Potential of school-based preventive treatment to reduce Plasmodium falciparum transmission

In areas where malaria remains entrenched, novel transmission-reducing interventions are essential for malaria elimination. Here we report the impact of screening-and-treatment of asymptomatic schoolchildren (N=705) on gametocyte - the parasite stage responsible for human-to-mosquito transmission - carriage and use concomitant household-based surveys to predict the potential reduction in transmission in the surrounding community. Among 179 students with gametocyte-containing infections, 84% had positive malaria raid diagnostic tests. While gametocyte burden remained constant in untreated children, treatment with artemether-lumefantrine reduced the gametocyte prevalence (p<0.0001) from 51.8% to 9.7% and geometric mean gametocyte density (p=0.008) from 0.52 to 0.05 gametocytes/microliter. Based on these estimates, the gametocyte burden in the community could be reduced by 25-55% depending on the season and the measure used to characterize gametocyte carriage. Thus, school-based interventions to treat asymptomatic infections may be a high-yield approach to not only improve the health and education of schoolchildren, but also decrease malaria transmission.


Introduction 45
In the last two decades, many regions have made substantial progress toward elimination of 46 malaria. Recently, however, progress has stalled and malaria remains entrenched in some highly 47 endemic areas like Malawi. 1 In these areas, current interventions (e.g., vector control, universal 48 access to effective testing and treatment, and preventive treatment of high risk populations 2 ) may 49 not adequately interrupt human-to-mosquito Plasmodium falciparum transmission.  infections in individuals at lower risk of disease often remain untreated, creating a silent reservoir 51 that may perpetuate transmission. 3 To achieve malaria elimination, interventions must not only 52 prevent and treat disease but also decrease parasite transmission. 4 53 54 School-age children bear an under-appreciated burden of P. falciparum infection and may be a key 55 transmission reservoir. 5 School-age children frequently have higher prevalence of infection than 56 younger children and adults 6-10 and their infections more often contain gametocytes, the parasite 57 stage required for human-to-mosquito transmission. 8,11 When bitten, infected school-age children 58 and young children are similarly infectious to mosquitoes. 12 However, school-age children have 59 larger body surface area and less bed net use, increasing availability for bites. 13-16 These factors 60 together suggest school-age children are dominant sources of human-to-mosquito P. falciparum 61 transmission. [16][17][18] Indeed, expanding community-based seasonal malaria chemoprevention from 62 only young children to include school-age children was associated with a 20% decrease in clinical 63 malaria in older community members who did not receive the intervention. 19  We enrolled 786 students in school-based cohorts: 405 in the rainy season and 381 in the dry 87 season. Complete follow-up was obtained for 616 students (78% of enrolled students) 88 (Supplemental Figure S1). Among the 705 students with baseline data, the mean age was 10.4 89 years, 51% were female, and 47% reported sleeping under a net the previous night. The weighted 90 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229336 doi: medRxiv preprint prevalence of RDT-positivity among students at baseline was 42%. An additional 8% of students . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229336 doi: medRxiv preprint

Determinants of gametocyte burden in school-based cohorts at baseline 105
Overall, at baseline, 28% of students had gametocyte-containing infections (8-50% across schools 106 and seasons; Figure 1). To disentangle predictors of having an infection containing gametocytes 107 from those of simply having any infection, we evaluated predictors among only students with P. 108 falciparum infection. Among the 253 students with P. falciparum infection by PCR at baseline, 109 70% contained gametocytes. Infections were more likely to contain gametocytes in the rainy 110 season and in younger students, although the age association was not statistically significant (Table  111 1). Associations were comparable in multivariable models with or without non-significant 112 variables. 113 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229336 doi: medRxiv preprint * Obtained in a survey weighted logistic regression that analyzed the variables shown in the table ITN = insecticide treated net; OR = Odds Ratio; CI = Confidence Interval a Univariate association for school, all other associations include school. Prevalence setting: Bvumbwe-low, seasonal; Ngowe-moderate, seasonal; Maseya-high; Makhuwira-high. b Mean age of students with infections containing gametocytes was 10·9 years compared to 11·6 years among those whose infections did not contain gametocytes. c Hemoglobin <11·0 g/dL; 1 missing observation d Measured temperature ≥37·5ºC at visit or reported in the last two weeks e Reported in the last two weeks; 2 missing observations f 5 missing observations

114
The crude weighted geometric mean gametocyte density among those with gametocytes was 0.34 115 gametocytes/µl (95% CI: 0.19-0.60 gametocytes/µl; range 0.002-661 gametocytes/µl). 116 Gametocyte density was higher in the rainy season and decreased with age (Table 2). Overall, 117 5.3% of students had high-density infections (≥10 gametocytes/µl) at baseline. Among students 118 with gametocyte-containing infections, 19% had high density infections. In addition to season and 119 age, odds of high density gametocyte infection were significantly higher in children with fever and 120 in the lowest prevalence school (Bvumbwe; Supplemental Table S3). 121 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020.

Gametocyte-containing infections missed by RDT screening 123
A key factor in the success of screen-and-treat interventions is the test sensitivity for detecting 124 infections of interest, i.e., gametocyte-containing infections for transmission reduction. Sixteen 125 percent of all students gametocyte-containing infections and 9% with high-density gametocyte-126 containing infections were RDT-negative and thus not treated. RDTs were less likely to detect 127 gametocyte-containing infections at lower densities (Table 3). After adjusting for density, RDTs 128 also missed gametocyte-containing infections more often in the lowest prevalence school 129 (Bvumbwe) than other schools (OR for failure 6.6, 95% CI:1.9-24, p=0.004). 130 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020.  infections among treated students decreased from 52% (95% CI: 45-58%) at baseline to 11% (95% 136 CI: 7-14%) after two weeks, a 79% reduction. Gametocyte prevalence remained low after six 137 weeks [10% 95% CI: 6-14%)]. Among untreated students (RDT-negative), the prevalence of 138 gametocyte-containing infections remained unchanged from baseline [9% (95% CI: 5-14%)] to 139 six weeks [12% (95% CI: 7-17%)]. Gametocyte density followed the same trend, with 90% 140 reduction in the geometric mean of gametocyte density after treatment versus 59% increase in 141 untreated children. Two weeks after screening and treatment, only one high-density gametocyte 142 infection was detected in an untreated student who had been RDT negative at baseline. Six weeks 143 after treatment there were four high-density gametocyte-containing infections, two in students that 144 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229336 doi: medRxiv preprint were not treated and two in students that received treatment, corresponding to a 90% reduction in 145 the prevalence of high-density gametocyte infections. 146 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020. The screen-and-treat intervention reduced gametocytemia in our school-based cohorts. 169 Furthermore, school-age children substantially contributed to the population of gametocyte-170 carriers in the surrounding communities; 46% of all gametocyte-containing infections and high-171 density gametocyte-containing infections were in school-age children, who comprised only 35% 172 of the population (Supplemental Results and Figure S2). If the screen-and-treat intervention were 173 extended to all school attendees, we estimate it could result in at least six weeks of reductions in 174 the community prevalence of gametocyte-containing infections as large as 26% in the rainy and 175 34% in the dry season ( Figure 3A and 3B). The total gametocyte burden (sum of gametocyte 176 densities) in the community would be reduced by 33% in the rainy season and 25% in the dry 177 season ( Figure 3C and 3D). The number of infections containing ≥10 gametocytes/µl would be 178 reduced by 44% and 55% in the rainy and dry seasons, respectively ( Figure 3E and 3F). 179 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229336 doi: medRxiv preprint Following school-based screening-and-treatment at baseline, the estimated impact on gametocyte 182 prevalence (A in rainy and B in dry seasons), total gametocyte burden (C in rainy and D in dry 183 seasons), and number of infections containing ≥10 gametocytes/µl (E in rainy and F in dry seasons) 184 in the communities surrounding the schools are predicted at one, two, and six weeks after the 185 intervention. Color indicates the proportion of the gametocyte measure by age group: school-age 186 children (6-15y) -black; younger children (6-71m) -light grey; adults (>15y) -dark grey. Total 187 gametocyte burden is the sum of gametocyte densities in individuals in each age group. These 188 calculations assume treatment is not provided to young children, adults, or school-age children 189 who test negative by RDT when the intervention is implemented. Reduction is calculated as the 190 proportional difference between the baseline and six-weeks post intervention. 191 192 193 194 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229336 doi: medRxiv preprint The high prevalence of gametocyte-containing infections in school-age children in this study is 207 consistent with prior work by our group and others. 8,11,16 However, to our knowledge, this is the 208 first study to simultaneously determine gametocyte burden in schools and surrounding 209 communities to estimate the potential impact of school-based treatment on transmission. In 210 Uganda, school-based intermittent preventive treatment was associated with reduced parasite 211 prevalence in the surrounding community in a large cluster randomized trial. 23 Although the 212 community-level effect was significant, the magnitude was limited by low intervention coverage. 213 Because this intervention was conducted as a clinical trial, parents had to come to the school to 214 consent for each student, creating logistical barriers. Thus, further estimates of the full potential of 215 this intervention are needed. 216 217 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229336 doi: medRxiv preprint There are limitations to our analysis. We did not use RDTs to detect infections in the communities 218 surrounding schools and had to extrapolate from the relationships of PCR-and RDT-detected 219 infections in the school-based cohort to estimate the number of RDT-positive children in the 220 community. However, the distribution of gametocyte prevalence in the school-based and 221 community-based surveys was comparable, supporting the estimate. Furthermore, we used 222 sensitive molecular methods to detect and quantify gametocytes but did not directly measure 223 infectiousness using feeding assays, nor did we measure other factors that contribute to 224 transmission likelihood, such as gametocyte sex ratio, transmission-reducing immunity, and 225 heterogeneity of mosquito biting. However, our data still likely represent the relative contribution 226 of different age groups to transmission because prior studies have shown that school-age children 227 and younger children are more infectious to mosquitos than adults 12 and school-age children are 228 more likely than younger children to be bitten by competent vectors. 16 Lastly, our estimates of the 229 impact of the intervention in the community assume universal coverage in students. In Malawi, 230 school-based deworming programs routinely report coverage of >90% of school-age children. First, the impact of clearing gametocyte-containing infections in school-age children is expected 235 to be amplified because competent vectors probably feed more often on school-age children and 236 adults who have larger body surface than younger children 15,27 and use bed nets less frequently. 14-237 16,27 Second, in our community-level predictions, we conservatively assumed that gametocyte-238 containing infections in non-treated age groups would remain constant. However, the decreased 239 pool of infectious school-age children should also lead to fewer new infections and fewer 240 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. One concern is that risk of infection and disease could increase once students leave school or if 258 the intervention ceased. A prior study of chemoprophylaxis in younger children demonstrated a 259 transient increase in clinical infection when the intervention was discontinued. 28 However, this 260 "rebound effect" was not observed in all chemoprophylaxis trials, nor has it been found after 261 intermittent preventive treatment of infants. 29-31 In highly endemic settings where school-based 262 treatment interventions are most needed, schoolchildren have acquired partial immunity and often 263 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229336 doi: medRxiv preprint have sub-clinical infections. Continued exposure should maintain some naturally acquired 264 immunity. Another concern about the widespread use of preventive treatment is the potential for 265 drug resistance. While this concern is important, it should be weighed against the direct student 266 health and potential indirect community benefits when evaluating the approach. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229336 doi: medRxiv preprint School-based cohort studies and household-based cross-sectional surveys were conducted at the 287 end of the rainy season (April-May) and during the dry season (September-October) of 2015. Two 288 sites with consistently high (>40%) parasite prevalence in school-age children -Maseya and 289 Makhuwira -and two with lower, seasonally varied transmission (>two-fold seasonal prevalence 290 difference) -Bvumbwe and Ngowe -were selected from 30 previously studied sites in southern 291 Malawi. 6 Long-lasting insecticide treated nets were distributed through a national campaign in 292 2012. Rapid diagnostic tests (RDTs) and treatment with artemether-lumefantrine were generally 293 available in local government-operated health facilities. 294 295 School-based cohorts: Students present on a sampling day were assigned numbers. Fifteen 296 students per grade-level were sampled using a random number generator. Students were excluded 297 if they: had no parent/guardian available to provide consent, would not attend school throughout 298 the 6-week study, or had a known artemether-lumefantrine allergy. Students enrolled in the rainy 299 season cohort were excluded from the dry season cohort. 300 301 Enrolled students were interviewed at baseline, one-, two-, and six-week visits about bed net use 302 the night prior, current or recent illness, and antimalarial treatment. At baseline, a finger-prick 303 blood sample was obtained for detection of P. falciparum by RDTs and hemoglobin was measured 304 by portable photometer (Hemocue, Angelholm, Sweden). RDT-positive students received weight-305 based treatment with artemether-lumefantrine (Novartis Pharma AG or Ajanta Pharma Ltd). At 306 baseline and all follow-up visits, finger-prick blood was obtained for molecular detection of any-307 stage parasite and gametocytes. At the final visit, parents were interviewed and health passports 308 (individual portable medical records) reviewed to identify intercurrent fever or malaria treatment. 309 . 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(which was not certified by peer review) preprint
The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229336 doi: medRxiv preprint For school-based cohorts at baseline, logistic regression models were used to assess determinants 353 of gametocyte prevalence and linear models (log10-transformed) were used to assess determinants 354 of gametocyte density y among infections with > 0 gametocytes. We used logistic regression 355 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229336 doi: medRxiv preprint The funders of the study had no role in study design, data collection, data analysis, data 408 interpretation, or writing the report. The corresponding author had full access to all data and took 409 final responsibility for the decision to submit for publication. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229336 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

Figure 1: Baseline P. falciparum infection prevalence (×) and proportion of students with
The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229336 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229336 doi: medRxiv preprint Following school-based screening-and-treatment at baseline, the estimated impact on gametocyte 557 prevalence (A in rainy and B in dry seasons), total gametocyte burden (C in rainy and D in dry 558 seasons), and number of infections containing ≥10 gametocytes/µl (E in rainy and F in dry seasons) 559 in the communities surrounding the schools are predicted at one, two, and six weeks after the 560 intervention. Color indicates the proportion of the gametocyte measure by age group: school-age 561 children (6-15y) -black; younger children (6-71m) -light grey; adults (>15y) -dark grey. Total 562 gametocyte burden is the sum of gametocyte densities in individuals in each age group. These 563 calculations assume treatment is not provided to young children, adults, or school-age children 564 who test negative by RDT when the intervention is implemented. Reduction is calculated as the 565 proportional difference between the baseline and six-weeks post intervention. 566 567 568 569 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229336 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229336 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229336 doi: medRxiv preprint Maseya-high; Makhuwira-high. b Mean age of students with gametocyte containing infections not detected by RDT was 11.5 compared to 10.8 years for students with gametocyte containing infections detected by RDT. c Among students with gametocyte-containing infections not detected by RDT, the mean log gametocyte density was -2.417 compared to a mean log gametocyte density of -0.825 among students with gametocyte-containing infections detected by RDT. d Hemoglobin <11.0 g/dL; 1 missing observation e Measured temperature ≥37.5ºC at baseline visit or reported in the last two weeks f Reported in the last two weeks; 1 missing observation g Adjusted OR for Bvumbwe compared to all other schools in the multivariable model is 6.6 [95%CI: 1.9-24], p=0.004 h 4 missing observations 574 575 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229336 doi: medRxiv preprint