some differences in statistical significances potentially due to small sample sizes. The increased variability in the Brisbane studies likely reflects heterogeneity in study design (ie, antimalarials, doses, and objectives), whereas the Nijmegen data were from a single transmission study.
Included studies were either designed to evaluate efficacy of candidate antimalarials or transmission, and only included Brisbane studies published up to 2021. Two additional transmission studies from Brisbane have been published [8, 9], whereby all 10 participants had transmission as assessed by eMFA, with high infectivity rates (percentage of mosquitoes positive for oocyts) of median 86% (min–max, 22%–98%). These studies further support the utility of the CHMI model for evaluating transmission-blocking agents.
This analysis did not consider the effects of different antimalarials on gametocyte commitment, gametocyte health, or infectivity of gametocytes to mosquitoes. To mitigate the effect of drugs with known or suspected effects on gametocyte viability or transmission potential, data for transmission analyses were censored after administration of either artemether-lumefantrine [35], atovaquone-proguanil [36], primaquine [37] or sulfadoxine-pyrimethamine [3]. This study utilized data from highly controlled conditions, with feeding experiments during early days of an infection, indicating that transmission is efficient at low gametocyte densities. This has implications for defining minimal parasite densities that need to be considered in interventions evaluated in endemic settings that aim to include all potentially infectious individuals. However, the gametocyte and infectivity data in this study were over an infection lasting up to 30 days, substantially shorter than the course of P falciparum infections that can last many months in endemic settings [38]. Therefore, within the context of elimination, results from this study are informative but not comprehensive.
Several factors relevant to onward transmission in more natural settings were not explicitly investigated. These include gametocyte maturity [39], duration of gametocytemia, changes in gametocyte health over time, successful sporogony in the mosquito [40], host inflammatory state (C-reactive protein is negatively correlated with transmission intensity [8]), host immunity, and parasite genetic factors.
This study used longitudinal data from 91 participants in 9 IBSM studies to investigate factors influencing human-to-mosquito transmission at low gametocyte levels. Factors contributing to the likelihood of onward transmission include both host and parasite factors as well as study design factors, including timing of administration and choice of drugs used to clear asexual parasitemia, assays to quantify gametocyte levels, and the type and timing of feeding assay used to evaluate transmission. The limited accuracy of the models to predict onward transmission based on sex-specific densities suggests the role of additional undiscovered factors. With the modest number of observations, our data indicate a minimum gametocyte density of approximately 1000 female and male gametocytes per milliliter, below which transmission is unlikely. As many naturally infected individuals carry gametocytes at these low densities, these findings help define who may contribute to transmission among the gametocyte-positive population.
Supplementary Material
Notes
Author contributions. L. M.-W., P. O., and J. M. conceptualized the study. K. C., M. A., K. L., T. B., and J. M. generated data. L. M.-W., S. L., P. O., and L. W. prepared data for analyses. L. M.-W. and P. O. performed statistical analyses. L. M.-W., K. C., M. A., K. L., P. O., B. B., T. B., and J. M. reviewed and interpreted the results. L. M.-W., P. O., B. B., T. B., and J. M. drafted the manuscript. All authors contributed to the final manuscript with final responsibility for the decision to submit the report for publication.
Acknowledgments. We thank all volunteers who participated in the studies. For the QIMR Berghofer clinical trials, we thank the staff at QIMR Berghofer Medical Research Institute who managed and prepared the Plasmodium falciparum 3D7 inoculum; the clinical study team at Q-Pharm who conducted the trial; Claire Y. T. Wang and staff at the Queensland Paediatric Infectious Diseases laboratory for qPCR analysis; and Hayley Mitchell, Melanie Rampton, and Matthew Adams for the mosquito infection and dissection work. We thank Sam McEwan for assistance with data preparation and assistance with initial analyses. For the Nijmegen clinical trial, the authors thank staff from the Radboud University Medical Center Technology Center Clinical Studies; Geert-Jan van Gemert, Wouter Graumans, and Rianne Stoter for performing mosquito feeding assays; and Karina Teelen for contributing to molecular analyses.
Data availability. Data are available for the Nijmegen study at https://doi.org/10.5061/dryad.vq83bk3nq [26]. The data from the studies undertaken at QIMR Berghofer that support the findings of this study are available from the corresponding author upon reasonable request.
Financial support. The clinical trials undertaken in Brisbane were funded by the Medicines for Malaria Venture through a Wellcome Trust grant (reference no. 095909/Z/11/Z), a grant by the Global Health Innovation and Technology Fund (grant no. G2014-108), and by funding from the Gates Foundation. J. M. was funded by a National Health and Medical Research Council Investigator Grant (grant no. 2016396). The clinical trial undertaken in Nijmegen was supported by PATH's Malaria Vaccine Initiative (grant OPP1108403) and by the European Research Council (fellowship to T. B. from grant ERC-2014-StG 639776). T. B. is supported by a fellowship from The Netherlands Organisation for Scientific Research (Vici fellowship NWO project number 09150182210039).
All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
Contributor Information
Louise Marquart-Wilson, School of Public Health, University of Queensland, Brisbane, Queensland,Australia; Infection and Inflammation Program, QIMR Berghofer, Brisbane, Queensland, Australia.
Manon Alkema, Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlands.
Katharine A Collins, Infection and Inflammation Program, QIMR Berghofer, Brisbane, Queensland, Australia; Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlands.
Kjerstin Lanke, Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlands.
Peter O’Rourke, Infection and Inflammation Program, QIMR Berghofer, Brisbane, Queensland, Australia.
Stacey Llewellyn, Population Health Program, QIMR Berghofer, Brisbane, Queensland, Australia.
Lachlan Webb, Population Health Program, QIMR Berghofer, Brisbane, Queensland, Australia.
Bridget E Barber, Infection and Inflammation Program, QIMR Berghofer, Brisbane, Queensland, Australia.
Teun Bousema, Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlands.
James S McCarthy, Infection and Inflammation Program, QIMR Berghofer, Brisbane, Queensland, Australia; Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria,Australia; Infection and Global Health Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.
Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Citations
- Bousema T, Alkema M, Reuling IJ, et al. A randomized clinical trial to compare P. falciparum gametocytaemia and infectivity following blood-stage or mosquito bite induced controlled malaria infection. Dryad [preprint, not peer-reviewed]. Posted online 30 March 2022, Available at: 10.5061/dryad.vq83bk3nq. Accessed 13 April 2022. [DOI]