Non-adherence to long-lasting insecticide treated bednet use following successful malaria control in Tororo, Uganda.

Indoor residual spraying (IRS) and long-lasting insecticide-treated bednets (LLINs) are common tools for reducing malaria transmission. We studied a cohort in Uganda with universal access to LLINs after 5 years of sustained IRS to explore LLIN adherence when malaria transmission has been greatly reduced. Eighty households and 526 individuals in Nagongera, Uganda were followed from October 2017 -October 2019. Every two weeks, mosquitoes were collected from sleeping rooms and LLIN adherence the prior night assessed. Episodes of malaria were diagnosed using passive surveillance. Risk factors for LLIN non-adherence were evaluated using multi-level mixed logistic regression. An age-matched case-control design was used to measure the association between LLIN non-adherence and malaria. Across all time periods, and particularly in the last 6 months, non-adherence was higher among both children <5 years (OR 3.31, 95% CI: 2.30-4.75; p<0.001) and school-aged children 5-17 years (OR 6.88, 95% CI: 5.01-9.45; p<0.001) compared to adults. In the first 18 months, collection of fewer mosquitoes was associated with non-adherence (OR 3.25, 95% CI: 2.92-3.63; p<0.001), and, in the last 6 months, residents of poorer households were less adherent (OR 5.1, 95% CI: 1.17-22.2; p = 0.03). Any reported non-adherence over the prior two months was associated with a 15-fold increase in the odds of having malaria (OR 15.0, 95% CI: 1.95 to 114.9; p = 0.009). Knowledge about LLIN use was high, and the most frequently reported barriers to use included heat and low perceived risk of malaria. Children, particularly school-aged, participants exposed to fewer mosquitoes, and those from poorer households, were less likely to use LLINs. Non-adherence to LLINs was associated with an increased risk of malaria. Strategies, such as behavior change communications, should be prioritized to ensure consistent LLIN use even when malaria transmission has been greatly reduced.

Introduction

Long lasting insecticide-treated bednets (LLINs) are a mainstay of malaria prevention [1]. LLINs provide both individual protection from mosquitoes and a broader community effect via vector control. Despite increasingly robust programs for the universal distribution of LLINs, the impressive declines in malaria transmission achieved from 2000 to 2015 have recently stalled and may be reversing, especially in high burden African countries [2]. This stall in progress in malaria reduction has highlighted the importance of other tools for vector control such as indoor residual spraying of insecticides (IRS).

Evidence as to whether the addition of IRS to LLINs has a beneficial effect on reducing transmission is mixed. A recent comprehensive review of randomized trials concluded that caution was warranted, since some studies showed an additive effect, but other studies did not [3]. For example, a study in Gambia reported no significant difference in the density of indoor biting vectors caught in light traps in households receiving both LLINs and IRS compared to households receiving LLINs alone [4]. Similar findings have been reported in Benin [5]. A subgroup analysis from the comprehensive review, however, demonstrated that in populations with high LLIN adherence (reported use > 50%), adding IRS was associated with a substantial reduction in parasite prevalence, with a risk ratio of 0.47 (95% confidence interval [CI]: 0.33 to 0.67) compared to populations without IRS. Thus, the benefits of combining IRS with LLINs may depend on the levels of LLIN adherence.

The decision to use an LLIN, when one is available, is up to the individual or their parent/guardian. This is in contrast to decisions about IRS, which are in the hands of government officials as long as people consent to spraying of their households. There are a variety of well-described barriers to LLIN ownership and use that affect individual decision making, such as knowledge of malaria transmission, perceptions of risk and a sense of individual agency [6,7]. In addition, it is well documented that LLIN use changes in response to environmental factors such as seasonal rainfall and changes in temperature [8-10]. Less well described is how LLIN use changes over time and what factors are most determinative of individual decisions to use LLINs when transmission of malaria begins to decline in settings of intense malaria control. Identifying these dynamics in LLIN use and the mechanisms through which they act are crucial for policy makers to design effective LLIN promotion strategies to sustain malaria control once achieved.

Given that LLIN adherence may play a central role in the effectiveness of IRS and that successful IRS campaigns may have an effect on subsequent LLIN behaviors, we designed this study to explore LLIN non-adherence in a cohort living in an area of Eastern Uganda with historically high transmission intensity where transmission was dramatically reduced following two rounds of universal LLIN distribution and over five years of sustained IRS implementation [11]. Longitudinal measures of mosquito exposure, LLIN use and malaria episodes were used to 1) describe how LLIN use changed over time, 2) identify household, entomological and individual characteristics associated with non-adherence to LLINs, 3) estimate how non-adherence affected mosquito biting and the odds of being diagnosed with malaria, and 4) describe perceptions of malaria risk among participants.

Materials and methods

Study setting

The study took place in Nagongera sub-county in the Tororo District of Eastern Uganda. As part of two national campaigns, universal distribution of free LLINs was conducted in the district in November 2013 and repeated in May 2017. IRS with the carbamate bendiocarb was first initiated in December 2014 –January 2015, with additional rounds administered in June-July 2015 and November-December 2015. In June-July 2016, the formulation of insecticide used for IRS was changed to the organophosphate pirimiphos-methyl (Actellic), with repeated rounds in June-July 2017, June- July 2018, and March-April 2019. These malaria interventions coincided with a significant reduction in malaria transmission, from an estimated entomological inoculation rate (EIR) of 238 infective bites per person per year prior to IRS being implemented, to an EIR of only 0.43 after 5 years of IRS [14].

Study design and participants

Full details on enrollment and follow-up procedures for the cohort used in this study have been described previously [14]. In brief, 80 households and all of their inhabitants were recruited in October 2017 and followed through October 2019. The cohort was dynamic such that over the course of the study any permanent residents that joined the household were enrolled and residents leaving the household were withdrawn. All 80 enrolled households remained enrolled until the end of the cohort. Four hundred and sixty-six (466) participants were enrolled initially, with 65 individuals added (either born into or establishing residency in a cohort household) and 62 participants either dying or moving away, resulting in a total of 469 participants at the study end. At enrollment, household characteristics were assessed for the creation of a wealth index, as described elsewhere [12]. All designated sleeping rooms and sleeping areas were mapped and enumerated. All cohort households were provided additional LLINs at enrollment to ensure coverage of all sleeping spaces, and LLINs were provided on demand during monthly clinic visits for any households needing a new or replacement LLIN. Cohort study participants were encouraged to come to a dedicated study clinic open 7 days per week for all their medical care. As part of the broader cohort activities, routine visits were scheduled every 4 weeks for clinical assessments, malaria surveillance and measurement of other malaria risk factors, including a standardized evaluation for any overnight travel outside of the sub-county. Study participants found to have a tympanic temperature > 38.0°C or history of fever in the previous 24 hours at the time of any clinic visit had a thick blood smear read urgently. If the thick smear was positive, the patient was diagnosed with malaria and managed according to national guidelines. Study subjects who missed their scheduled routine visits were visited at home and requested to come to the study clinic as soon as possible.

Data collection

Entomological surveillance

Mosquito collections were conducted every 2 weeks in all cohort study households. In each room where cohort study participants slept, a miniature CDC light trap (Model 512; John W. Hock Company, Gainesville, Florida, USA) was positioned 1 m above the floor. Traps were set at 7 PM and collected at 7 AM the following morning. Female Anopheles mosquitoes were subsequently identified taxonomically and dissected, with each mosquito classified as either blood fed, not blood fed or unable to assess (for example due to damage in processing).

LLIN adherence measures

Every two weeks, on the morning after the CDC light traps were collected, a structured questionnaire was administered to an adult respondent in each household to gather information about reported LLIN use for each study participant the prior night, and where the participant slept the previous night (for assigning individual mosquito exposure) ().

Exit interviews

In November-December 2019, at the conclusion of the study, a semi-structured questionnaire was administered to the remaining enrolled participants (469) to inquire about their perceived risk of malaria, knowledge about malaria transmission, community norms in relation to LLIN use, and indications for and barriers to LLIN use. Children under 12 years were aided by their parents in responding to this survey based on their perceptions and habits. Attempts were made to administer this exit interview to all cohort participants with multiple home visits, but, since the main cohort study had come to a close, logistical barriers resulted in only 459 of the total 469 participants being located for this final questionnaire (.

Study endpoints

Factors associated with non-adherence

For the analysis of risk factors for non-adherence, LLIN use for each individual was defined as reported use or non-use the prior night. The following factors were identified as potentially associated with LLIN non-adherence and included in the model: age and gender of the individual, household wealth index (in tertiles) and the total number of anopheles mosquitoes captured from the room where the individual slept during the night for which LLIN use was reported. Based on effect modification by calendar time on associations between our risk factors of interest and LLIN non-adherence, the analysis was stratified into two time periods: November 2017 through April 2019 (18 months) and May 2019 through October 2019 (6 months). Based on the distribution of the data, plausible categorization of differences in LLIN use behaviors and for aid in interpretation, age was stratified into three categories: under 5 years, 5 to 17 years and ≥ 18 years. A household wealth index was generated based on ownership of various assets using principal components analysis and LLIN non-adherence in the poorest households (lowest tertile) was compared to the least poor households (all other households). Finally, based on the distribution of the data and association with the outcome, the total number of female Anopheles mosquitoes captured from the room where an individual slept was categorized as either 0 to 2 versus 3 or more.

Association between LLIN non-adherence and number of captured mosquitoes having taken a blood meal

Mosquito bites are an intermediate link between LLIN non-adherence and acquiring malaria. We hypothesized that the number of mosquitoes captured from participant rooms classified as blood fed, i.e. representing a potentially infectious bite, would be higher during a night when residents in the room reported non-adherence to LLINs. We chose to use blood fed mosquitoes as a marker of potential infection, and not the more traditional sporozoite rate, because we identified only nine mosquitoes with sporozoites out of a total of 15,780 collected. Such a low number would not have provided adequate power to support our inquiry. By room and date, we generated a variable representing the number of total blood fed mosquitoes, after adjusting for mosquitoes unable to be assessed for blood fed status due to damage in processing. We classified LLIN adherence, by room and date, as either complete LLIN adherence by all participants or any non-adherence reported by a room participant. Finally, since we reasoned that more people would represent both a higher potential lure for meal seeking mosquitoes and more available targets for biting, we created a variable accounting for the number of people sleeping in the room.

Case-control design for associations between LLIN non-adherence and risk of malaria episodes

A case-control design was employed which identified age-matched controls for each case of malaria based on the date of diagnosis for each episode of malaria included in the analysis. A total of 38 cases of malaria were diagnosed over the 2 year follow-up period. No participant had more than one case of malaria. Malaria cases were excluded when prior LLIN use could not be assessed (n = 6), persistent asymptomatic parasitemia preceded the diagnosis of malaria (n = 6), and travel outside of the district was reported in the prior month (n = 4). These exclusions resulted in 22 cases of malaria included in the analyses. All age-matched controls available on the date malaria was diagnosed were included. Whenever possible, controls were matched based on the year of age of the case. For three of the cases among older individuals (17, 29 and 38 years of age respectively), this constraint was relaxed to +/- 2 years in order to identify an adequate number of controls for each case. A mean of 22 age-matched controls (range 10 to 35) were identified per case. The main exposure of interest was individual LLIN use reported at the biweekly household visits and aggregated over various time windows. To account for a minimum incubation period of 7 days from an infectious mosquito bite to the onset of clinical symptoms of malaria, time windows for assessment of LLIN adherence were defined as 1–5 weeks, 1–9 weeks and 1–13 weeks prior to the date when a case/control was identified. The mean number of mosquitoes captured from the participant’s room during the biweekly CDC light trap collections was included as a covariate and calculated similarly to LLIN use for the corresponding time windows.

Statistical analysis

LOWESS smoothing was used to visually display trends in LLIN adherence and vector density over time. For the analysis of risk factors associated with LLIN non-adherence, a multi-level mixed effects logistic regression model was fit to account for both the multiple measures at the household and individual levels and the clustering of participants within the same households. In assessing the association between blood fed mosquitoes and LLIN non-adherence, we utilized a multi-level mixed effects negative binomial regression model, accounting for multiple measures at the room level, the hierarchical clustering of rooms within households and adjusting for the number of participants in the room. The measure of association is reported as a risk ratio with 95% confidence intervals (CIs) comparing the prevalence of blood fed mosquitoes, of the total captured, in rooms with and without any reported LLIN non-adherence. For the case-control analysis, conditional logistic regression models were fit for each time window of LLIN non-adherence exposure and adjusted for mean mosquito exposure. Measures of associations for this analysis and the risk factors associated with LLIN adherence were reported as odds ratios (ORs) with 95% CIs. All statistical analyses were conducted at the 5% significance level and were performed using STATA 14 (StataCorp LP. 2015. College Station, TX).

Ethics approval and consent to participate

Written informed consent for participation in the cohort study was obtained in the appropriate language from the adults and children. In addition, children above 8 years also provided assent for study participation. For children under 8 years, consent was provided by the parents or guardians. Additional verbal consent was obtained prior to the exit interview questionnaire. Study ethical approval was obtained from Uganda National Council for Science and Technology (UNCST), Makerere University School of Medicine Research Ethics Committee, University of California, San Francisco Committee for Human Research and the London School of Hygiene and Tropical Medicine Ethics Committee.

Results

Characteristics of study participants

A total of 80 households were enrolled, with 526 individuals providing data on LLIN adherence. There was an average of 5.8 individuals per household (SD: 1.3), 1.8 rooms per household used for sleeping (SD: 0.6) and 1.7 sleeping spaces per room (SD: 0.7). At enrollment, 98% of households owned at least one LLIN, with an average of 3.5 (SD: 1.6) LLINs per household. The number of LLINs was not measured after enrollment, but was assumed to be adequate to cover all sleeping areas due to on demand distribution to cohort participants. Fifty-two percent of participants were female and the average age at enrollment was 15.9 years (SD: 16.1; range: 1 month to 76 years). At enrollment, 34% (177/526) of participants were under five years of age, 38% (201/526) were 5–17 years of age, and 28% (148/526) were 18 years or older.

Changes in mosquito density, malaria episodes and LLIN use over time

Over the two year study period, a total of 15,780 female Anopheles mosquitoes were collected during the biweekly collections, resulting in an average density of 2.1 mosquitoes per room per night. Ninety-nine percent of mosquitoes collected were identified as Anopheles arabiensis by PCR and one percent were Anopheles gambiae sensu stricto. Each year there was a large peak in mosquito density following the long rainy season, which occurs from April-June, and a smaller peak following the short rainy season, which occurs from October-November. Following the short rainy seasons, there were periods when almost no mosquitoes were collected. This period extended from December-April during the second year, possibly because the last round of IRS was administered early, in March 2019. There were a total of 38 malaria cases diagnosed over the 2 year follow-up period. Small clusters of cases were evident following the two large peaks in mosquito density ().

During the first year of follow-up, mean LLIN adherence fluctuated from 50–85%, generally decreasing during periods of lower mosquito density and increasing during periods of higher mosquito density. From November 2018 to February 2019, mean LLIN adherence decreased precipitously, falling below 10%. This marked decline corresponded with an extended 6-month period when almost no female Anopheles mosquitoes were collected. During the last 6 months of follow-up, mean LLIN adherence increased, reaching over 40%, and corresponding with a sharp rise in mosquito density ().

Since LLINs are used differently by different age groups, changes in mean reported LLIN adherence over time were stratified by age categories (i.e. under 5 years, 5 to 17 years and ≥18 years). The three age groups had a qualitatively similar pattern over time, but, over the entire study period, LLIN adherence was consistently higher among adults and lowest among school-aged children (5 to 17 years). Reported adherence among children under five years of age was higher than among school age children, but persistently less than that for adults across the entire study. All three groups had a similar nadir in LLIN adherence in February 2019, but as LLIN use began to rise in May 2019, there is evidence that adults were more likely to use LLINs than children, reaching a peak of 65% use, compared to 45% in children under 5 years, and only 30% in children 5 to 17 years of age ().

Factors associated with LLIN non-adherence

During the first 18 months of follow-up, age was significantly associated with the odds of reported LLIN non-adherence after adjusting for mosquito exposure, household wealth and gender. Compared to adults, both children under 5 years and those aged 5 to 17 years were less likely to be adherent: OR 1.47 (95% confidence interval (CI): 1.27 to 1.70; p<0.001) and 2.67 (95% CI: 2.34 to 3.06; p<0.001), respectively. In this time period, individuals sleeping in rooms where 0–2 mosquitoes were captured per night had 3.25 (95% CI: 2.92 to 3.63; p<0.001) the odds of non-adherence compared to those sleeping in rooms where 3 or more mosquitoes were captured per night. There was no association between household wealth and non-adherence during the first 18 months of follow-up (). In the final six months, differences in the odds of LLIN non-adherence according to age increased, such that, compared to adults, children under 5 years had 3.31 times the odds (95% CI: 2.30 to 4.75; p<0.001) and children 5 to 17 years had 6.88 times the odds (95% CI: 5.01 to 9.45; p<0.001) of LLIN non-adherence. There was no association between vector density and non-adherence in the final six months of the study, but individuals living in the poorest tertile of households had 5.09 (95% CI: 1.17 to 22.2; p = 0.03) the odds of non-adherence compared those living in wealthier households. There was no association between gender and non-adherence in either the first 18 months or final six months of follow-up.

Association between LLIN non-adherence and blood-fed mosquitoes

The prevalence of blood fed mosquitoes was 6.3% (95% CI: 5.3% to 7.4%) in rooms where everyone reported LLIN adherence compared to 12.0% (95% CI: 10.5% to 12.4%) in rooms with any reported non-adherence (p<0.001). In the mixed effects model, adjusting for multiple measures and the number of people sleeping in the room, any reported LLIN non-adherence was associated with 2.2 times (95% CI: 1.8 to 2.7; p>0.001) the prevalence of blood fed mosquitoes. Interestingly, after adjusting for LLIN adherence, the number of participants in the room showed no linear trend in increasing prevalence of blood fed mosquitoes captured (p = 0.99).

Associations between prior LLIN non-adherence and malaria episodes

The results of the case-control study show that any reported LLIN non-adherence was associated with increased odds of being diagnosed with malaria after adjusting for mosquito exposure. The strength of this association increased when non-adherence was assessed over longer windows of time preceding the date malaria was diagnosed (). Compared to those who always reported LLIN use, the odds of being diagnosed with malaria was 3 times higher (OR 3.12, 95% CI: 1.06–9.21, p = 0.04) in individuals who reported not using their LLIN at least once over a one month period (from 1 to 5 weeks prior to the date of diagnosis), and 15 times higher (OR 15.0, 95% CI: 1.95–114.9, p = 0.009) among those who reported not using their LLIN at least once over a 2 month period. When adherence was assessed over a three month period, the odds of being diagnosed with malaria could not be estimated because none of the cases (0/22) reported full adherence, whereas 26.4% (121/458) of controls were fully adherent at all measurements.

Knowledge and perceptions of malaria risk in the cohort

As noted above, after multiple home visits after the end of the cohort study in an attempt to reach all cohort participants, only 459 of the total 469 participants were administered the LLIN adherence questionnaire. When interviewed at the end of the study, 93.7% (430/459) of individuals reported they thought that the malaria risk had decreased in the community compared to the prior year. Knowledge of malaria transmission was heterogeneous, with 93.2% (428/459) of individuals identifying mosquitoes as a potential cause of malaria, but only 31.8% (146/459) of participants reporting that only mosquitoes can transmit malaria. Despite reported LLIN adherence being lower in children than adults, knowledge of priority groups for LLIN use was high; 96.1% of participants identified children under 5 years of age, 94.1% pregnant women and 80.1% school-aged children as individuals who should use an LLIN every night (). Many individuals knew how to use an LLIN correctly (88.7%). Reasons that an individual might not use an LLIN that is hung above a sleeping space, included ‘too hot’ (85.6%), no mosquitoes around (28.8%) or no malaria (6.3%). Interestingly, 24.6% of individuals reported the presence of bedbugs/fleas as a potential barrier to individual LLIN use. When asked about recent experience with LLIN non-adherence, participants reported that they simply forgot (35.2%), were travelling (18.5%), it was too hot (16.8%), there were no mosquitoes (7.4%), and that there was no malaria (2.8%).

Discussion

In this cohort of households from a district in Uganda experiencing more than a 500-fold reduction in malaria transmission following universal LLIN distribution and 5 years of sustained IRS, we identified various important findings in relation to how LLIN use has changed over time. First, a marked decline in individual-reported LLIN use was observed: only 20% of individuals reporting LLIN use from February to June 2019, compared to an average of 60% during the same time period in the prior year. LLIN non-adherence was significantly higher in children compared to adults, and highest in school-aged children, despite widespread reported knowledge of the importance of ensuring nightly LLIN use by children. The finding of poor LLIN use among school age children has been well-described elsewhere [13], but the consistently low adherence among children under five years of age was surprising. In addition to age, other factors identified as associated with lower LLIN adherence included lower household wealth and the presence of fewer mosquitoes in the room in which an individual slept. Any reported LLIN non-adherence was associated with 2.2 times the prevalence of mosquitoes captured that had taken a blood meal, establishing an intermediate link between poor LLIN adherence and increased risk of potentially infectious mosquito bites. Finally, using an age-matched case control design, not using an LLIN was associated with increased odds of having a malaria episode, confirming the importance of using LLINs even with very low levels of malaria transmission.

The steep decline in reported LLIN use in 2019 was remarkable given that all study participants had access to LLINs and that malaria transmission had not changed much compared to the prior year. Whether a drop of this magnitude in LLIN use is unusual is not clear, as most data on changes in LLIN use come from repeated cross-sectional studies rather than the prospective cohort design that we employed [14,15]. A cohort study in Kenya reported how LLIN use impacted mortality over time, but did not report serial measures of LLIN use [16]. Other studies in the context of free universal distribution campaigns have also noted sharp declines in reported use over time, but have often attributed this to a decline in ownership of LLINs [17]. Changes in ownership should not have significantly affected our cohort, since access to an LLIN was universal. Anecdotal reports from health officials in the district noted an outbreak of bedbugs leading people to use their LLINs less, and 25% of cohort participants reported bedbugs as a potential reason for not using an LLIN. Associations between household insect infestations, IRS and LLIN use have been reported previously, but the evidence is mixed on the direction of associations with LLIN adherence, as concerns regarding bedbugs have been identified as both facilitators [18] and barriers [19] to LLIN use.

The use of LLINs during successful IRS campaigns is important, as there is evidence of an interaction between LLINs and IRS [20], and additive effects could depend on levels of LLIN adherence [3]. Additionally, when IRS campaigns have been stopped, marked resurgence in the burden of malaria has been described despite universal LLIN distribution [21]. There is interest in promoting LLIN use at the time of stopping IRS, and in introducing next generation LLINs with novel synergists or insecticides, as an “exit strategy” [22]. In this study, it appears that perceptions of risk of malaria transmission decreased as community burden and mosquito densities declined. Along with this decrease in actual and perceptions of risk, it is possible that other salient barriers to use, such as the heat (referenced by 85.6% of respondents), bedbugs (24.6%), not liking the smell (11.5%) or the bednet being too dirty (9.6%), overcome other incentives to consistently use LLINs and lead to lower adherence.

Even when malaria transmission, and overall mosquito populations are significantly decreased [14], by a combined strategy of IRS, universal access to LLINs and case management with artemisinin-based combination therapies, LLIN adherence still has an important effect on an individual’s risk of malaria. In this cohort, even when malaria transmission was reduced to historically low levels, not using an LLIN at every biweekly measurement over two months was associated with a 15-fold increase in the odds of being diagnosed with malaria, after controlling for age and adjusting for mosquito density. Thus, maintaining high and consistent LLIN use should remain an important priority for individual protection from malaria, especially for high risk groups.

Of particular interest in this cohort was the association between age and LLIN non-adherence. Not surprisingly, school age children (age 5 to 17) were consistently poor users of LLINs, as has been demonstrated elsewhere [8,23,24]. In addition, though nearly everyone in the cohort understood that children under five should use an LLIN every night, adults used LLINs significantly more than children. In the final six months of the study, this difference was multiplied two-fold. This is particular interesting given that it might be expected that children under five will often use their bednets under the supervision of adults in the household. The discordance between LLIN use by adults and young children deserves special attention in future efforts to understand who is responsible for choices related to bednet use in Ugandan households. Given their heightened risk of severe malaria, the lower use of LLINs by young children in this study highlights the necessity of more robust social and behavior change campaigns that can reach beyond knowledge and address actual behaviors.

Another finding of interest was that individuals from the poorest households were less likely to use an LLIN in the last six months of the study, when LLIN use was generally lower. The effect of wealth on LLIN use is mixed and may be context specific. While most studies show that wealth is inversely associated with LLIN use [25,26], other studies have not shown this effect [10]. One plausible mechanism for decreased LLIN use among poorer households, and one that may have implications for IRS settings, could be LLIN attrition. As malaria risk declines, the value of LLINs to the owner may decline as well, incentivizing the sale or trade of the LLIN. This seems unlikely in this study, however, as the households had free access to LLINs.

This study had important limitations. Extending our conclusions to other populations is limited by the fact that the households were enrolled in a specialized cohort, with access to free medical care, access to free LLINs on demand and frequent household visits, which may have affected the incidence of malaria, knowledge and behaviors compared to the general population. For the questionnaire related to LLIN adherence, due to logistical constraints at the end of the cohort study, only 459 of the total 469 participants were administered the exit interview. This could have introduced bias, although multiple home visits were attempted and there is no reason to suspect that the households who were administered the survey would differ systematically from the overall cohort in respect to their perceptions about LLINs. The use of LLINs was reported every two weeks by one adult in the household for all other members of the household. This may have introduced biases in relation to reported LLIN use, but longitudinal measures of use are likely more reliable than one time cross sectional measures, as used in standard Malaria Indicator Surveys and most other methods of assessing LLIN use. Social desirability bias may also have been a factor, though it might be expected to have inflated reported use, whereas in this study LLIN use was reported to be quite low. In particular, social desirability bias does not seem plausible as the driver of low relative LLIN use for children, because presumably it would be socially undesirable to report non-use in children.

Conclusion

As malaria transmission declined in our study setting with sustained IRS, LLIN use declined as well. Despite understanding the importance of LLIN adherence among children, adults were more likely to use LLINs than children and non-adherence was strongly associated with a higher odds of malaria. Future research should focus on the dynamic interplay between LLIN adherence and changing perceptions of malaria risk. Social and behavior change communication programs and other strategies for sustaining high levels of LLIN use should be prioritized to ensure consistent LLIN adherence, even in the setting of successful malaria control and reduced transmission.

Biweekly household questionnaire.

(DOC)

Click here for additional data file.

Exit interview instrument.

Exit interview questionnaire relating to bednet adherence.

(DOCX)

Click here for additional data file.

24 Sep 2020

PONE-D-20-27114

Non-adherence to long-lasting insecticide treated bednet use following successful malaria control in Tororo, Uganda

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**********

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5. Review Comments to the Author

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Reviewer #1: General

This is a longitudinal cohort study aimed to explore non-adherence of long-lasting insecticide treated bed-net (LLIN) use by measuring mosquito exposure, and malaria episodes in a malaria high transmission area which the cases significantly reduced after LLIN distribution and IRS implementation. It is an interesting study and quite appreciative for the authors conducting a comprehensive study related to behavior, perceived risk of malaria transmission, entomology and history of illness in this study. However, there are several significant methodological problems that need to be discusses carefully, which could potentially affect the transparency, accuracy and reliability of the findings (details can be found in the comments below).

Introduction

The introduction well written. The aim of this study clearly stated. However, authors need to further review existing literature and provide examples that relevant with association between the use of LLIN and number of captured mosquitoes. Authors could add and cite factors association between the non-adherence of LLIN and risk of malaria transmission papers and provide more explanation or justification on the novelty and rationale of this study – to make this introduction stronger and convincing and explain how their study will provide additional insight to the effectiveness LLIN implementation program.

What is the significance or expected benefit of the findings of this study? Please add.

Method

The authors are suggested to re-organized the sections and would be good if they could provide flowchart illustrating steps of analyses. There is critical point in the methods – the authors need to carefully describe how to choose respondent for the exit interviews as it was mentioned cohort enrollment was dynamic.

Specific comments:

• Authors could provide flowchart illustrating cohort participants enrollment including for the exit interviews.

• Please describe activities on routine visits, why did scheduled every 4 weeks? Please explained.

• It is good to present the methods by heading and sub-headings. Study setting, Study design and participants, data collection with sub-headings entomological survey, risk factors associated with LLIN non-adherence, exit interviews, and then move to statistical analysis.

• It should be stated the species of mosquitoes captured in the study based on vector or suspected vector malaria

• Why was age stratified into three categories: under 5 years, 5 to 17 years and ≥ 18 years? Age range should be considered when determining adherence. it should be used adult age range.

• Authors may cite / add literature to categorize the number of persons sleeping within LLIN (0-2 Vs 3 or more)

• Why LLIN non-adherence was measured on the basis of the number of mosquitoes caught having taken a blood meal instead of those positive for sporozoites? Please explain

Result

• Authors should compile the section of headings more concise and systematic. For example: characteristics of the study participants, factors associated between non-adherence of LLINs use, mosquito biting and malaria episodes; predictors of non-adherence of LLINs Use

• Line 310: Authors should be opening by explaining the total sample outcome for the exit interview, since the number for the cohort was different from the total sample, please add.

Discussion

It would be better if the authors would opening/introductory paragraph by describing the main result of study.

Conclusions

Authors should be more specific to address regarding changing perception of malaria risk.

Reviewer #2: Review of the paper by Rek et al Non-adherence to long-lasting insecticide treated bednet use following successful malaria control in Tororo, Uganda

Non adherence to bed net could reduce the effectiveness of LLINs. My comments on the manuscript are as follows

Method

The author say the classify household as poor or not poor but they didn’t explain which criteria they used to assess the household wealth this information need to be added.

Line 121 – 122 In addition to EIR data, also add information on anopheline species in the study area (composition and density)

Line 148 to 152 « Every two weeks, on the morning after the CDC light traps were collected, a structured questionnaire was administered to an adult respondent in each household to gather information …» Please add the questionnaire as an additional file

Line 154 to 158 « Exit interviews

In November-December 2019, at the conclusion of the study, a semi-structured questionnaire was administered to each participant inquiring about perceived risk of malaria, knowledge about malaria transmission, community norms in relation to LLIN use, and indications for and barriers to LLIN use. » please add questionnaire as an additional file

Line 188 - 192 « A total of 38 cases of malaria were diagnosed over the 2 year follow-up period. Malaria cases were excluded when prior LLIN use could not be assessed (n=6), persistent asymptomatic parasitemia preceded the diagnosis of malaria (n=6), and travel outside of the district was reported in the prior month (n=4). These exclusions resulted in 22 cases of malaria included in the analyses. » Did the authors registered people having two or more malaria attacks during the study period and how did they proceeded with the analysis

Page 11 line 258 « Since LLIN behaviors vary by age, changes in mean reported LLIN adherence … » Please what is « LLIN behaviour » ?

Results

The authors should mention anopheline species collected

The authors need to say whether they had cases of people who drop out from the study and give the attrition rate.

Table 1

« N » is placed for what ? please explain

First line Age in years

The age range « 5 - <18 » (is it 18 years) Also the author used the sign « < » does this means that children of 1 day old were included in the study?

Table 2

The first line 4/22 and 17/22 does not sum to 22/22 please verify your calculations

Table 3

First line N refer to what ?

Discussion

The fact that non adherence to LLINs use was associated to more blood fed mosquitoes indoor and increase risk of malaria transmission does it mean no effect of IRS on mosquito populations ? and what could be the influence of insecticide resistance can the authors discuss this further in the discussion section.

**********

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11 Oct 2020

“Non-adherence to long-lasting insecticide treated bednet use following successful malaria control in Tororo, Uganda”.

Response to Reviewers:

Journal Requirements

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As requested, we have made adjustments based on the referenced style templates. In particular, we have removed the key words, funding, word count and author contributions from the title page. We have also retitled the main sections to reflect the template (i.e. Background is now Introduction). We have also indented all paragraphs and reformatted the abstract to be unstructured.

2. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information.

Thank you for this request, which we note was also the subject of Reviewer 1’s comments. We have provided additional information in the Materials and Methods section (page 6) in regards to the procedures for identifying respondents to the exit survey, explanations for the discrepancy in numbers as was a concern of Reviewer 1 (459 exit interview respondents versus 469 remaining cohort participants) and clarification of the role of parents in aiding their children in responding to the questions. We have also clarified in the ethics approval section (page 9) that additional verbal consent was obtained from cohort participants for this questionnaire. Finally, we have added the verbal consent script and exit interview questionnaire relating to bednet adherence that was administered to the cohort study participants as a supplementary file (S2).

Reviewer Comments

1. “…authors need to further review existing literature and provide examples that relevant with association between the use of LLIN and number of captured mosquitoes.”

Thank you for this suggestion. As requested, we have added two additional citations that highlight the uncertain additive effects of IRS in addition to LLINs on vector density measured, as in our study, with CDC light traps. We have added the following text on page 3:

“For example, a study in Gambia reported no significant difference in the density of indoor biting vectors caught in light traps in households receiving both LLINs and IRS compared to households receiving LLINs alone [4]. Similar findings have been reported in Benin [5].”

2. “Authors could add and cite factors association between the non-adherence of LLIN and risk of malaria transmission papers…”

To bolster our discussion of additional factors associated with non-adherence to LLINs, we have added an additional sentence and two more citations that highlight some of the other well-described barriers to LLIN use (page 3; line 60):

“There are a variety of well-described barriers to LLIN ownership and use that affect individual decision making, such as knowledge of malaria transmission, perceptions of risk and a sense of individual agency [9,10].”

3. “…[P]rovide more explanation or justification on the novelty and rationale of this study…[w]hat is the significance or expected benefit of the findings of this study? Please add.”

Thank you for pointing out that we could do better at highlighting the added value from this study. We have edited the sentence on the top of page 4 to read:

“Less well described is how LLIN use changes over time and what factors are most determinative of individual decisions to use LLINs when transmission of malaria begins to decline in settings of intense malaria control. Identifying these dynamics in LLIN use and the mechanisms through which they act are crucial for policy makers to design effective LLIN promotion strategies to sustain malaria control once achieved.”

4. “There is critical point in the methods – the authors need to carefully describe how to choose respondent for the exit interviews as it was mentioned cohort enrollment was dynamic.”

We appreciate this concern and have added in the methods section a more complete description of the procedures for identifying the subjects for the exit interviews (page 6; line 122):

“…a semi-structured questionnaire was administered to the remaining enrolled participants to inquire about their perceived risk of malaria, knowledge about malaria transmission, community norms in relation to LLIN use, and indications for and barriers to LLIN use. Children under 12 years were aided by their parents in responding to this survey based on their perceptions and habits. Attempts were made to administer this exit interview to all cohort participants with multiple home visits, but, since the main cohort study had come to a close, logistical barriers resulted in only 459 of the total 469 participants being located for this final questionnaire.”

We have additionally added to the limitations section (page 19; line 371)a discussion of any potential bias this may represent:

“For the questionnaire related to LLIN adherence, due to logistical constraints at the end of the cohort study, only 459 of the total 469 participants were administered the exit interview. This could have introduced bias, although multiple home visits were attempted and there is no reason to suspect that the households who were administered the survey would differ systematically from the overall cohort in respect to their perceptions about LLINs.”

5. “Authors could provide flowchart illustrating cohort participants enrollment including for the exit interviews.”

We appreciate that the full study flowchart is useful for reference. Given that this study was based on a subset of the total cohort activities, we opted to refer to a recently published article in which the full details are described, including a flowchart illustrating enrollment and the dynamic nature of the cohort (see reference #14: Nankabirwa et al. Malaria Transmission, Infection, and Disease following Sustained Indoor Residual Spraying of Insecticide in Tororo, Uganda. Am J Trop Med Hyg. 2020 Jul 20. doi: 10.4269/ajtmh.20-0250). To make clear the flow of participants we added on page 5, line 97, a brief description of the dynamic cohort:

“All 80 enrolled households remained enrolled until the end of the cohort. Four hundred and sixty-six participants were enrolled initially, with 65 individuals added (either born into or establishing residency in a cohort household) and 62 participants either dying or moving away, resulting in a total of 469 participants at the study end.”

6. “Please describe activities on routine visits, why did scheduled every 4 weeks? Please explained.”

We have added additional information on activities at the routine visits and why they were scheduled every 4 weeks on page 5, line 101. We do not elaborate on activities that were performed outside the scope of the current study and instead reference to the full details of the cohort activities published elsewhere (see above):

“As part of the broader cohort activities, routine visits were scheduled every 4 weeks for clinical assessments, malaria surveillance and measurement of other malaria risk factors, including a standardized evaluation for any overnight travel outside of the sub-county.”

7. “It is good to present the methods by heading and sub-headings. Study setting, Study design and participants, data collection with sub-headings entomological survey, risk factors associated with LLIN non-adherence, exit interviews, and then move to statistical analysis.”

As requested, in the Materials and methods section we have retitled our headings and sub-headings to clearly separate study setting, study design, data collection, study endpoints and statistical analysis.

8. It should be stated the species of mosquitoes captured in the study based on vector or suspected vector malaria

Thank you for noting this important omission. We have added an additional line on page 10, line 218:

“Ninety-nine percent of mosquitoes collected were identified as Anopheles arabiensis by PCR and one percent were Anopheles gambiae sensu stricto.”

9. “Why was age stratified into three categories: under 5 years, 5 to 17 years and ≥ 18 years? Age range should be considered when determining adherence. it should be used adult age range.”

On reviewing the data and identifying plausible categories for age ranges, we felt that these three categories capture the data best and map well with other studies of age determinants of LLIN use. Our categories include the less than 5 year old, pre-school children, the five to 17 year old, “school-age children” and adults who are 18 years old and older. These seem to us to allow the best interpretation of how activities with age-associations track with LLIN use behaviors.

10. “Authors may cite / add literature to categorize the number of persons sleeping within LLIN (0-2 Vs 3 or more)”

Thank you for identifying this important point. We typically think of the number of individuals using an LLIN as a measure of availability of nets when supply is limited. The nature of our cohort study, where all households had free access on demand to LLINs at any time to cover all sleeping areas, made this distinction less important in our minds. For that reason, we did not measure the number of individuals under each net, and thus were not able to categorize then as suggested. We believe that since we are assuming that all sleeping areas have bednets and we have individualized reports of LLIN use, the number of individuals under each LLIN is less important to our overall outcomes such as blood-fed mosquitoes and malaria episodes.

11. “Why LLIN non-adherence was measured on the basis of the number of mosquitoes caught having taken a blood meal instead of those positive for sporozoites? Please explain”

Thank you for this question. Unfortunately, due to the extremely low levels of malaria transmission in our cohort, we did not identify adequate numbers of sporozoite-positive mosquitoes to support our analysis. We added on page 7, line 155:

“We chose to use blood fed mosquitoes as a marker of potential infection, and not the more traditional sporozoite rate, because we identified only nine mosquitoes with sporozoites out of a total of 15,780 collected. Such a low number would not have provided adequate power to support our inquiry.”

12. “Authors should compile the section of headings more concise and systematic. For example: characteristics of the study participants, factors associated between non-adherence of LLINs use, mosquito biting and malaria episodes; predictors of non-adherence of LLINs Use”

Thank you for this helpful suggestion. We have changed some of the sub-headings to make them more clear. For example we changed “Demographics” to “Characteristics of study participants” (page 10, line 209) and “Association between LLIN non-adherence and blood-fed mosquitoes” (page 12; line 265).

13. “Line 310: Authors should be opening by explaining the total sample outcome for the exit interview, since the number for the cohort was different from the total sample, please add.”

This is another helpful suggestion. Besides the additional information on the exit interview procedure that we have added to the methods and limitations sections, already discussed above, we have also added in this section a reminder of why the total number administered the questionnaire differ from the total in the cohort (page 15, line 289):

“As noted above, after multiple home visits after the end of the cohort study in an attempt to reach all cohort participants, only 459 of the total 469 participants were administered the LLIN adherence questionnaire.”

14. “It would be better if the authors would opening/introductory paragraph by describing the main result of study.”

Thank you for requesting this clarification. We have added a phrase highlighting the main findings of the study in the first paragraph of the discussion (page 15, line 305):

“In this cohort of households from a district in Uganda experiencing more than a 500-fold reduction in malaria transmission following universal LLIN distribution and 5 years of sustained IRS, we identified various important findings in relation to how LLIN use has changed over time. First, a marked decline in individual-reported LLIN use was observed: only 20% of individuals reporting LLIN use from February to June 2019, compared to an average of 60% during the same time period in the prior year.”

15. “Authors should be more specific to address regarding changing perception of malaria risk.”

Thank you for this suggestion. However, since the exit interviews were only administered once at the end of the cohort study, we are unable to make clear conclusions about how perceptions of malaria risk may have changed over time. We can hypothesize that the reason for the decrease in use of LLINs may be a result of changes in perceptions, but we unfortunately are unable to make that conclusion based off only one measurement at the end of the cohort. Future work would ideally measure perceptions of risk at baseline and hope to capture changes in those perceptions in relation to the level of malaria transmission.

Reviewer 2

1. “The author say the classify household as poor or not poor but they didn’t explain which criteria they used to assess the household wealth this information need to be added.”

Thank you for identifying this omission. We have included a reference to a recent paper from our group which explains the components and procedures utilized to create the wealth index used for this study.

Page 5 line 98:

“At enrollment, household characteristics were assessed for the creation of a wealth index, as described elsewhere [15].”

2. “Line 121 – 122 In addition to EIR data, also add information on anopheline species in the study area (composition and density)”

Thank you for this suggestion. As also requested above, we have added information on the species of anophelines that were captured in the study on page 10, line 222:

“Ninety-nine percent of mosquitoes collected were identified as Anopheles arabiensis by PCR and one percent were Anopheles gambiae sensu stricto.”

3. “Line 148 to 152 « Every two weeks, on the morning after the CDC light traps were collected, a structured questionnaire was administered to an adult respondent in each household to gather information …» Please add the questionnaire as an additional file”

Thank you for this request. We have added the biweekly questionnaire as a supplementary file 1.

4. Line 154 to 158 « Exit interviews. In November-December 2019, at the conclusion of the study, a semi-structured questionnaire was administered to each participant inquiring about perceived risk of malaria, knowledge about malaria transmission, community norms in relation to LLIN use, and indications for and barriers to LLIN use. » please add questionnaire as an additional file”

We have added the exit interview questionnaire as supplementary file 2.

5. “Did the authors registered people having two or more malaria attacks during the study period and how did they proceeded with the analysis”

Thank you for this question. In fact, no participant had more than one malaria episode during the study. We have added an additional sentence to clarify this on page 8, line 163:

“No participant had more than one case of malaria.”

6. Page 11 line 258 « Since LLIN behaviors vary by age, changes in mean reported LLIN adherence … » Please what is « LLIN behaviour » ?

We have clarified that we mean LLINs are used differently by different age groups. We have altered the sentence to now say:

“Since LLINs are used differently by different age groups…”

7. “The authors need to say whether they had cases of people who drop out from the study and give the attrition rate.”

As above, we reference the publication with the full study details and also add on page 5, line 97 a brief description of the attrition rate:

“All 80 enrolled households remained enrolled until the end of the cohort. Four hundred and sixty-six participants (466) were enrolled initially, with 65 individuals added (either born into or establishing residency in a cohort household) and 62 participants either dying or moving away, resulting in a total of 469 participants at the study end.”

8. “Table 1 « N » is placed for what ? please explain”

We have adjusted the “N” to indicate “Observations” in Table 1, page 14.

9. “First line Age in years The age range « 5 - <18 » (is it 18 years) Also the author used the sign « < » does this means that children of 1 day old were included in the study?”

The first (5 to <18) indicates from 5 years up to just under 18 years of age, but not 18 years. Yes, the less than 5 year age group includes just born children. We think this is correct in the inequalities as written.

10. “Table 2. The first line 4/22 and 17/22 does not sum to 22/22 please verify your calculations”

Thank you for this close attention. There was a typographical error and the first should be 5/22 (=22.7%). We have adjusted this in Table 2 on page 14.

11. “Table 3. First line N refer to what ?”

To make this clearer, we have adjusted the “N” to indicate “Observations” in Table 3, page 16.

12. “The fact that non adherence to LLINs use was associated to more blood fed mosquitoes indoor and increase risk of malaria transmission does it mean no effect of IRS on mosquito populations?”

Thank you for this question. We have highlighted in the discussion that even with low mosquito populations and low malaria incidence there is still a significant impact of LLIN use. Despite historically low values in both of these metrics, LLIN use is still of value in individual protection from mosquito bites and malaria episodes.

“Even when malaria transmission, and overall mosquito populations are significantly decreased [14], by a combined strategy of IRS, universal access to LLINs and case management with artemisinin-based combination therapies, LLIN adherence still has an important effect on an individual’s risk of malaria.”

13. “what could be the influence of insecticide resistance can the authors discuss this further in the discussion section.”

Thank you for this question. This is obviously a very important consideration. However, we do not feel that this paper’s focus on LLIN use allows us to make conclusions about insecticide resistance, either to IRS nor to LLINs. Our findings indicate that even with very low community transmission and lower than historical mosquito densities, both findings which would not be expected if there was widespread and significant insecticide resistance, LLIN use still provides value for individual protection. Thus, we avoid making conclusions about insecticide resistance and instead focus our attention on the importance of understanding changes in LLIN adherence and how policymakers might perhaps address low LLIN adherence in particular groups where it still matters.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

26 Oct 2020

PONE-D-20-27114R1

Non-adherence to long-lasting insecticide treated bednet use following successful malaria control in Tororo, Uganda

PLOS ONE

Dear Dr. Krezanoski,

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Reviewers' comments:

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I appreciate the effort and response of the authors to my previous comments and suggestions. However, before considering the manuscript for publication, there are some questions that need be discussed. My notes below on the revised manuscript are:

• line 221: It should be Result section, please changed

• line 235: Authors could discuss more rigorously regarding the findings "general compliance with LLIN decreases during periods of lower mosquito density and increases during periods of higher mosquito density" in line with the findings (line 303): the reason one might not use LLIN is 28.8 % answered that there were no mosquitoes compared to 'too hot' (85.6%). Please explain… considering understanding the importance of LLIN compliance is strongly associated with a higher likelihood of malaria

• Line 318: Author should add in the discussion section regarding non-adherence the used of LLINs in the age group under 5 years old. Considering the age range is still under the supervision of parents or adults, therefore I was clarified on my previous review note why was age range under 5 years old, included.

Reviewer #2: (No Response)

**********

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Reviewer #1: Yes: Mara Ipa

Reviewer #2: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

10 Nov 2020

1) Line 221: It should be Result section, please change

As requested, we have corrected this typo and removed “and discussion” from line 221.

2) Line 235: Authors could discuss more rigorously regarding the findings "general compliance with LLIN decreases during periods of lower mosquito density and increases during periods of higher mosquito density" in line with the findings (line 303): the reason one might not use LLIN is 28.8 % answered that there were no mosquitoes compared to 'too hot' (85.6%). Please explain… considering understanding the importance of LLIN compliance is strongly associated with a higher likelihood of malaria

Thank you for highlighting this important behavioral point that highlights individuals’ reported reasons for the observed behaviors. We have edited line 235 to read:

“In this study, it appears that perceptions of risk of malaria transmission decreased as community burden and mosquito densities declined. Along with this decrease in actual and perceptions of risk, it is possible that other salient barriers to use, such as the heat (referenced by 85.6% of respondents), bedbugs (24.6%), not liking the smell (11.5%) or the bednet being too dirty (9.6%), overcome other incentives to consistently use LLINs and lead to lower adherence.”

3) Line 318: Author should add in the discussion section regarding non-adherence the used of LLINs in the age group under 5 years old. Considering the age range is still under the supervision of parents or adults, therefore I was clarified on my previous review note why was age range under 5 years old, included.

This is another important point and we agree that more discussion of the intrahousehold behaviors related to age and bednet use is warranted. We have added the following text on line 318:

“This is particular interesting given that it might be expected that children under five will often use their bednets under the supervision of adults in the household. The discordance between LLIN use by adults and young children deserves special attention in future efforts to understand who is responsible for choices related to bednet use in Ugandan households.”

Submitted filename: Response to reviewers.docx

Click here for additional data file.

19 Nov 2020

Non-adherence to long-lasting insecticide treated bednet use following successful malaria control in Tororo, Uganda

PONE-D-20-27114R2

Dear Dr.  Krezanoski,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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24 Nov 2020

PONE-D-20-27114R2

Non-adherence to long-lasting insecticide treated bednet use following successful malaria control in Tororo, Uganda

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Table 1

Risk factors for LLIN non-adherence stratified by calendar time.

Risk factor	Categories	November 2017 –April 2019 (18 months)				May 2019 –October 2019 (6 months)
		Observations	Non-adherence	OR (95% CI)	p-value	Observations	Non-adherence	OR (95% CI)	p-value
Age in years	≥ 18	5155	37.7%	reference group		1668	51.6%	reference group
	< 5	4772	47.6%	1.47 (1.27–1.70)	<0.001	1263	65.6%	3.31 (2.30–4.75)	<0.001
	5 - <18	7358	54.6%	2.67 (2.34–3.06)	<0.001	2568	75.5%	6.88 (5.01–9.45)	<0.001
Vector density a	≥ 3	2711	32.1%	reference group		1433	64.5%	reference group
	0–2	14574	50.5%	3.25 (2.92–3.63)	<0.001	4066	66.5%	0.99 (0.80–1.23)	0.91
Household wealth b	Least poor	11017	47.5%	reference group		3509	60.0%	reference group
	Poorest	6268	47.8%	1.03 (0.54–1.97)	0.94	1990	76.5%	5.09 (1.17–22.2)	0.03
Gender	Male	8344	48.7%	reference group		2583	66.6%	reference group
	Female	2879	46.6%	0.98 (0.88–1.10)	0.76	2916	65.4%	1.15 (0.87–1.52)	0.34

a Number of female anopheles captured using CDC light traps the prior night in the room the participant was sleeping.

b Wealth index stratified into the poorest households (lowest tertile) and the least poor households (all other households).

Table 2

Association between LLIN non-adherence and odds of malaria episode.

Period of assessmenta	Reported LLIN adherenceb	Proportion among cases (%)	Proportion among controls (%)	OR (95% CI)c	p-value
1 to 5 weeks prior	At each assessment	5/22 (22.7%)	196/458 (42.8%)	reference group
	Less than always	17/22 (77.3%)	262/458 (57.2%)	3.12 (1.06–9.21)	0.04
1 to 9 weeks prior	At each assessment	1/22 (4.6%)	163/458 (35.6%)	reference group
	Less than always	21/22 (95.5%)	295/458 (64.4%)	15.0 (1.95–114.9)	0.009
1 to 13 weeks prior	At each assessment	0/22 (0%)	121/458 (26.4%)	reference group
	Less than always	22/22 (100%)	337/458 (73.6%)	Unable to estimate	N/A

a Relative to date of diagnosis of case (or comparable control).

b Measured every 2 weeks.

c Controlling for mean vector density during period of assessment from room where participant slept.

Table 3

Knowledge and perceptions of malaria risk relating to LLIN adherence.

Question	Options	Observations	Proportion
Which of these groups should use a bednet every night?a	Children <5 years	441/459	96.1%
	Pregnant women	433/459	94.3%
	School age children	371/459	80.1%
	Adults	365/459	79.5%
Do you feel that you know how to use a bednet correctly?	Yes	407/459	88.7%
	No	50/459	10.9%
	Unsure	2/459	0.4%
What are some important reasons why someone would not use a bednet that is hung above their sleeping space?a	Too hot	393/459	85.6%
	No Mosquitoes	132/459	28.8%
	Bed bugs/fleas	113/459	24.6%
	Forgot	76/459	15.6%
	Don't like smell	53/459	11.5%
	Net too dirty	44/459	9.6%
	Itching, rashes, allergic reaction	36/459	7.8%
	Net too old/too many holes	30/459	6.5%
	No malaria	29/459	6.3%
	Don't know how to use	15/459	3.2%
	Net no longer kill insects	12/459	2.6%
	Inconvenient to use	6/459	1.3%
	Others (spread infection; fire risk)	9/459	2.0%
	Unsure	8/459	1.7%
Please bring to mind the last night you recall not using a bednet. Can you tell me why you did not use a bednet during that night?	I just forgot	162/459	35.3%
	I was travelling	85/459	18.5%
	It was too hot	77/459	16.8%
	Not applicable, I never miss a night	74/459	16.1%
	There were no mosquitoes around	34/459	7.4%
	There is no malaria here	13/459	2.8%
	Not sure	14/459	3.1%

a Multiple responses accepted.