CD44-rs353630 and CHI3L2-rs684559) were reported to be associated with survival after PDAC resection. We attempted to replicate the associations in 1856 PDAC patients (685 resected with stage I/II) from the PANcreatic Disease ReseArch (PANDoRA) consortium. We also analysed the combined effect of the two genotypes in order to compare our results with what was previously reported. Additional stratified analyses considering TNM stage of the disease and whether the patients received surgery were also performed. We observed no statistically significant associations, except for the heterozygous carriers of CD44-rs353630, who were associated with worse OS  among patients with stage I disease. This association is in the opposite direction of those reported previously, suggesting that data obtained in such small subgroups are hardly replicable and should be considered cautiously. The two polymorphisms combined did not show any statistically significant association. Our results suggest that the effect of CD44-rs353630 and CHI3L2-rs684559 cannot be generalized to all PDAC patients.Although pancreatic ductal adenocarcinoma (PDAC) survival is poor, there are differences in patients’ response to the treatments. Detection of predictive biomarkers explaining these differences is of the utmost importance. In a recent study two genetic markers ( PDAC is, therefore, projected to be the second cause of cancer-related deaths in the USA by 20302 with a similar figure in most Western countries. One of the reasons of this meager survival is the lack of specific symptoms and of biological markers for early diagnosis and risk stratification. Moreover, treatment options are poor, and surgery in the setting of multimodal treatments is the only possible cure. However, there are marked differences in patients’ responses to the treatments, which cannot be explained by the traditional prognostic factors such as tumour size, lymph node involvement and distal metastasis3. Thus, investigation of biomarkers able to predict the tumour behaviour is of the utmost importance.Pancreatic ductal adenocarcinoma (PDAC) is a lethal tumour type, with an increasing incidence over the past decades and a five year survival rate still as low as 9%4. Several germline mutations, associated with well described hereditary syndromes, increase the risk of developing PDAC and subjects carrying such mutations are offered surveillance in the context of research protocols5. Besides these high penetrance germline mutations, there are overwhelming evidences on the role of germline genetic polymorphisms in the development of the disease, identified through genome-wide association studies (GWAS) or through large scale case–control studies21. However, the possible association between genetic variants and patients’ outcome in PDAC, both in terms of survival and response to treatments, has been poorly investigated with limited success29. The genetic contribution to PDAC survival has, however, been studied with GWAS. For example, a small-scale GWAS done on 252 PDAC cases, with subsequent validation done on 261 and 572 sets of patients, respectively, reported an association between a single nucleotide polymorphism (SNP) on chromosome 12 and overall survival (OS) of PDAC cases (p = 1.72 × 10−7 in the combined dataset)27. Innocenti et al. reported on a GWAS on OS of 351 pancreatic cancer patients treated with gemcitabine in the context of a randomized clinical trial. This study showed an association of a SNP in IL17F with OS (p = 9.51 × 10−7)29. Wu et al. performed the largest GWAS on OS to date in 1005 PDAC cases (10), of whom 642 were of European descent (from prospective cohort studies), used as discovery phase, and 363 retrospectively collected cases of Chinese descent used for replication25. In the first stage 131 SNPs at 28 loci showed association with OS of the PDAC cases (p < 10−5). Combining the discovery and the replication phases, a locus on chromosome 11 near the SBF2 gene was the most significantly associated with OS of PDAC patients (p = 1.72 × 10−7). It is worth noticing that none of the findings of the above mentioned GWAS reached genome-wide significance level, conventionally set at p < 5 × 10−8, and that only a few loci were successfully replicated across multiple reports. In one of the few studies of this kind, we genotyped 44 SNPs proposed by the GWAS by Wu et al. in one of the largest study so far consisting in 1722 PDAC cases, in the context of the PANDoRA consortium25. We validated associations of one SNP in the CTNNA2 gene (rs1567532) and one SNP in the RUNX2 gene (rs12209785) with OS26. The lack of replicability of the PDAC survival loci is in striking contrast with the situation observed for GWAS-identified risk loci, which are generally confirmed in multiple independent studies, to some extent even in populations of diverse ethnic background.Genetic variability accounts for a large part of the risk to develop PDAC30. The authors used a dense SNP array and investigated more than 2 million SNPs in 331 PDAC patients in a two-phase approach. The main findings consisted in association of CD44-rs353630 and CHI3L2-rs684559 with PDAC survival. The diplotype combination of the two alleles of the two SNPs associated with better survival  reached genome-wide significance  in the merged population consisting of the two phases.Recently, Dimitrakopoulos and colleagues carried out a genome-wide screening of functional variants aimed at identifying novel markers associated with PDAC survival after pancreatic resectionOne of the reasons behind the lack of success of studies investigating genetic variants as biomarkers of disease outcome is that most studies so far were underpowered and therefore prone to statistical fluctuation, and/or that a proper replication phase in an independent adequately sized population was lacking. Considering the capricious nature of association studies, we aimed at validating the findings by Dimitrakopoulos and colleagues in a large cohort of 1856 PDAC cases among which 685 resected patients with tumour stage I or II from the PANcreatic Disease ReseArch (PANDoRA) consortium.The population investigated in the present study consisted overall of 1856 PDAC patients: 105 with stage I, 717 with stage II, 348 with stage III and 686 with stage IV at the time of diagnosis. The median age was 66 and both sexes were represented with a slight majority of males (56%). 685 patients with stage I or II were operated with curative intent. A description of the population is shown in Table CD44-rs353630 and 96% for CHI3L2-rs684559, the concordance between the duplicated samples was higher than 99%. All the SNPs resulted to be in HWE equilibrium with non-significant p values (p > 0.05) when considering all the individuals together or dividing them by country of origin. The minor frequency (MAF) observed in PANDoRA is 25.62% for CD44-rs353630 and 36.07% for CHI3L2-rs684559, in line with what observed in the European populations in the 1000 Genomes project: 25.35% for CD44-rs353630 and 36.88% for CHI3L2-rs684559.The genotyping call rate was 95% for CD44-rs353630 of having a worse survival compared with the T/T homozygous . We also performed stratified analysis by disease stage and when analysing the individuals that presented stage I disease at diagnosis we observed a statistically significant association for the heterozygous C/T carriers of CD44-rs353630 and worse OS compared with the reference . The analysis considering only the 685 patients who received surgery did not show any statistically significant associations. We also performed an analysis considering the combination of the two markers and we observed no statistically significant result. The frequencies and distribution of the genotypes, the HR and 95% CI for the association with PDAC survival are shown in Table p = 0.64), according to the combined genotypes of the two SNPs is showed in Fig. CHI3L2-rs684559 SNP, in comparison with the homozygotes for the common allele .Analysing the whole PDAC case population and adjusting for age, sex and stage we observed no statistically significant association between the two SNPs under investigation and OS of PDAC patients, with the best finding consisting in a non-significant trend for the C/C genotype of the CD44-rs353630: the weak (p = 0.00025) association between the A allele and a decreased expression of the CD44 gene in the tibial nerve. GTEx predicts that CHI3L2-rs684559 is an eQTLs for the CHI3L2, DENND2D, CHIAP2 and CHIA genes across seven tissues, although none of them in the pancreas . For CD44-rs353630 RegulomeDB showed a rank of 4 that is the third lowest and indicates that the polymorphism may be situated in a possible transcription factor binding site. On the other hand, for CHI3L2-rs684559 RegulomeDB showed a rank of 1b indicating a very high degree of supporting data in the database and high probability of functional relevance. Haploreg showed no SNPs in LD with CD44-rs353630 and no eQTLs, while six SNPs  in LD with CHI3L2-rs68455 and 14 eQTLs in lymphoblastoid cell lines and in the whole blood.The GTEx portal identified only one eQTL for 37, the involvement of genetic variability in PDAC survival has been investigated with limited success. The most reliable factors in determining PDAC patient survival are, therefore, clinical parameters such as TNM stage, tumour grade, margins of resection and pre- and postoperative levels of C-reactive protein (CRP) and CA-19-938. Unfortunately, these factors have limited accuracy in predicting the outcome of PDAC patients and their response to treatments. Therefore, the identification of additional biological markers would be of the utmost importance as a basis to elaborate novel decisional algorithms aimed at optimizing treatment and improving patients' outcomes. Additionally, discovering new PDAC survival genetic loci may give insight to the molecular mechanisms of survival and suggest, directly or indirectly, new therapeutic targets.In the last decades genetic epidemiological approaches, especially those performed through genome-wide association studies in the context of multicentric studies, have identified thousands of SNPs and have shed light on the biology of many cancer types. As far as regards PDAC, around 30 risk loci showed a clear association with the development of the disease. However, differently from other cancer types such as breast, lung, prostate, colorectal cancers, and multiple myeloma29. Recently two novel candidates, identified through a GWAS approach, have been proposed. A total of 331 PDAC patients were investigated by means of a dense SNP array, encompassing more than 2 million SNPs, finding an association between CD44-rs353630 and CHI3L2-rs684559 and PDAC survival in resected patients of whom 90.9% with stage I or II. The diplotype combination of these two alleles was, indeed, associated with longer survival after surgery30. We genotyped these two SNPs and analysed them, separately or in combination, in a large set of PDAC cases belonging to the PANDoRA consortium. Considering the paucity of evidences on markers related to PDAC survival this attempt to generalize the results of the two SNPs was necessary. We had more than 99% power to replicate the associations, but we observed none when analysing the whole population or when considering only the resected patients. The most clinically relevant result of the study by Dimitrakopoulos and colleagues is the association of the two combined polymorphisms with better survival in 331 patients resected with intent of radicality. Availability of such a biomarker might be extremely relevant as it would help selection of patients to consider for upfront surgery or neoadjuvant chemotherapy, thus contributing to a personalized medicine approach for PDAC. However, in our subgroup analysis on 685 patients resected with stage I or II disease (therefore with a much larger sample size and a power to detect the association greater than 99%) the same SNP combination did not show any statistically significant association (the best association we observed had a p = 0.48).Several SNPs have been proposed to be possible prognostic markers in PDAC patients, given the association with survival. However, with only a few exceptions, none has been consistently replicated in following studiesCD44-rs353630 C/T individuals and worse OS among PDAC cases diagnosed with stage I. The estimate is very high (HR > 5) and it goes in the opposite direction of the ones reported by Dimitrakopoulos, which all showed association of the effect alleles/genotypes with better OS. These findings arise from an analysis conducted in a very small subgroup of individuals and only comparing heterozygous with the reference homozygous  and therefore must be taken with caution. The analysis conducted to assess the functional relevance of the SNPs clearly shows that for CD44-rs353630 there is little or no evidence of its involvement in gene expression, while CHI3L2-rs684559 is possibly an eQTL with regulatory potential in several tissues, but not in the pancreas. It is therefore difficult to link the SNPs to a functional role in pancreatic cancer progression or outcome.Further stratifying the population by stage, we observed a statistically significant association between CHI3L2-rs684559 SNP. We note that this association is not significant if we consider a significance threshold adjusted for multiple testing . Considering also the relatively small number of cases used for this analysis (18 patients diagnosed with stage I and 16 in stage II), and the difficulty of explaining why the polymorphism should be associated with OS only in Hungarian patients, we are inclined to think that this result is due to statistical fluctuation and does not reflect a real association. A possible limitation of the present study is the lack of information on chemotherapeutic regimens administered to the patients, either before or after surgery. This prevents us to explore association between the genetic variability and drug response that may help personalization of treatments. In addition, the included patients were not consecutively enrolled in each center, and that could add a potential bias to the findings. We believe, however that given the samples size this bias should be diluted and unlikely to be responsible for the lack of statistically significant association that we observed.Analyses stratified by country of origin did not show anything remarkable, with the exception of a weak association with longer OS in Hungarian patients diagnosed in stage I or II for heterozygous for the 9.The PANDoRA consortium consists of a multicentric study that includes 12 European countries  and Japan and has been described in detail elsewhere39, and patients were categorized as stage I , stage II , stage III , stage IV . The patients that underwent surgery were categorized by the stage at the time of operation.Briefly, all cases included in the consortium population are defined by a confirmed diagnosis of pancreatic cancer and were collected with a non-consecutive sampling approach in 21 different centers, including departments of surgery, endoscopy and gastroenterology. In addition to pancreatic cancer cases, controls have been selected among the general population, blood donors and among hospitalized subjects with different diagnosis excluding cancer. In the present study, 1856 PDAC cases of European descent were included, for whom information on country of origin, sex, age at diagnosis, overall survival (OS) and disease stage assessed by TNM stage was available. Disease stage was defined according to American Joint Committee on Cancer TNM system 7th versionAll subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Commission of the Medical Faculty of the University of Heidelberg (project identification code: S-565/2015). A description of the population is shown in Table CD44-rs353630 and CHI3L2-rs684559 that were proposed recently by Dimitrakopoulos and colleagues as associated with survival of PDAC patients that received surgery. For each subject DNA was extracted from whole blood using a QIAamp 96 DNA QIAcube HT Kit on QIAcube 96 instrument and kept frozen at − 20° till use. Genotyping was conducted using TaqMan  allelic discrimination technology following the manufacturer’s recommendation (assay id C____779802_10 for CD44-rs353630 and assay id C___3138574_20 for CHI3L2-rs684559). For each reaction 10 ng of dried DNA were used. The genotyping was conducted in 384-well plates and duplicated samples (8%) were included in each plate for quality control purpose. The identity of the samples was not known to the personnel performing the lab work. Genotype attribution was made using QuantStudioTM 5 Real-Time PCR system  and QuantStudio software.We selected two germline variants, CD44-rs353630 and A for CHI3L2-rs684559). In addition, considering that the most statistically significant association observed by Dimitrakopoulos was the one combining the two genotypes, we also performed this analysis defining two groups: the individuals with the CD44-rs353630 T/T and/or CHI3L2-rs684559 A/A as the reference category and all the other genotype combinations as the other group. For this analysis we considered only individuals with 100% call rate. All analyses were adjusted for sex, age and TNM stage. We also performed an analysis adjusting only for disease stage, since Dimitrakopoulos and colleagues did not adjust for sex and age.Deviation from Hardy–Weinberg equilibrium (HWE) was tested for both polymorphisms in the overall population and dividing for country of origin using the Pearson corrected chi square test. Survival analysis was conducted by Cox proportional hazard models with OS as endpoint, computing hazard ratios (HR) and 95% confidence intervals (CI), using an additive and a codominant inheritance models and setting the same alleles used by Dimitrakopoulos and colleagues as the reference category (T for As the vast majority (90.9%) of the patients in the study by Dimitrakopoulos and colleagues were resected with stage I or II, stratified analysis was conducted considering patients showing TNM stage I, TNM stage II, and TNM stage I and II together. We also performed a stratified analysis considering TNM stage I and II for patients who received surgery, as well as by country of origin. The probability of survival was calculated by means of a Kaplan–Meier curve.CD44-rs353630 and CHI3L2-rs684559 we used several bioinformatic tools. In particular, the Genotype-Tissue Expression (GTEx) project portal  was used to identify potential cis-acting expression quantitative trait loci (eQTLs) in order to establish whether the two SNPs could be involved in the expression of nearby genes (accession date 8th May 2020)40. We analysed all the tissues present in the database. We have used Regulome DB 2.0 (https://www.regulomedb.org/regulome-search/) to evaluate the effect of the SNPs with regulatory regions of the human non-coding genome41. Regulome DB 2.0 assigns to each SNP a rank consisting of 14 steps, that reflect the volume of the supporting data on the functional relevance of the SNP, going from 6 (lowest amount of evidence) to 1a (highest amount of evidence). Finally, we used Haploreg v4.1  to link the SNPs to functional annotations of the genome42. Haploreg is designed to explore the possible mechanistic interaction of the candidate SNPs  with regulatory motifs.To test the possible functional relevance of The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Commission of the Medical Faculty of the University of Heidelberg (project identification code: S-565/2015).CD44-rs353630 and CHI3L2-rs684559 in the pancreatic tissue. Our results strongly support the need to consider with caution findings on genetic variables as prognostic markers in PDAC, in the absence of replication in large and independent datasets or of compelling in vitro mechanisms to support the associations.In conclusion, we attempted to replicate the results of a genome-wide investigation on genetic polymorphisms and survival of PDAC patients, with a well-powered study. Our findings suggest no involvement in the PANDoRA population of the two variants reported by Dimitrakopoulos and colleagues and highlight a lack of functional or regulatory role of Moreover, it would be desirable for future studies to have information on the therapy and to include this information in the analyses. This could make possible to evaluate the different genetic predisposition in drug response, and further our knowledge of this deadly disease.


The scale-up of malaria control efforts has led to marked reductions in malaria burden over the past twenty years, but progress has slowed. Implementation of indoor residual spraying (IRS) of insecticide, a proven vector control intervention, has been limited and difficult to sustain partly because questions remain on its added impact over widely accepted interventions such as bed nets. Using data from 14 enhanced surveillance health facilities in Uganda, a country with high bed net coverage yet high malaria burden, we estimate the impact of starting and stopping IRS on changes in malaria incidence. We show that stopping IRS was associated with a 5-fold increase in malaria incidence within 10 months, but reinstating IRS was associated with an over 5-fold decrease within 8 months. In areas where IRS was initiated and sustained, malaria incidence dropped by 85% after year 4. IRS could play a critical role in achieving global malaria targets, particularly in areas where progress has stalled. Indoor residual spraying (IRS) of insecticide is one of the primary malaria vector control initiatives, but implementation is limited. Here, the authors show that discontinuation of IRS in Uganda was associated with increased malaria incidence, and introduction of IRS was associated with decreased incidence. However, global progress has slowed in recent years, particularly in sub-Saharan Africa, which accounted for 94% of the world’s 215 million cases in 20192. Long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) of insecticide are the primary vector control interventions used for the prevention of malaria. The World Health Organization recommends universal coverage of LLINs for at-risk populations in sub-Saharan Africa, where the proportion of households owning at least one LLIN is estimated to have increased from 47% in 2010 to 72% in 2018. Pyrethroids are the only class of insecticides widely use in LLINs and, given the emergence of widespread pyrethroid resistance4, there is concern that the effectiveness of LLINs may be diminishing, leading to the development of new LLIN formulations including pyrethroid synergists and non-pyrethroid nets. Unlike LLINs, IRS has the advantage of utilizing multiple different classes of insecticides and combining IRS with LLINs may improve malaria control and slow the spread of pyrethroid resistance. However, few controlled trials have evaluated the effect of adding IRS to communities using LLINs and the evidence is mixed, with a few studies showing benefits when IRS included “non-pyrethroid-like” insecticides5. Other barriers to IRS delivery—including cost, logistics, and community acceptance—have limited its use6, such that less than 2% of the population at risk in sub-Saharan Africa was protected by IRS in 2019, a decrease from over 10% coverage in 20102.Over the past 20 years the scale-up of malaria control efforts has led to marked reductions in morbidity and mortality2. Malaria control efforts in Uganda have primarily focused on LLINs. In 2013–14 it became the first country to implement a universal LLIN distribution campaign, which was repeated in 2017–18. In 2018–19, Uganda had the highest coverage of LLINs in the world, with 83% of households reporting owning at least one LLIN7. In contrast to LLINs, the implementation of IRS in Uganda has been focal and limited. In 2006, IRS was reintroduced into Uganda for the first time since the 1960s. In 2007–09, the IRS program was shifted to ten high burden districts in the north, leading to large reductions in malaria burden9. In 2014, the IRS program was relocated from these ten northern districts to 14 districts in the eastern part of the country, where it has been sustained. The discontinuation of IRS in the ten northern districts was followed by a marked resurgence in malaria cases11, prompting the implementation of a single round of IRS in these ten districts in 2017.Uganda is illustrative of a country where the burden of malaria remains high and progress has slowed in recent yearsIn this study, we use data from a network of health facility-based malaria surveillance sites to evaluate the impact of different IRS delivery scenarios in 14 districts in Uganda. This study has three objectives: (1) to estimate the impact of withdrawing IRS after 5 years of sustained use on the burden of malaria in three sites in Northern Uganda; (2) to estimate the impact of restarting IRS with a single round 3 to 4 years after IRS was discontinued on the burden of malaria in nine sites in Northern Uganda; and (3) to estimate the impact of 5 years of sustained IRS on the burden of malaria in five sites in Northern and Eastern Uganda.12. DDT or pyrethroids were initially used but in 2010 the insecticide was changed to a carbamate (bendiocarb) due to concern regarding the spread of pyrethroid resistance. Rounds of bendiocarb were repeated approximately every 6 months until 2014 when the IRS program was discontinued, so that resources could be shifted to other high burden districts. In 2017, these ten districts in northern Uganda received a single round of the organophosphate pirimiphos-methyl (Actellic 300CS®) following reports of malaria resurgence after IRS has been discontinued in 2014. Between 2014 and 2015, IRS with bendiocarb was implemented in 14 districts in the Northern and Eastern part of the country. Rounds of bendiocarb were repeated approximately every 6 months until 2016 when the formulation was changed to Actellic 300CS®, which continues to be administered once a year.This study utilized data from 14 health facilities located in 14 districts in Northern and Eastern Uganda  due to concerns of pyrethroid resistance. During the latter distribution, all districts included in this analysis received conventional pyrethroid insecticides due to prior concerns of antagonism between PBO LLINs and Actellic 300CSWe assessed the impact of stopping IRS after sustained use (objective 1), reinitiating IRS for a single round after stopping 3 years prior (objective 2), and initiating and sustaining IRS in areas that had not received the intervention prior (objective 3) on changes in malaria case counts relative to a baseline before to starting or stopping IRS. Figure Across the three sites included in the analysis, a total of 224,859 outpatient visits were observed Table . During Monthly adjusted IRRs and 95% confidence intervals (CI) for the three sites combined are presented in Fig. A total of 858,380 outpatient visits were recorded across the analysis period for the nine sites Table . Mean moMonthly adjusted IRRs and 95% CI for the nine sites combined are presented in Fig. In total, 574,587 outpatient visits were observed across the five sites included in the analysis Table . During Monthly adjusted IRRs and 95% CI for the five sites combined are presented in Fig. 14. However, despite this success, the burden of malaria remains high in much of the country. Uganda had the third highest number of malaria cases reported in 2019, with reported case incidence increasing since 20142. If Uganda is to achieve the goals established by the World Health Organization’s Global Technical Strategy for malaria including reducing malaria case incidence by at least 90% by 2030 as compared with 201515, additional malaria control measures will be needed. This report highlights the critical role of IRS in substantially reducing the burden of malaria in areas where transmission remains high despite deployment of LLINs. Withdrawing IRS after 5 years of sustained use in three districts in northern Uganda was associated with a more than fivefold increase in malaria cases within 10 months. Restarting IRS with a single round in nine districts in Northern Uganda ~3 years after IRS had been stopped was associated with a transient but important (more than a fivefold) decrease in malaria cases within 8–12 months, returning to pre-IRS levels after 23 months. Initiating and sustaining IRS in five districts in Eastern Uganda was associated with a gradual reduction in malaria cases reaching almost a sevenfold reduction after 4–5 years.Uganda has been exceptionally successful in scaling-up coverage of LLINs. Following the mass distribution campaigns to deliver free LLINs in 2013–14 and 2017–18, 90 and 83% of households, respectively reported ownership of at least one LLINP. falciparum malaria by almost half16. However, there is concern that the effectiveness of LLINs may be diminishing due to widespread resistance to pyrethroids which until recently were the only class of insecticides approved for LLINs. Similar to many other African countries, high-level resistance to pyrethroids among the principle Anopheles vectors has been reported recently throughout Uganda19. In addition, behavioral changes in vector biting activity following the introduction of LLINs have been reported which could present new challenges for malaria control22. Finally, the effectiveness of LLINs may be further compromised by poor adherence and waning coverage in the setting of free distribution campaigns done intermittently. In Uganda, less than 18% of households reported adequate coverage (defined as at least one LLIN per two residents) 3 years after the 2013–14 distribution campaign23 and adequate coverage decreased from 71% to 51% between 6 and 18 months following the 2017–18 distribution campaign24. Although the World Health Organization recommends mass distribution campaigns every 3 years, mounting evidence suggests that LLINs should be distributed more frequently to sustain high coverage31.Robust evidence supports the widespread use of LLINs for malaria control. In a systematic review of clinical trials conducted between 1987 and 2001, insecticide treated nets reduced all cause child mortality by 17% and the incidence of uncomplicated 32, it is surprising that the evidence base from contemporary controlled trials on the impact of adding IRS to LLINs for vector control is limited. A recent systematic review of cluster randomized controlled trials conducted in sub-Saharan Africa since 2008, reported that adding IRS using a “pyrethroid-like” insecticide to LLINs did not provide any benefits, while adding IRS with a “non-pyrethroid-like” insecticide produced mixed results5. Among the four trials comparing IRS plus LLINs with LLINs alone, three evaluated IRS with a carbamate (bendiocarb) and one evaluated a long-lasting organophosphate, pirimiphos-methyl (Actellic 300CS®)36. Only two trials (both using bendiocarb) assessed malaria incidence; one from Sudan found a 35% reduction when adding IRS to LLINs34, while another from Benin found no benefit of adding IRS33. All four trials assessed parasite prevalence, with an overall non-significant trend towards a lower prevalence when adding IRS to LLINs 5. However, when the analyses were restricted to include only the two studies with LLIN usage over 50%, adding IRS reduced parasite prevalence by over 50% 5. Of note, none of the trials that evaluated the impact of adding IRS with a “non-pyrethroid-like” insecticide assessed outcomes beyond 2 years. More recently, a number of observational studies have reported benefits of using IRS with pirimiphos-methyl (Actellic 300CS®). In the Mopti Region of Mali, delivery of a single round of IRS with Actellic 300CS® was associated with a 42% decrease in the peak incidence of laboratory-confirmed malaria cases reported at public health facilities37. In the Koulikoro Region of Mali, villages that received a single round of IRS with Actellic 300CS® combined with LLINs observed a greater than 50% decrease in the incidence of malaria compared to villages that only received LLINs38. In the Northern Region of Ghana, districts that received IRS with Actellic 300CS® reported 26–58% fewer cases of laboratory-confirmed malaria cases reported at public health facilities over a 2-year period, compared to districts that did not receive IRS39. In Northern Zambia, implementation of IRS with Actellic 300CS® targeting only high burden areas over a 3 year period was associated with a 25% decline in parasite prevalence during the rainy season, but no decline during the dry season40. In Western Kenya, the introduction of a single round of IRS with Actellic 300CS® was associated with a 44–65% decrease in district level malaria case counts over a 10 month period compared to pre-IRS levels41. In addition, several recent reports have documented dramatic resurgences of malaria following the withdrawal of IRS with bendiocarb in Benin42, and the withdrawal of IRS with Actellic 300CS® in Mali and Ghana39.Given concerns about the current effectiveness of pyrethroid-based LLINs and the persistently high burden of malaria despite aggressive scale-up of LLINs in countries like Uganda, additional malaria control measures are needed. IRS is an attractive option. Historically, IRS programs were used to dramatically reduce and even eliminate malaria in many parts of the world. Thus, while there is some evidence for the impact of IRS in the absence of LLINs11. In this study we expand on these findings by including data from three districts and covering a 31-month period following the withdrawal of IRS. We were able to quantify more than a fivefold increase in malaria cases which was sustained over the 10–31 months following the withdrawal of IRS. This marked resurgence occurred despite the fact the first universal LLIN distribution campaign was timed to occur right after IRS was withdrawn. Given the dramatic nature of the resurgence, the Ugandan government was able to procure funding for a single round of IRS with Actellic 300CS® ~3 years after IRS was withdrawn in 10 districts of Northern Uganda. In this study, we assessed the impact of this single round in nine of these districts. This single round was associated with over a fivefold decrease in malaria cases after 8–12 months, with malaria cases returning to pre-IRS levels after almost 2 years. These data suggest that IRS with longer-acting formulations such as Actellic 300CS® administered every 2 years could be considered as a strategy for mitigating the risk of resurgence following sustained IRS and/or enabling countries to expand coverage when resources are limited, but formal assessment and a cost-effectiveness analyses are needed. This study also evaluated the impact of 5 years of sustained IRS in five districts of Eastern Uganda, starting first with bendiocarb and then switching to Actellic 300CS® after 18 months. Rounds of IRS were initially associated with marked decreases in malaria cases followed by peaks before subsequent rounds until the fourth and fifth years after IRS was initiated when there was a sustained decrease of almost sevenfold compared to pre-IRS level. Given the before-and-after nature of our study design, it is not clear whether the maximum sustained benefits of IRS seen after 4–5 years were due to the cumulative effect of multiple rounds of IRS, the switch from bendiocarb to Actellic 300CS®, improvements in implementation  at 70 level III/IV public health facilities across Uganda. At each MRC, individual-level data from standardized registers for all patients presenting to the outpatient departments are entered into a Microsoft Access (v16.0) database by on-site data entry officers. Variables include patient demographics, results of laboratory testing for malaria (rapid diagnostic test [RDT] or microscopy), diagnoses given, and treatments prescribed. Emphasis is placed on ensuring that patients with suspected malaria undergo testing, by either RDT or microscopy.Enhanced malaria surveillance was established by UMSP in 2006This study utilized data from 14 MRCs located in districts that either previously had IRS or have ongoing IRS campaigns. We estimated the impact of withdrawing IRS using data from three sites in Northern Uganda that had at least 6 months of data preceding the final round of IRS administered in 2014. To estimate the impact of restarting IRS with a single round administered in 2017, we used data from nine sites in Northern Uganda. To estimate the impact of sustained IRS over 5 years, we used data from five sites in Eastern Uganda where IRS had been implemented since 2014–15. For all analyses, we accessed raw, individual-level data, and aggregated to monthly-level data.The exposure was specified as an indicator variable for each month since IRS was withdrawn or initiated relative to a baseline period Fig. . All basThe primary outcome was the monthly count of laboratory-confirmed malaria cases at each MRC. The case count was corrected for testing rates by multiplying the number of individuals with suspected malaria but not tested each month by the TPR  for that month and adding the result to the number of laboratory-confirmed positive cases. As a sensitivity analysis, we re-specified the models including only laboratory-confirmed case counts as the outcome.47, indicator variables for month of the year , the proportion of tests that were RDTs in that month (vs. microscopy), and the number of individuals who attended the health facility but were not suspected of having malaria in that month .We adjusted for time-varying variables that impact malaria burden and malaria case detection at the health facility. These variables included monthly rainfall at the health facility lagged by 1 month extracted from the Climate Hazards Infrared Precipitation with Stations databaseFor each objective, we specified mixed effects negative binomial regression models with random intercepts for health facility. Coefficients for the exposure variable were exponentiated to represent the incidence rate ratio (IRR) comparing the incidence of malaria in the month of interest relative to the baseline period. This method assumes that the underlying population has remained constant over the study period. All analyses were carried out in R v3.6 and Stata v14.Ethical approval for study procedures and data collection was provided by ethics committees of University of California San Francisco (REF 250046), the School of Medicine College of Health Sciences at Makerere University (REF 2019-087), and Uganda National Council of Science and Technology (REF HS 2659). Written informed consent was not required by the ethical review committees due to the routine, de-identified nature of the data.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Titanium alloy (Ti6Al4V) is one of the most prominent biomaterials for bone contact because of its ability to bear mechanical loading and resist corrosion. The success of Ti6Al4V implants depends on bone formation on the implant surface. Hence, implant coatings which promote adhesion, proliferation and differentiation of bone-forming cells are desirable. One coating strategy is by adsorption of biomacromolecules. In this study, Ti6Al4V substrates produced by additive manufacturing (AM) were coated with whey protein isolate (WPI) fibrils, obtained at pH 2, and heparin or tinzaparin (a low molecular weight heparin LMWH) in order to improve the proliferation and differentiation of bone-forming cells. WPI fibrils proved to be an excellent support for the growth of human bone marrow stromal cells (hBMSC). Indeed, WPI fibrils were resistant to sterilization and were stable during storage. This WPI-heparin-enriched coating, especially the LMWH, enhanced the differentiation of hBMSC by increasing tissue non-specific alkaline phosphatase (TNAP) activity. Finally, the coating increased the hydrophilicity of the material. The results confirmed that WPI fibrils are an excellent biomaterial which can be used for biomedical coatings, as they are easily modifiable and resistant to heat treatments. Indeed, the already known positive effect on osteogenic integration of WPI-only coated substrates has been further enhanced by a simple adsorption procedure. Ti6Al4V is a well-known biomaterial for orthopaedic implants widely used due to its high specific strength and high corrosion resistance. One of the advantages of this material is that it can be produced using AM techniques, allowing the generation of implants with complicated shapes while keeping production costs reasonably low . The staPrevious studies demonstrated that WPI increases cell proliferation and osteogenic differentiation in soluble form both in cell culture medium  and as aIn this study, we demonstrated the possibility of coating Ti6Al4V with heparin-enriched WPI fibrils in order to combine the positive effects of all the components, resulting in a system that is easily sterilizable and can lead to the production, via AM, of complex-shaped prostheses that are highly biocompatible. In addition, we evaluated the influence of the molecular weight of heparin on its ability to modulate the growth of hBMSC by comparing the effects of sodium heparin  and tinzaparin . For this purpose, we studied cell proliferation via metabolic activity by a metabolic assay and osteogenic differentiation via the activity of the tissue non-specific alkaline phosphatase enzyme (TNAP) at days, in culture after two and 11 days. The fluorescence emission of Thioflavin T (ThT) is an indication of the presence of fibrils since the molecule binds to β-sheets that are present in the protein . The intThe effective presence of the fibrils has been confirmed by the scanning electron microscopy (SEM) images shown in According to statistical analysis , each coX-ray photoelectron spectroscopy (XPS) results  showed tThe MTS assay measures metabolic activity of cells as an indication for cell number and cell viability . After tFor evaluation of the osteoinductive potential of different Ti6Al4V coatings, the activity of the TNAP enzyme in hBMSC cultured for 11 days was analyzed. Besides the deposition of calcium phosphate as a final differentiation marker for osteoblasts, TNAP activity is frequently used as an early osteogenic differentiation marker that indicates the potential of hBMSC to form hydroxyapatite ,19. ThusWPI fibrils were prepared according to the protocol described by Keppler et al. ): WPI  additively manufactured in an A2 ARCAM EBM machine  were coated with fibrils by adsorption from the suspension. The substrates were left in contact with 1 mL of the fibril solution (2.5 wt%) for one hour, then rinsed three times with Milli-Q water to remove excess coating and left to air dry. For samples with heparin (or tinzaparin), the protocol was repeated on the fibril-coated samples using a 10 wt% heparin (or tinzaparin) solution.The sterilization  was performed after the coating procedure with a Bench top Autoclave. Addition of heparin or tinzaparin by adsorption from solutions was performed in sterile conditions under a laminar flow hood using syringe  and filters .X-ray photoelectron spectroscopy (XPS) was performed to analyze the surface chemical composition on the samples using an Axis Supra spectrometer  with a monochromatic Al Kα source (1.487 keV). Samples were mounted using carbon tape on a sample holder. An internal flood gun was used for neutralizing charging effects. Wide scans were recorded at a pass energy of 160 eV and a step size of 1 eV. Samples were measured at an emission angle of 0° , power of 225 W (15 kV × 15 mA) and an analysis area of 700 × 300 µm. Three different locations on each coating’s type were analyzed. Spectra were analyzed with CasaXPS software . All binding energies were referenced to the C-C component of the C1s spectrum at 284.8 eV to compensate for the surface charging effects. The curve fitting procedure of the components was performed using Gaussian-Lorentzian function and a linear background.Contact angle measurements were achieved with a home-built system consisting of a light, a stand and a camera connected to a computer. The images were analysed with ImageJ software with the drop analysis plugin .The SEM characterization was performed as follows: samples were mounted on standard aluminum pin stubs using double sided conductive carbon adhesive dots. They were subsequently sputter coated with approximately 5 nm of gold  using a system by Quorum Technologies Ltd., Lewes, UK, Q150RES. Finally, samples were imaged using a Jeol JSM-7800F Field Emission Scanning Electron Microscope (FEG-SEM) using the lower secondary electron detector.hBMSC were isolated from bone marrow aspirates obtained from donors at the Bone Marrow Transplantation Center of the University Hospital Dresden. The cells were characterized as described in the work by . The don2). Two hours after plating, DMEM was added to cover the samples with medium. At day four after plating, cell culture medium was replaced by osteogenic differentiation medium  as described previously [For the in vitro experiments, 5000 hBMSC were seeded in an 80 µL-droplet of cell culture medium , supplemented with 10% heat-inactivated fetal calf serum and antibiotics  onto the surface of each sample  at day two after plating. The cell culture medium was replaced by fresh medium containing 10% of MTS dye solution. After 2 h of incubation at 37 °C in a humidified CO2 incubator, 80 µL of medium was transferred into a 96-well plate and the absorbance of the formed formazan dye was measured photometrically at 490 nm.The metabolic activity of hBMSC cells was determined by the MTS assay  with p-nitrophenylphosphate  as a substrate, as previously described [At day 11 after seeding, hBMSC were analysed for TNAP enzyme activity. TNAP enzyme activity was determined from cell lysates  each in duplicate. The results were presented as mean ± standard error of the mean (SEOM). Statistical significance was analyzed with GraphPad Prism 8.4 software  by ANOVA analysis with Bonferroni’s post-test. The contact angle was measured on four water drops for each sample. One-way ANOVA with Tukey’s post-test was carried out with the software IBM SPSS Statistics Version 27.Cell experiments were performed with cells from three different donors (Coating Ti6Al4V with WPI fibrils obtained at pH 2 enriched with heparin and tinzaparin proved to be a successful strategy to create a viable substrate for hBMSC. This substrate for the cells promotes osteogenic differentiation by improving the quality of the differentiated cells, as evidenced by the increase in the TNAP activity. In particular, this work showed that enriching the coating with heparin and tinzaparin improves the aforementioned effect considerably. Specifically, it seems that tinzaparin has the highest impact on the TNAP activity. In any case, further investigations of the molecular mechanism are needed to further elucidate this behavior. As far as the coating protocol is concerned, one hour of adsorption time was sufficient to successfully coat Ti6Al4V substrates. However, the presence of some uncoated areas was detected. Additionally, XPS can be used as a method to evaluate the presence of heparin and proteins on the surface of the material. Nonetheless, it seems that this is not a suitable method for the detection of tinzaparin, and it advisable to use a different approach in future work. The coating increased the hydrophilicity of the material with a higher extent when enriched with tinzaparin compared to heparin. This behavior, together with the statistically significant difference seen between the heparin and tinzaparin coated samples in the TNAP activity test, provides further evidence of the presence of the LMWH molecule.


What are the trends in homeschooling rates after the elimination of nonmedical exemptions to childhood immunizations for school entry in California?In this cross-sectional study of students in kindergarten through grade 8 in California, elimination of nonmedical exemptions to mandatory childhood immunizations for school entry was not associated with an increase in homeschooling rates.The findings of this study suggest that elimination of nonmedical exemptions to the requirement of childhood immunizations for school entry is not associated with increases in the population of homeschooled children. This cross-sectional study evaluates changes in homeschooling rates among kindergarten through grade 8 students before and after California’s Senate Bill No. 277 eliminated nonmedical exemptions to vaccination requirements in the state. In 2015, California passed Senate Bill No. 277 (SB 277) and became the first state in more than 30 years to eliminate nonmedical exemptions to mandatory childhood immunizations for school entry. One concern that emerged was that the law created an incentive for parents to remove children from brick-and-mortar schools to bypass the immunization requirements.To assess the trends in homeschooling rates after the elimination of nonmedical exemptions to the requirement of childhood immunizations for school entry.This preintervention-postintervention cross-sectional study calculated homeschooling rates as the number of students in kindergarten through grade 8 (K-8) enrolled through each of California’s 3 homeschooling mechanisms  divided by all K-8 students enrolled in the same academic year. Data on homeschooling rates were obtained from the California Department of Education. Interrupted time series analyses were conducted using a linear regression model in which the outcome variable was the percentage of students enrolled in a homeschool program before and after SB 277. Data were collected and analyzed from October 3, 2012, to October 2, 2019.Passage of SB 277, which eliminated nonmedical exemptions to childhood immunizations for school entry.Homeschooling rates for K-8 students.Among the students included in the analysis, the homeschooling enrollment for K-8 students in California increased from 35 122 students (0.8%) during the 2012-2013 school year to 86 574 students (1.9%) during the 2019-2020 school year; however, the implementation of SB 277 was not associated with an increase in the percentage of students enrolled in homeschooling programs in California beyond the secular trend. The increase in homeschooling was greatest for the lower grade levels: kindergarten homeschooling enrollment increased from 2068 students (0.4%) in the 2012-2013 school year to 10 553 students (1.9%) in the 2019-2020 school year, whereas the grade 8 homeschool enrollment rate increased from 5146 students (1.0%) in the 2012-2013 school year to 10 485 students (2.0%) in the 2019-2020 school year. Independent study programs accounted for 20 149 students (45.3%) of homeschooling enrollment, private school affidavits accounted for 19 333 students (43.5%), and private school satellite programs accounted for 4935 students (11.1%) during the 2015-2016 school year.The findings of this study suggest that legislative action to limit nonmedical exemptions for compulsory vaccination for school entry is not associated with removal of students from classroom-based instruction in brick-and-mortar institutions. The passing of SB 277 made California the first state in more than 30 years to eliminate nonmedical exemptions,3 thereby providing an opportunity to better understand both the intended and unintended consequences of such a policy change. School entry mandates and vaccine exemptions have been an active area of state policy and legislation,8 and many states have looked to California as an example when drafting similar legislation.The so-called Disneyland measles outbreak of 2015 followed a decade-long decline in childhood immunization coverage rates in California.10 showed that the law proved effective in increasing vaccine uptake, with kindergarten immunization rates increasing from 92.8% in the 2015-2016 school year to 95.1% in the 2017-2018 school year.11 However, investigators were concerned that the law created an incentive for parents who could no longer obtain a nonmedical exemption to either substitute a medical exemption12 or to homeschool their children and thereby circumvent school entry immunization requirements (a new provision of SB 277).13 In fact, the medical exemption rate for kindergarteners almost tripled from the 2015-2016 to 2017-2018 school years14; when many of these exemptions were suspected of being fraudulent, the legislature passed Senate Bill No. 276 in 2019 to strengthen the requirements to obtain a medical exemption.15 Although removal of unvaccinated children from congregate settings such as school and daycare may reduce the risk of highly transmissible vaccine-preventable diseases, it may also have consequences on children’s social and emotional development, given differing opportunities for peer interaction.16 The purpose of this study was to evaluate the second potential mechanism to bypass vaccination by describing the changes in homeschooling rates before and after SB 277.After the implementation of SB 277, data from the California Department of Public Health17 We herein describe our approach to estimating enrollment for each homeschooling mechanism from the 2012-2013 to 2019-2020 school years. Data were collected for this study from October 3, 2012, to October 2, 2019. The study did not require institutional review board approval because we used only public databases from the California Department of Education (CDE) with deidentified information; furthermore, we had no interaction or intervention with the population being studied. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.18Parents choosing to homeschool in California may do so by one of several mechanisms, including independent study programs (ISPs), private school satellite programs (PSPs), or a private school affidavit (PSA) .17 We he20 Parents may choose an ISP because their child has competing priorities  or requires an adjusted curriculum , among other reasons.19 Students enrolled in an ISP are required to meet with a credentialed teacher regularly and take school- and state-administered tests.20 To identify ISP programs in California, we obtained a list of all past and current public schools from the CDE.21 The CDE obtains enrollment data for all schools for kindergarten through grade 8 (K-8) on Census Day, or the first Wednesday in October. Independent school programs were identified as schools that offered any combination of grades inclusive of K-8 in at least 1 school year from 2012-2013 to 2019-2020 and that were either primarily virtual  or exclusively virtual .22An ISP is a program of the public school system in California. Compared with other homeschooling mechanisms, an ISP offers the greatest support to parents, by providing either a standardized curriculum and educational material or by providing funding so that parents may purchase their own curriculum.23; this allows the family’s residence to become a standalone school with the parent acting as administrator and teacher. In this capacity, parents keep detailed records of attendance, coursework, and grades and provide instruction in a variety of core subjects.23 Private school affidavits receive no funding, curricular materials, or instructional support24 but afford a substantial degree of autonomy.23 Although the CDE publishes data on private school enrollment, these data are censored for schools with fewer than 6 students. To obtain the censored data, we filed a California Public Records Act request with the CDE for a deidentified listing of all schools with 5 or fewer enrolled students. We created 2 estimates of homeschooling enrollment via PSA: the low estimate, which included schools that had an enrollment of only 1 student, and the high estimate, which included schools that had enrollment of 5 or fewer students .Another mechanism for homeschooling in California is by filing a PSA26 Private school satellite programs may require parents to serve as teachers and/or administrators and provide administrative, curricular, and community support  in exchange for a membership fee.27 Some PSPs are denominational.26 California maintains a database of all private schools in the state28; however, the database does not distinguish between a brick-and-mortar private school and a PSP. To identify PSPs, we compiled a list from PSP directories maintained on the websites of 3 homeschooling networks . To confirm that the listed schools were PSPs, we individually reviewed each school’s website and/or contacted the school administrators via telephone or email. Of note, 15 PSPs did not exist in the CDE database. An additional 9 PSPs were brick-and-mortar schools with a PSP offshoot; these were excluded because the proportion of the total enrollment that was accounted for by the PSP is unclear. To address uncertainty in PSP classification, we created 2 estimates of PSP enrollment: the low estimate, which included only schools that were confirmed as PSPs via website, telephone call, or email communication with a school official, and the high estimate, which also included schools that could not be confirmed as PSPs.A PSP is a private school that has filed an affidavit and whose main function is to support home-based instruction.21), the private school database with 5 or fewer students , and the private school database with greater than 5 students (available on the CDE private school website28). The CDE databases also included the category ungraded elementary, which represented students who did not fit into a particular grade level; these were included in the numerator for ISP, PSA, and PSP counts and in the denominator for total enrollment.The total number of students enrolled in grades K-8 in California was calculated by combining the count data from 3 separate databases: the public school database  rates. Rates were calculated for both low and high estimates of PSA and PSP enrollment as described earlier.29 and so it was believed they would be unlikely to provide a different answer.To assess the trends in homeschooling rates after the elimination of nonmedical exemptions to childhood immunizations for school entry, we conducted interrupted time series analyses using linear regressions, in which the outcome variable was the percentage of children enrolled in a homeschool program. Periods were defined as pre–SB 277 (academic school years 2012-2013 to 2015-2016) and post–SB 277 , given the 2016 implementation date of the law. Other variables included in the model were academic year as well as a composite variable of homeschooling mechanism  and grade level (K-8), which was used as a random intercept to account for previous trends in homeschool rates. To ensure that variance was not underestimated, robust SEs were used in the analysis. The significance level was set at .05 and the hypothesis tests were 2 sided. Of note, stronger methods  were considered and would be relevant if other states’ homeschooling rates were declining; however, homeschooling rates are increasing nationally30 Inferential analyses were conducted in Stata, version 16.1 (StataCorp LLC).The outcomes for the interrupted time series analyses were as follows: low and high estimates for all children enrolled in all homeschool programs, low and high estimates for children enrolled in all homeschool programs by grade , all K-8 children enrolled in ISPs, and children enrolled in ISPs by grade. Restriction of the sample to K-8 enrollment in ISPs alone evaluated the hypothesis that parents seeking to homeschool their children merely to bypass vaccination would be more likely to choose the option with the greatest degree of curricular or financial support. Restriction to kindergarten enrollment evaluated only the hypothesis that any effects of the elimination of nonmedical exemptions would be seen at the main entry point into schooling. Students already enrolled with a nonmedical exemption before SB277 were grandfathered in subsequent grades.Total homeschooling enrollment for K-8 students in California increased from 35 122 students or 0.8% of all enrolled K-8 students in the state in the 2012-2013 school year to 86 574 students or 1.9% during the 2019-2020 school year for the low estimate (42 379 students [0.9%] to 97 316 students [2.1%] for the high estimate). The results are presented in The increase in homeschooling in California was greatest for the lower vs the upper grade levels ; for exaThe mechanisms responsible for most homeschooling during the school year when SB 277 was enacted (2015-2016) included the following from most common to least common: for ISP, 20 149 students or 45.3% (20 149 students or 38% for high estimate); for PSA, 19 333 students or 43.5% (27 989 students or 53% for high estimate); and for PSP, 4733 students or 11.1% (4935 students or 9% for high estimate).We found no significant increase in the percentage of children enrolled in homeschooling programs in California associated with the implementation of SB 277 in 2016 . However3 This may indicate that legislative action to limit exemptions to compulsory vaccination for school entry is not associated with removal from classroom-based instruction in brick-and-mortar institutions nor with restricted opportunities for peer interactions.To our knowledge, this study is the first to estimate homeschooling rates in California. We found no evidence that SB 277’s elimination of nonmedical exemptions to mandatory childhood immunizations for school entry before the 2016-2017 school year was associated with an increase in homeschooling rates for K-8 students in California. This finding suggests that SB 277 had minimal unintended consequences related to homeschooling.14 Another drastic mechanism for avoiding SB 277’s restrictions—moving out of the state—is difficult to document as a result of SB 277. Finally, parents may have avoided homeschooling as a solution to SB 277 simply because schools did not enforce SB 277 consistently. In previous work, we found evidence to suggest that the law was variably interpreted, implemented, and enforced across school districts, given vague regulatory language.31 One study found that the number of students overdue for vaccination more than quadrupled,3 suggesting that some parents were being allowed to enroll their children in brick-and-mortar schools despite being undervaccinated or unvaccinated.Our results are consistent with multiple explanations of parents’ responses to SB 277. One possibility is that vaccine-hesitant or vaccine-refusing parents who would have pursued a nonmedical exemption if available decided that the effort of homeschooling outweighed their opposition to vaccination. Alternatively, parents who would have pursued a nonmedical exemption, if available, may have instead pursued a medical exemption, an outcome consistent with the observed tripling of the medical exemption rate from the 2015-2016 school year (before SB 277) to the 2017-2018 school year (after SB 277), documented by our team.3 In 2015, state and local health departments in California additionally began an effort to ensure proper application of the state’s conditional school entrance criteria for students not up to date with vaccinations.3 If this was the case, increases in homeschooling rates may have occurred for several years rather than immediately after the implementation of SB 277, creating the possibility of a type II error in our analysis. However, we find this explanation unlikely given that before SB 277 was implemented, children who were homeschooled via either public or private homeschooling options were subject to the same school entry immunization mandates and had the same nonmedical exemptions options available to them.It is important to note that other legislative, regulatory, and surveillance responses to increasing nonmedical exemptions in California may have created pressure to homeschool before SB 277. For example, Assembly Bill 2109 (passed in 2012 and implemented in 2014) tightened the criteria for obtaining a nonmedical exemption.This study has an important limitation: Although we are confident that the ISP and PSA data are complete and accurate, PSP data are subject to reporting biases inherent in crowdsourced listings on websites. Additional possible sources of error include PSPs that were either not registered with the CDE (and had no publicly available data) or PSPs that were affiliated with brick-and-mortar private schools (and had merged data). Given the relatively small contribution of PSPs to overall homeschooling rates (approximately 10%), we do not believe the uncertainties regarding PSP enrollment had a significant impact on the analysis.The findings of our evaluation of homeschooling trends in California before and after SB 277 suggest that the elimination of nonmedical exemptions to mandatory childhood immunizations for school entry is not associated with an increase in homeschooling rates. This conclusion is predicated on the continued ability of vaccine-refusing parents to enroll children in brick-and-mortar schools, either by substituting with a medical exemption or by residing in a local school district with poor implementation of the law. States looking to eliminate nonmedical exemptions to childhood immunizations can learn from California’s example to better understand the unintended consequences of elimination of nonmedical exemptions to childhood immunizations; they may then use this insight to craft legislation capable of realizing the largest gains in vaccination rates with minimal unintended consequences.


A subgroup of those with bipolar spectrum disorders experience ongoing mood fluctuations outside of full episodes. We conducted a randomised, controlled feasibility study of a Dialectical Behavioural Therapy-informed approach for bipolar mood fluctuations (Therapy for Inter-episode mood Variability in Bipolar [ThrIVe-B]). Our study aimed to examine the feasibility and acceptability of a future definitive trial evaluating the clinical and cost effectiveness of the ThrIVe-B programme. Participants were required to meet diagnostic criteria for a bipolar spectrum disorder and report frequent mood swings outside of acute episodes. They were randomised to treatment as usual (control arm) or the ThrIVe-B intervention plus treatment as usual (intervention arm). Follow-up points were at 3, 6, 9 and 15 months after baseline, with 9 months as the primary end point. To evaluate feasibility and acceptability we examined recruitment and retention rates, completion rates for study measures, adverse events and feedback from participants on their experience of study participation and therapy.Of the target 48 participants, 43 were recruited , with a recruitment rate of 3.9 participants per month. At 9 months 74% of participants engaged in research follow-up assessment, exceeding the pre-specified criterion of 60%. There were no serious concerns about the safety of the research procedures or the intervention. On one of the four candidate primary outcome measures, the 95% CI for the between-group mean difference score excluded the null effect and included the minimal clinically important difference, favouring the intervention arm, whilst on no measure was there evidence of deterioration in the intervention arm relative to the control arm. Attendance of the intervention  was below the pre-specified continuation criterion of 60%, and qualitative feedback from participants indicated areas that may have hampered or facilitated engagement.It is broadly feasible to conduct a trial of this design within the population of people with frequent bipolar mood swings. Changes should be made to the therapy to increase uptake, such as simplifying content and considering individual rather than group delivery.Trial registration ISRCTN: ISRCTN54234300. Registered 14th July 2017, http://www.isrctn.com/ISRCTN54234300The online version contains supplementary material available at 10.1186/s40345-021-00226-4. Within bipolar spectrum disorders, ongoing instability of mood (henceforth referred to as bipolar mood instability [BPMI]) is characteristic of Cyclothymic Disorder, yet can also be experienced by individuals with Bipolar disorder I or II outside of acute episodes of mania or depression  as a bespoke approach for those with BPMI. In this approach participants are invited to reflect upon their behavioural response to extreme mood and activation states day-to-day, modifying this where necessary, congruent with evidence that unhelpful behavioural response to mood is associated with bipolar symptom exacerbation  . Our objectives were to: (i) establish recruitment pathways and trial teams in two sites; (ii) establish the number of participants initially identified, approached, consented, randomised and completed; (iii) establish the acceptability and experience of the trial process to participants, including randomisation and completion of outcome measures; (iv) further assess the acceptability of the treatment via qualitative interviews and, based on input from trial participants and clinicians, to further refine and develop the treatment manual and the procedures for training, supervising and assessing the competence of trial therapists; (v) assess the performance of selected candidate primary outcome measures for a future definitive trial with respect to level of acceptability to participants and participant-perceived relevance and value; (vi) measure data completeness at follow-up, SD of the likely primary outcome measure, and describe the variability of the comparator condition, treatment as usual (TAU), across individuals and sites; (vii) pilot a measure of resource use and to assess the feasibility and acceptability of candidate health outcome measures for economic evaluations; and (viii) identify, measure and cost the resources used and needed to deliver the intervention.We also aimed to evaluate whether the following continuation criteria were met prior to planning a future definitive trial: (i) trial participation does not lead to serious negative consequences (unexpected serious adverse reaction) for participants; (ii) any serious concerns about the acceptability and feasibility of the trial procedures can be rectified prior to a full trial; (iii) follow-up data at 9 months are available for at least 60% of participants; (iv) at least 60% of patients in the intervention group complete treatment (attend at least 50% of possible sessions).We conducted a feasibility study with a two-arm, randomised, parallel, controlled trial design. Participants were randomised in a 1:1 ratio to TAU only (control arm) or TAU plus the ThrIVe-B programme (intervention arm). Outcome measures were administered at baseline and at 3, 6, 9 and 15 months after randomisation. The primary end point was predetermined to be at 9 months. The full trial protocol is reported elsewhere  or Cyclothymic Disorder, according to the criteria in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-V)  completed a telephone screening interview prior to the eligibility assessment to increase the likelihood that those invited for a time-consuming face-to-face assessment would be eligible. At the eligibility assessment written, informed consent was sought, eligibility criteria were checked and baseline measures completed.On the basis of our previous work  was delivered in addition to TAU. The content of the ThrIVe-B programme is described in detail in the logic model in Additional file Therapy was delivered by four qualified mental health professionals with previous specialist training in cognitive behavioural therapy and related approaches (two therapists per group). A 5 day bespoke, face-to-face training programme was provided by the main author of the ThrIVe-B therapy protocol, which covered background information about Bipolar disorder, the therapeutic stance within DBT-informed approaches, therapy components, and skills in group facilitation. Weekly supervision occurred throughout therapy delivery. Before each group session participants completed the Beck Depression Inventory  between months 3 and 9. As per the intervention arm, participants could set number of alerts per day from 0 to 10. Other than the experimental intervention no treatments were withheld.Primary outcome measures. Numbers of patients identified, approached, consented, randomised and completed were used to assess feasibility, as was participant attrition from the trial and from treatment. Acceptability was assessed by questionnaires at baseline, 9 month follow-up and on ineligibility or withdrawal from the study. Items with quantitative ratings at 9 month follow-up were rated on a five point scale and asked about acceptability and satisfaction  and likelihood of recommending the trial to eligible friends and family. To assess patient valuation of outcomes, at the 9-month follow-up point participants were asked to rank the four candidate outcome measures (see below) and a measure of anxiety symptoms, the Generalised Anxiety Disorder 7-item scale (GAD-7)  was defined as all 15 group sessions, booster session and initial and final individual sessions .The ThrIVe-B app was used to gather data on momentary mood in order to assess mood stability. All participants were invited to use the app for 1 week at baseline and at 9-month follow-up, to give comparable 1 week periods of mood stability data that did not overlap with periods of therapeutic app use. Mood stability measured by the ThrIVe-B app is a potential secondary outcome in a definitive trial, therefore we tested the feasibility and acceptability of gathering these data, as indicated by completion rates and participant feedback.Secondary outcome measures. The following were candidate primary outcome measures for a future definitive trial and were completed at baseline and all follow-up points: the Patient Health Questionnaire 9-item scale (PHQ-9)   and local departmental research ethics committees. Assessments were face to face, by telephone, by post or online, dependent upon participant preference. In-person contact was required at baseline and at months 9 and 15 in order to complete the interview-based measures. At 9 months the questionnaire on experiences of the study was completed and a subset of participants completed the semi-structured interview.Participants were randomised on a 1:1 ratio, with minimisation by trial site and medication status (any medication for Bipolar disorder prescribed: yes/no). To ensure concealment, eligible participants were randomised via a validated password website hosted by Exeter Clinical Trials Unit. The first ten participants were allocated using simple randomisation; the remainder were allocated using the minimisation procedure, maintaining a stochastic element to the algorithm to allow concealment. Participants were randomised prior to the start of each therapy group, and informed by an unblinded researcher.Researchers conducting follow-up assessments were blind to allocation and participants were reminded of this at each follow-up contact. All unblindings were recorded and where possible, researchers who remained blinded to that participant’s status conducted future follow-up assessments. All statistical analyses were performed by a statistician using groups indicated by an anonymised code.The primary analyses were performed after 9-month follow-up. Further analyses were performed after the final 15-month follow-up. No interim analyses were conducted. All analyses were on an intention-to-treat basis; included outcomes were reported according to randomised allocation, regardless of the treatment actually received. No attempts to address missing data, such as multiple imputation, were made. The economic analysis took the NHS and social care perspective.At 9-month follow-up only, an inferential analysis was performed for continuous outcomes only, reporting the relevant 95% CI for the between-group mean difference (intervention minus TAU), but no p value. Inferential analyses included the randomisation covariates of site and baseline medication status.Qualitative interviews and written responses on the feedback questionnaires were analysed using a framework approach  and 1.3 per month in Cumbria (nine participants over 7 months).Of those randomised 33 (77%) were informed about the study by their GP, 5 (12%) through contact with local mental health services, 4 (9%) through media and social media campaigns and 1 (2%) unknown.Of those randomised to the intervention arm, 8 were allocated to the first Devon group, 9 to the second, and 5 to the group in Cumbria.The demographic and clinical characteristics of the sample are given in Table At study entry participants reported mean depression and anxiety levels in the moderate range, and low levels of current mania symptoms Table . Mean quWe distinguish between research follow-up attrition and therapy attrition, as it was possible for participants to complete therapy and then opt not to complete research follow-up assessments, and vice-versa.At 9-month follow-up 74% of participants  provided at least partial follow-up data; 11/43  were lost to follow up. Five participants withdrew in the period between randomisation and final follow-up ; none were withdrawn by the research team. Figure Of the 22 participants allocated to receive the intervention, 17 attended at least three sessions .Data completion for the smartphone app was low: at baseline, 16 participants provided data for at least 7 days . Between months 3 and 9, 10 participants  used the app at least once; at 9 months 3 TAU participants used the app at least once (no participants in the intervention group used the app at 9 months).Quantitative acceptability data were available from 17 participants  at 9 months. Of these, 16 found the research process at least moderately acceptable, with 14/17 at least moderately satisfied. Of the eight participants in the therapy arm who responded, seven rated therapy as “very” or “extremely” acceptable, and were at least “moderately” satisfied with therapy; one found therapy “slightly” acceptable/satisfactory.Qualitative acceptability data were synthesised from four sources: acceptability surveys at 9 month follow-up (n = 17), qualitative interviews at 9 month follow-up , surveys of participant experience after the baseline assessment (n = 19) and surveys of those who declined to take part or were not eligible (n = 9).Making a contribution to society, learning more, and having the chance to access a new therapy emerged as key motives for participation. With regard to the experience of being involved, some participants found the volume of measures challenging; participants also highlighted how important it was that the measures felt relevant to them. Having a positive relationship with the research team was seen as important and was associated with a positive experience of interviews. Participants in the control group expressed some disappointment at not being offered therapy, whilst appreciating of the need for a control group in trials. Barriers to taking part in the study included cost of travel and childcare, and the level of commitment required. The smartphone app was used as part of both the research and therapy aspect of the study. Interruption of activities due to the alerts and direction of attention to negative feelings whilst making mood ratings were seen as downsides and several suggestions were made for improvement. Some participants reported technical issues with the app which may have contributed to low rates of data return.Analysis of qualitative feedback on the therapy itself  was organised under three main themes: interpersonal aspects of therapy (subdivided into group process and therapist style); therapy content and delivery modes, and where/how the therapy helped. A summary of themes and sub-themes with exemplar quotes is given in Additional file Participants valued the closed nature of the group (that the same group of people met each session) and the mutual support it generated, although some struggled with particular group dynamics. Both the group and the therapist were instrumental in enabling participants to feel held and understood, which was key to a positive therapeutic experience. Therapist personality and style was seen as very important; authenticity was particularly valued and helped participants to feel that the material was coming from a person rather than from a textbook: “Yeah, [therapist] would give examples from her life which you felt were very genuine … it felt very genuine, and I could get on with that” . The amount of information delivered was seen to be high; some found this problematic and were worried about overload, but reported that it was helped by the use of multiple modes of delivery and tools for repeating and retaining information, such as handouts and booster sessions. Participants noted the importance of feeling that the material was relevant and tailored to them, and that it was offered at the right time of life. Participants noted several areas of benefit from the therapy, including greater understanding of the self, including re-evaluation of the self in a more positive or accepting way; greater understanding between the self and others; changes in behavioural responses to moods (even if the mood did not change), and broadening their range of activities.Local clinicians (n = 8) saw DBT-informed therapy as appropriate for the client group, similarly the length of treatment and point of delivery in the care pathway. Clinicians queried whether a group based approach could be delivered and accessed successfully in the health service, as patients might initially be put off by the prospect of a group-based approach, and whether extreme mood states might interfere with group work. Feedback from therapists (n = 4) revealed overall high satisfaction with the techniques and materials, and with the potential to help this patient group, but there was also a sense of having a great deal of information to communicate to patients.With regard to rankings of personal importance of the candidate primary outcome measures and GAD-7, the mean rankings were highest for the BRQ and ALS and lowest for the PHQ9. The GAD7 was ranked number 1 by the greatest proportion of participants 8/30: 27%), followed by the BRQ and ALS (both 7: 23%). The median smallest meaningful change values for each of the five measures are given in Table 7%, folloOver the study period there were 48 adverse events . These included 32 instances related to risk of suicide or self-harm , six reports of distress during or after study procedures, eight physical health events requiring treatment and two psychiatric admissions. Five were serious adverse events, none of which were unexpected. It was judged that trial procedures (completion of questionnaires) may possibly have contributed to one of these events ; the other serious adverse events were judged unrelated to the trial.Mean scores on candidate primary and secondary outcome measures at follow-up are given in Table At 9-month follow-up, the between group mean difference for the Kentucky Inventory of Mindfulness Skills was 13.1 (95% CI 4.9 to 21.3), indicating higher self-reported mindfulness skills in the intervention group, with the 95% CI excluding the null treatment effect. The remaining nine secondary outcomes all yielded a 95% CI that included the null treatment effect.Completion rates were similar between EQ-5D-3L and SF-36 measures, but lower for the resource use measure. Table This randomised, controlled feasibility trial is the first to focus specifically upon people with ongoing mood instability within Bipolar Spectrum Disorders, and represents an important step in the development and testing of a novel psychological intervention for this population. In accordance with the study aims, we established recruitment pathways and trial teams in two sites and gathered information on patient recruitment and attrition rates. The overall rate of randomisation of 3.9 participants per month supports the feasibility of a larger trial, recruiting across a longer period and across multiple sites. This could be maximised by learning from experiences in the current trial, where one site recruited more successfully than the other. In particular, rural sites face greater challenges than do more urban ones when aiming to fill a therapy group with patients who have a relatively low-prevalence condition. In this study we initially sought to recruit patients from primary care only; whilst this was the case for the majority of participants, a small number were in secondary care. Setting out to work with a larger number of GP practices per site, selecting sites with a track record of primary care mental health recruitment and including a generally publicised self-referral option should be a priority for future studies in this population.All four candidate primary outcome measures were seen as similarly valuable by participants and had similar rates of completion: on this basis any of these could be suitable as a primary outcome measure in future trials. Based on participant feedback about key areas of perceived change, it may be appropriate to select two primary outcome measures, one reflecting mood variability and another functioning or recovery. Rates of completion were similar across health economics measures.Continuation criterion one (no unexpected serious adverse reactions to the therapy or research procedures) was met. Completion rate of measures met continuation criterion three (follow up data at 9 months are available from at least 60% of participants), with no evidence of greater attrition in the control arm. Nevertheless some participants in both arms reported finding the self-report measures onerous; this could potentially be improved by reducing the number of measures, using a more sophisticated online data collection platform, and making more use of participant payment. Whilst participants reported many positive aspects of the therapy, overall therapy attendance rates were numerically lower than the previous open trial  and feedback surveys (n = 8).Additional file 5. Table displaying intervention costing.Additional file 6. Table displaying unit cost of services.Additional file 7. Table displaying cost of services by participant (average number of contacts).