ZP4 ablation impairs fertility in female rabbits. Ovulation, fertilization and in vitro development to blastocyst were not affected by ZP4 ablation. However, in vivo development is severely impaired in embryos covered by a ZP4-devoided zona, suggesting a defective ZP protective capacity in the absence of ZP4. ZP4-null ZP was significantly thinner, more permeable, and exhibited a more disorganized and fenestrated structure. The evolutionary conservation of ZP4 in other mammals, including humans, suggests that the structural properties conferred by this protein are required to ensure proper embryo sheltering during in vivo preimplantation development.Zona pellucida (ZP), the extracellular matrix sheltering mammalian oocytes and embryos, is composed by 3 to 4 proteins. The roles of the three proteins present in mice have been elucidated by KO models, but the function of the fourth component (ZP4), present in all other eutherian mammals studied so far, has remained elusive. Herein, we report that  The egg cells of mammals, called oocytes, are encased in a protective layer called the zona pellucida. This layer is made from proteins called ZP1 to 4. Most studies of the zona pellucida use mice, which do not have ZP4. This means that the research community have limited knowledge of what ZP4 does in humans and other mammals.Scientists can now use a technique called CRISPR to selectively modify the genetics of living things to help us to understand what specific genes and proteins do. The ZP4 protein can be eliminated from rabbit oocytes using CRISPR to help understand its role in egg cell fertilization and development.Lamas-Toranzo et al. examined the effect of losing ZP4 from rabbit oocytes. Without ZP4 the zona pellucida becomes thinner, irregular and more flexible. However, the loss of ZP4 did not affect ovulation (i.e. the release of egg cells from an ovary), fertilization, or the early stages of development of embryos when studied in the laboratory. However, rabbits without ZP4 were much less fertile. Indeed, only one out of 10 female rabbits without ZP4 was able to deliver pups because in most cases the development of embryos in the womb failed.These findings show that ZP4 has a structural role in the zona pellucida. Without ZP4 fertility is reduced. This work lays the ground for further investigation of the role of ZP4. It could also offer new insights into the causes of infertility. Mammalian oocytes and early preimplantation embryos are surrounded by a glycoprotein shell termed zona pellucida (ZP) that serves important functions during folliculogenesis, species-specific fertilization and early development. In eutherian mammals, this glycoprotein shell is solely composed of 3 to 4 proteins depending on the species. The differences in ZP composition between eutherian mammals is the consequence of duplication and/or pseudogenization events during evolution. In all eutherian mammals studied so far ZP always contain ZP2 and ZP3 proteins, but ZP1 or ZP4 may be absent, as both are paralogous genes formed by duplication of a common ancestry gene (reviewed by Zp2 knock-out (KO) females after mating  present in mouse ZP have been elucidated by gene ablation experiments. ZP1 serves a structural function, being dispensable for sperm binding or fertilization . ZP1 nulr mating . Finallyr mating . Given tproteins  or an heproteins  have uncMus musculus), the only species where KO models were readily available, has precluded the study of the role of this protein by of loss-of-function experiments. Gene ablation experiments are essential to unequivocally know the function of a gene, but were largely restricted to mice, given the technical difficulties to perform site-specific genome modification in other mammalian species , resulting in the delivery of 12 pups . Genotyping was performed on ear biopsies and blood samples from each pup. Genome edition rates were assessed by sequencing a PCR product containing the target site. This initial screening revealed that all 12 pups were edited at the target site, but it did not allow to identify each mutated allele harbored by each individual. For this aim, clonal sequencing was performed, revealing that all pups but one male were mosaic, that is contained more than two alleles. This phenomenon appears when DNA replication precedes genome edition and has been commonly observed following zygote microinjection in different mammalian species , which were also detected in the solubilized extract of ZP4 KO ZP  by non-homologous end joining (NHEJ) . As someP4 KO ZP . This anZP4 ablation on female fertility was initially tested by crossing WT, Hz or KO females with fertile WT males. WT and Hz females delivered normal litter sizes, whereas only one out of 10 KO females, delivered a single litter of reduced size  are able to reach the blastocyst stage in vitro . Normal pronuclear formation was noted for embryos derived from ZP4-null oocytes, showing no effect of ZP4 ablation on the in vivo fertilization process  by Scanning Electron Microscopy (SEM). SEM images evidenced notable differences in textural properties between WT and ZP4 lacking ZP: while the former exhibited a smooth and compact texture, the latter displayed an uneven, rougher and porous aspect  compared to WT ZP . Furthers aspect . This le9 WT ZPs .Despite its essential functions during folliculogenesis, fertilization and preimplantation embryo development, ZP is composed of just 3 to 4 proteins, depending on the species. Gene ablation experiments have deciphered the functions of the three proteins present in mouse ZP, but the role of the fourth component (ZP4) has remained controversial. Previous studies have suggested that ZP4 may play a role during fertilization. Rabbit ZP4 was found to bind to rabbit sperm acrosome , althougZp1 KO were less severe than those caused by ZP4 ablation, as litter size was halved in Zp1 KO female mice compared to WT mice, whereas ZP4 ablation abolished almost completely embryo development. A plausible explanation for this difference is that mouse embryos may demand a lesser degree of ZP protection during preimplantation development than other domestic mammals, including rabbits. The early blastocyst hatching occurring in mouse  and a reduced hydrostatic pressure in female reproductive tract motivated by its small body size may be partly responsible for this difference.ZP plays an essential protective role during preimplantation embryo development, as despite the exception of a single case report where zona-free embryos were developed in vitro up to blastocyst before transfer , zona deZP4 coding region leading to premature stop codons (PTCs), which are the most evident loss-of-function mutations  were housed individually maintaining visual and olfactory contact with others to allow social interactions. Temperature was controlled to 20–24°C and light cycle was 14:10.All animal protocols were approved by INIA Animal Welfare Committee and Madrid Region authorities (authorization PROEX040/17). Rabbits . ZP4 KO were generated by CRISPR/Cas system. A single guide RNA (sgRNA) was designed at the beginning of the coding region of rabbit ZP4 gene using MIT CRISPR design tool  collected 14 hr after mating, fixed in 10% formaldehyde for 24 hr, processed and paraffin embedded. Rehydrated sections of 5 µm were incubated in 0.3% hydrogen peroxide in TBS to block endogenous peroxidase activity and then in primary antibody solution  dilution 1:2000 in TBS-1% BSA solution) for 1 hr at room temperature in a moist chamber. After a brief wash in TBS, sections were incubated in secondary antibody solution  for 30 min at 37°C and immunoreaction was revealed with 3–3´diaminobencidine. Finally, sections were counterstained with Harry´s hematoxylin (Thermo Scientific), dehydrated, cleared and mounted. Positive immunoreaction was identified as a dark-brown precipitated. The images were collected with a Leica DM 6000 microscope and Software Leica Application Suite.Proteomics was performed on 50 grown oocytes/group recovered by slicing ovaries in PBS. ZP was solubilized by incubation at 75°C for 45 min  and anal2 and 5% O2 water saturated atmosphere. Embryonic cleavage and development to blastocysts were assessed on Days 1 and 5 of culture, respectively.All experiments were performed following natural mating with WT males of proven fertility. Ovulation in rabbits is induced by mating, allowing precise determination of embryo developmental timing. Zygotes were collected post-mortem 14 hr after mating by oviductal flushing with Dulbecco´s PBS media supplemented with 1% BSA (DPBS) in five independent replicates. Ovulation rates were assessed by counting corpora lutea. Embryo culture took place on 25 µl drops of TCM-199 media (Sigma) supplemented with 5% FCS at 38.5°C in a 5% COFollicular counts were performed in histological sections of ovaries collected 14 hr after mating, fixed and stained with Haematoxylin-Eosin as detailed above . Pronuclear formation rates were analysed in the zygotes obtained from three females/group fixed 20 hr following mating as described previously . PronuclTo analyse in vivo development to Day 4 blastocyst, three females/group were sacrificed 4 days after mating and oviducts (where no structures were recovered) and uteri were independently flushed with DPBS. The number of structures recovered were counted and blastocysts were observed and measured under light estereomicroscopy (Zeiss V20 coupled with Hammamatsu Orca Flash 4.0 camera). A similar approach was followed to analyse in vivo development to Day 6 blastocysts, but in this case four females/group were used and only uteri were flushed. Corpora lutea and the number of structures were counted to determine recovery rates. The structures recovered were observed and measured under light estereomicroscopy (Zeiss Stemi 305 coupled with Izasa CMOS 1080P camera).Zona pellucida thickness was analysed in 30 fresh embryos/group 40–43 hr post-mating by inverted light microscopy (Nikon Eclipse) using the measurement tool of the software NIS (Nikon).Scanning Electron Microscopy (SEM) was performed on five zygotes/group fixed in 2% glutaraldehyde for 2 hr at 4°C, washed in PBS and postfixed in 1% osmium tetroxide for 1 hr. After washing, the zygotes were dehydrated in increasing concentration of acetone and air dried. Finally, samples were sputter coated with gold and studied in Jeol-6100 scanning electron microscope.ZP permeability test was performed by incubating fresh zygotes (3 replicates of 3 embryos/group) in 0.005% (1:1000 dilution) solutions of carboxylate-modified 505/515 Fluospheres (Invitrogen) of different sizes  for 30 min, using the culture media and atmosphere conditions detailed above. Immediately following incubation and washing, zygotes were observed under an inverted fluorescent microscope to assess the penetration of fluorescent particles. This test yields a binary result: green fluorescence can be detected inside ZP (ZP is permeable to that size of particle) or fluorescence cannot be detected inside ZP (particles do pass through ZP).http://gnomad.broadinstitute.org/gene/ENSG00000116996), and a cvs file was downloaded and manipulated with R v3.5.1. Loss of function ZP4 polymorphisms passing QC filters were queried online at , where Chi-square test were used. All statistical tests were performed using the software package SigmaStat. Differences were considered statistically significant at p<0.05. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Michael Eisen as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Véronique Duranthon (Reviewer #2).Thank you for submitting your article "ZP4 confers structural properties to the zona pellucida essential for embryo development" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The zona pellucida is an essential oocyte structure for fertilization and subsequent embryonic development. Yet, the composition of this structure is species-specific, hence the difficulty to make assumptions from one animal model to the other, and to humans ultimately. ZP4 is a glycoproteic component of the zona pellucida that exists in most eutherian mammals, except the mouse. The authors report here the first ablation (CRISPR-Cas9-driven) of the Zp4 gene in rabbits and its effect on development. The zona pellucida structure appears affected in absence of ZP4, presenting as thinner and more permeable compared to wildtype. Interestingly, compared to other Zp genes, ZP4 is not absolutely required for oocyte fertilization, but has post-fertilization effects in vivo and is essential for female fertility, while being dispensable for in vitro preimplantation development.The reviewers agreed on the fact that this is an important work, with novel conclusions and interesting implication for humans. However, there was also consensus towards the conclusion that more work is needed to properly delineate the role of ZP4 for the embryo and that some of the conclusions are not fully supported by the data..eLife allows for revision. Personally, I think point 2 may be the more interesting – if equally feasible – to experimentally address, as it may point to the reason why embryonic development fails in absence of ZP4. But wherever experiments could not be carried out, conclusions should be toned down, according to the recommendations below.The reviewers are well aware that obtaining edited rabbits is challenging, so between essential points 1 and 2, a choice may have to be made, regarding the limited period of 2 months that Essential revisions:1) The lack of effect of the ZP4 mutation on the process of fertilization is not directly proven, only implied. The authors should be more cautious in their conclusion. Additional experimental work would be needed to exclude any effect on fertilization-related events per se, like sperm binding with the egg, acrosome reaction, sperm penetration etc. There are precedents in the literature of fertilization-related proteins whose role can be masked in vivo due to compensatory mechanisms. If more edited females can be obtained, in vitro fertilization needs to be performed and sperm binding, penetration, fertilization, and polyspermy quantified under clearly-defined conditions. If it is not feasible, then the conclusion that the fertilization step is not affected should be attenuated, by limiting the conclusion to a non-oriented statement of the results = cleavage is not affected and therefore ZP4 is not absolutely required for fertilization in vivo.2) The discrepancy between the in vivo and in vitro developmental outcome should be better addressed. Because blastocysts were lost at Day 6 of in vivo development, the authors concluded that the main function of ZP4 is to protect the preimplantation embryo. However, this is clearly not proven. If the authors want to prove that the phenotype at Day 6 is due to preimplantation loss, further investigations are required to show when the loss occurs and establish the phenotype of that loss. Moreover, the authors fail to mention the dynamics of the rabbit zona pellucida, which disappears in vivo between Day 3 and Day 4 to be replaced by a neozona . Such a modification does not occur in the in vitro culture conditions used in the paper . Additionally, during embryo transit in the oviduct, a mucin coat covers the developing embryo and is replaced by a gloiolemma at Day 5-6. The lack of development observed in vivo could equally result from effects of ZP4 on the formation of the mucinous coat, or, on the replacement of the zona by the neozona, none of these changes of the extracellular matrix being recapitulated in vitro. Whether ZP4 mutation may have rabbit specific effects due to these features should be discussed.3) About the part on human genetics of the ZP4 gene: the authors found 48 polymorphisms in ZP4 coding region leading to premature stop codons in human. They claim that ZP4 deficiencies could thus lead to infertility in human. As such, this conclusion seems overstated: only an enrichment for such mutations in infertile patients may have led to such a conclusion. This paragraph  should be moved in the Discussion section.4) Statistical and quantitative analyses should be strengthened, information is often missing or incomplete.- mention on each graph the numbers of embryos observed in each category and not only the percentages obtained .- Quantitative assessments on ovaries are needed to conclude that there was no effect on the development of oocytes.- Same for the analysis of embryonic development. For example, was there any effect on inner cell mass development?- Figure 2: Please mention in the legend the number of females involved. This is only mentioned in the Materials and methods section, which makes reading uneasy.- Only a single example of the nanosphere experiment is shown, and no quantification is reported. A quantitative, rigorous analysis is needed, with statistical tests.- The statistical analysis section of Materials and methods says analyses were done by ANOVA. However, ANOVA is not appropriate for only two groups , where a t-test should be used to compare means. ANOVA alone also cannot show comparisons among the treatment groups. A post-hoc test  is needed. This should be specified. Also, was it confirmed that the data were normally distributed and ANOVA was appropriate? This seems unlikely for proportion data like in Figure 2. Essential revisions:1) The lack of effect of the ZP4 mutation on the process of fertilization is not directly proven, only implied. The authors should be more cautious in their conclusion. Additional experimental work would be needed to exclude any effect on fertilization-related events per se, like sperm binding with the egg, acrosome reaction, sperm penetration etc. There are precedents in the literature of fertilization-related proteins whose role can be masked in vivo due to compensatory mechanisms. If more edited females can be obtained, in vitro fertilization needs to be performed and sperm binding, penetration, fertilization, and polyspermy quantified under clearly-defined conditions. If it is not feasible, then the conclusion that the fertilization step is not affected should be attenuated, by limiting the conclusion to a non-oriented statement of the results = cleavage is not affected and therefore ZP4 is not absolutely required for fertilization in vivo.We apologize for not being able to provide IVF data. Rabbit sperm capacitation is difficult to achieve in vitro and, hence, rabbit IVF is particularly challenging. We routinely perform IVF in different mammalian species , but to perform rabbit IVF we would need to develop a specific protocol which would not be ready to use in a reasonable time. As a result, we have toned down our conclusions by stating that these results prove that ZP4 is dispensable for fertilization in vivo (subsection “ZP4 ablation leads to severely impaired fertility” and Discussion), as similar pronuclear formation  and embryonic cleavage (oocyte activation following sperm penetration) rates were obtained following in vivofertilization of WT and KO females .2) The discrepancy between the in vivo and in vitro developmental outcome should be better addressed. Because blastocysts were lost at Day 6 of in vivo development, the authors concluded that the main function of ZP4 is to protect the preimplantation embryo. However, this is clearly not proven. If the authors want to prove that the phenotype at Day 6 is due to preimplantation loss, further investigations are required to show when the loss occurs and establish the phenotype of that loss. Moreoever, the authors fail to mention the dynamics of the rabbit zona pellucida, which disappears in vivo between Day 3 and Day 4 to be replaced by a neozona . Such a modification does not occur in the in vitro culture conditions used in the paper . Additionally, during embryo transit in the oviduct, a mucin coat covers the developing embryo and is replaced by a gloiolemma at Day 5-6. The lack of development observed in vivo could equally result from effects of ZP4 on the formation of the mucinous coat, or, on the replacement of the zona by the neozona, none of these changes of the extracellular matrix being recapitulated in vitro. Whether ZP4 mutation may have rabbit specific effects due to these features should be discussed.We agree that the failure on in vivodevelopment required further characterization. To exclude any possible effect attributable to the formation of neozona or to mucin coat substitution, we have recovered embryos fertilized and developed in vivoon Day 4. At that stage the original zona pellucida has disappeared and the embryo is only surrounded by mucin coat, as neozona will be formed later (Day 5). As it can be seen in the new Figure 3, embryos derived from WT or KO females were covered by mucin and ZP was dissolved in the developing embryos surrounded by KO ZP. ZP was present in degenerated embryos as ZP dissolution requires from embryo development . However despite mucin coating was normal and ZP without ZP4 could be dissolved, a clear reduction in development to blastocyst and in blastocysts size can be already noticed by that day (i.e. before mucin-gloiolemma exchange and neozona formation). Degenerated embryos and even some blastocyst trying to expand show a crunched appearance compatible with a mechanical damage occurring in the absence of ZP4. We have included a new figure with these results  and have modified the Results section (subsection “ZP4 ablation leads to severely impaired fertility”).3) About the part on human genetics of the ZP4 gene: the authors found 48 polymorphisms in ZP4 coding region leading to premature stop codons in human. They claim that ZP4 deficiencies could thus lead to infertility in human. As such, this conclusion seems overstated: only an enrichment for such mutations in infertile patients may have led to such a conclusion. This paragraph  should be moved in the Discussion section.ZP4 with women infertility.We have moved the paragraph to the Discussion section (third paragraph) and stated more clearly that we have not proved the association between the PTC mutations identified by our analysis in 4) Statistical and quantitative analyses should be strengthened, information is often missing or incomplete.- Mention on each graph the numbers of embryos observed in each category and not only the percentages obtained .eLife´s transparent reporting form, but it will be definitely more accessible to the readers in the columns. We have now included n in all columns of the figures and detailed its meaning on figure legends.We apologize for not providing earlier that information in the figures, it was included in the - Quantitative assessments on ovaries are needed to conclude that there was no effect on the development of oocytes.We have performed follicular counts on 4 females per group analysing 4 sections per ovary (16 sections/group). Similar numbers of antral and preantral follicles were obtained .- Same for the analysis of embryonic development. For example, was there any effect on inner cell mass development?in vitro produced blastocysts were morphologically similar in both groups, and all blastocysts develop ICM. However, in vivodevelopment was impaired: more than half of the embryos were not able to develop to blastocyst on Day 4. Those reaching the blastocyst stage in the KO group do develop ICM, but those ICM were smaller, as the rest of the embryo. Later on development (Day 6) the developmental impairment of the KO group was more evident, but again, those embryos developing to slightly expanded blastocyst do show embryonic disc, although it was less developed than that found in the WT group (as the rest of the embryo). In other words, it seems that the damage did not affect preferentially the ICM: both TE and ICM development were reduced when ZP4 was absent from ZP.- Figure 2: Please mention in the legend the number of females involved. This is only mentioned in the Materials and methods section, which makes reading uneasy.We have added this information to the figure.- Only a single example of the nanosphere experiment is shown, and no quantification is reported. A quantitative, rigorous analysis is needed, with statistical tests.We should have provided more details for this test. This assay yields a binary result: green fluorescence detected inside ZP (permeable) or no fluorescence detected inside ZP (not permeable). All WT embryos (9) analysed following incubation with 40 or 20 nm nanospheres lacked fluorescence inside the ZP and all KO embryos (9) showed fluorescence. We have modified the subsections “ZP4-devoided zona show structural abnormalities” and “Structural analyses” to explain better this test.- The statistical analysis section of Materials and methods says analyses were done by ANOVA. However, ANOVA is not appropriate for only two groups , where a t-test should be used to compare means. ANOVA alone also cannot show comparisons among the treatment groups. A post-hoc test  is needed. This should be specified. Also, was it confirmed that the data were normally distributed and ANOVA was appropriate? This seems unlikely for proportion data like in Figure 2.We have now performed t-test when 2 groups are compared. Regarding to ANOVA, the statistical software we use automatically perform ANOVA on ranks followed by Tukey´s when normalcy test fail, as it was the case for pregnancy data . We have modified figure legend and Materials and methods accordingly.


The era of biologic agents for the treatment of Crohn’s disease has brought about significant benefits for patients, and since the introduction of infliximab at the turn of the century, the entire field has moved on rapidly. Clinicians now have multiple agents at their disposal and a choice between several different anti-inflammatory mechanisms of action. This has allowed unprecedented improvements not only in symptoms and quality of life for patients previously refractory to conventional treatments but also for demonstrated healing of the intestinal mucosa and resolution of perianal fistulation. However, despite the undisputed efficacy of these agents, there remains a significant proportion of patients who fail to gain a meaningful benefit. Through years of studying infliximab and its counterpart anti-tumour necrosis factor (anti-TNF) agent, adalimumab, we now understand that strategies such as combining use with a conventional immunomodulator or measuring serum levels can help to optimise outcomes and reduce the proportion of patients for whom treatment fails. Work is ongoing to understand whether these principles apply to newer biologics such as vedolizumab and ustekinumab. In addition, novel approaches are being investigated in an attempt to maximise the benefit that these agents could offer. In this article, we summarise these new understandings and consider ways in which they could be integrated into clinical practice for the benefit of patients. In the two decades since the advent of infliximab (IFX) for the treatment of Crohn’s disease (CD), biologic therapies have delivered substantial improvements in outcomes for patients with inflammatory bowel disease (IBD). Not only can they improve symptoms  but they also resolve inflammation, judged objectively using endoscopic, radiological or biochemical measures. In addition, biologic therapies have significantly changed the way in which perianal CD is managed and are currently our most effective pharmacological class of drugs for this particularly debilitating manifestation. The US Food and Drug Administration’s approval of IFX for CD in 1998 was followed 9 years later by the approval of adalimumab (ADA) and certolizumab . For several years, these agents with their common anti-tumour necrosis factor (anti-TNF) mechanism formed the entirety of the licensed biologic options available for CD. However, in recent years, the range of agents and mechanisms of action has expanded. First, in 2014, the selective leukocyte adhesion molecule inhibitor, vedolizumab (VDZ), was approved for use in ulcerative colitis (UC) as well as CD. This was followed in 2016 by the approval (for CD only) of ustekinumab (UST), a monoclonal antibody that targets the p40 subunit of interleukin-12 and interleukin-23. In addition to these new agents, the number of licensed treatments has been further expanded by the growing range of IFX and ADA biosimilar agents now available.Whilst the large-scale registration trials by which the anti-TNF agents were granted their approvals clearly demonstrated their efficacy, it has taken many years and dedicated studies for us to gain a deeper understanding of how they should be used for maximum benefit. Examples include the benefit of their use in combination with a conventional immunosuppressant and introducing them earlier in the disease course as well as the use of therapeutic drug monitoring (TDM) for dose optimisation. Advances in our appreciation of these concepts have allowed practice to evolve to provide improved patient outcomes. Attention is now turning to investigating whether similar concepts will apply to newer biologic agents. In this review, we aim to draw together emerging data which provides new understanding of the optimal use of established agents and to consider how the impact of more novel biologics could potentially be optimised.Dosing regimen modification. Standard dosing for IFX induction is a 5-mg/kg intravenous infusion at weeks 0, 2 and 6 and then every 8 weeks thereafter. However, there are a number of ways that this dosing regimen can be modified to optimise an individual’s therapy. In patients with low IFX trough levels (and absent or low-titre anti-drug antibodies) during maintenance therapy, intensifying IFX dosing can improve clinical outcomes and increase the number of patients achieving clinical response1. This may be achieved either by increasing each infusion to 10 mg/kg or by shortening the dosing interval to either 4 or 6 weeks. Ideally, decisions regarding dose adjustment should be made with the benefit of TDM, inclusive of anti-drug antibody measurement. This is in view of the commonly encountered clinical scenarios for which dose intensification has less rationale. An example is active disease due to the development of high-titre antibodies with sub-therapeutic trough levels (immune-mediated pharmacokinetic failure) or adequate trough levels without antibodies (mechanistic/pharmacodynamic failure), which may warrant a change in therapy rather than dose intensification2.3. Again, this may be done by lengthening the inter-dose interval or reducing the concentration of the infusion (if previously receiving 10 mg/kg). The Trough Level Adapted Infliximab Treatment (TAXIT) study showed that dose reduction (targeting a trough level of 3 to 7 µg/mL) results in a similar proportion of patients in remission but with a 28% reduction in the associated drug costs1.At the opposite end of the spectrum, patients in deep remission on IFX maintenance with supra-therapeutic trough levels could de-escalate their dosing, as relapse rates have been demonstrated to be low4. Therefore, in the setting of a sustained remission, many clinicians recommend temporary discontinuation from this point until after delivery. However, where there is evidence of ongoing disease activity or in the setting of previously refractory or complex disease, the risk-benefit often favours continuing treatment throughout. Although the mechanisms which allow IFX and ADA to cross the placenta are efficient enough to result in up to fourfold higher levels in infant and cord blood compared with maternal levels6, this does not appear to adversely affect the developing infant in the short term7. Therefore, any putative benefit of reducing infant exposure should be balanced with the risks of an intra- or post-partum disease flare, of which there exists conflicting evidence. de Limaet al. reported no difference in relapse rates in women with sustained remission who stopped anti-TNF treatment before week 25 compared with those who continued therapy beyond week 30 8. By contrast, a study by Groupe d’Etude Thérapeutique des Affections Inflammatoires Digestives (GETAID) observed a relatively high intra-partum (14%) or early post-partum (32%) relapse rate in mothers who discontinued therapy before week 309. Where treatment is continued throughout pregnancy, recent evidence has shown that maternal IFX levels rise whilst ADA levels remain stable . It has therefore been suggested that TDM performed during the second trimester may help guide dosing during the third10.Pregnancy presents another situation in which the dosing regimen of biologic agents may be modified. In this scenario, the aim of modification is usually to maintain the beneficial effect of treatment while attempting to limit exposure to the infant. It is known that both IFX and ADA can cross the placenta from the latter part of the second trimester11. This is on the basis that rates of transportation of ADA across the placenta are lower12 and that it is cleared more quickly from the circulation of infants than IFX7. Nonetheless, it is important for clinicians and pregnant patients to be aware that European consensus recommends that live vaccines be avoided until after 6 months in infants exposed to anti-TNFin utero13. There are clearly far less data guiding how best to manage more novel biologics, such as VDZ and UST, during pregnancy. Although both agents are expected to cross the placenta in a manner similar to that of IFX and ADA, there has so far been no evidence of harm15. However, further studies are required to better understand their safety and pharmacokinetics during this period.On balance, for patients who have disease requiring biologic therapy and who are contemplating pregnancy, ADA appears preferable to IFXMeasuring and monitoring. As is now widely appreciated, trough levels of IFX have been shown to correlate with clinical response, mucosal healing, and clinical remission. The TAXIT study established that targeting IFX trough levels to 3 to 7 µg/mL resulted in more efficient use of the drug1. An analysis of trough-level thresholds showed a progressive reduction in the proportion of patients not achieving remission at lower levels. The rates decreased from 25% at a level of at least 1 µg/mL to 15% for those with a level of at least 3 µg/mL, 8% for levels of at least 5 µg/mL and 4% for at least 7 µg/mL. Taking into account these data in addition to several other studies, the American Gastroenterological Association (AGA) made a “conditional” recommendation that at least 5 µg/mL should be the target for IFX trough concentrations16. However, it should be noted that TDM comprises just one aspect of monitoring in an attempt to maintain tight disease control and that dosing regimen modifications should be tailored to the individual. Moreover, although many clinicians advocate proactive TDM17 , there currently exist little direct data to recommend this strategy. Indeed, the first published randomised trial  of proactive versus reactive  dose adjustment strategies found no difference between the two18.20. Yaruret al. performed a cross-sectional study showing that median IFX levels amongst patients with fistula healing were significantly higher than those without fistula healing (15.8 versus 4.4 µg/mL)20. When levels were stratified by quartiles, a linear association between IFX trough levels and fistula healing was observed. In addition, the absence of anti-drug antibodies was shown to correlate with healing. The optimal levels for fistula healing were at least 10 µg/mL and some patients even required levels of at least 20 µg/mL20. These findings suggest that trough levels that would usually be considered sufficient for the treatment of luminal disease may be inadequate to achieve fistula healing. Although current evidence does not suggest a relationship between drug exposure and adverse events21, the safety of such high trough levels has not yet been confirmed in large or longitudinal cohorts.One subset of patients that warrants separate discussion consists of the patients who have fistulating disease. It has been shown that higher IFX trough levels are associated with perianal fistula healing in both adults and childrenCombination with a conventional immunosuppressant. The immunogenicity of IFX and its clinical implications have been well established for some time. A recent review article analysing 114 studies reported that IFX immunogenicity rates ranged from 0 to 65.3% and were slightly higher for CD than UC22. In addition, the proportions of patients achieving and maintaining a response were lower in those patients with detected anti-drug antibodies. Other outcomes, including adverse event data  and trough IFX levels, were superior in those who did not develop anti-drug antibodies22.23. Recent observational and randomised studies, in abstract form, have shown that combination therapy reduces the rates of immunogenicity. A large, prospective, observational UK-wide study from the Personalised Anti-TNF Therapy in Crohn’s disease (PANTS) investigator consortium showed immunogenicity rates for IFX (Remicade) of 26% at week 54 and 42% at 3 years (and similar results were seen for the IFX biosimilar CT-P13). These rates were reduced with immunomodulator use (hazard ratio (HR) = 0.37,P <0.0001)24. Similarly, a recent randomised study showed that in those who failed ADA because of anti-drug antibody development, use of combination therapy (with azathioprine) when starting IFX significantly lowered the risk of immunogenicity25.Robust data generated by the Study of Biologic and Immunomodulator Naive Patients in Crohn’s Disease (SONIC) trial demonstrated that combination therapy with azathioprine achieves higher remission rates in CD26. This demonstrated that early combined immunosuppression was associated with a reduced rate of major adverse outcomes  compared with conventional management .Perhaps more compelling than short- and medium-term efficacy studies were the findings of the Randomized Evaluation of an Algorithm for Crohn’s Treatment (REACT) study, a cluster randomisation trial of treatment strategies27. In keeping with this, a subsequent prospective study observed that optimised IFX monotherapy is as effective as optimised combination therapy28. However, the IFX monotherapy group required significantly higher rates of treatment escalation, which makes this strategy unfavourable on a cost-effectiveness basis. In summary, the high rates of IFX immunogenicity observed in the literature and the associated poorer clinical outcomes appear to offer support for the use of combination therapy wherever possible. More recently, a post-hoc analysis of the SONIC trial suggested that the benefit of adding azathioprine to IFX could be explained solely by the resulting increment in IFX serum concentrations rather than the additive immunosuppressive effect of azathioprineDosing regimen modification. Currently, the approved dosing schedule for ADA is to give 160 mg followed by 80 mg two weeks later and then a maintenance dose of 40 mg every two weeks. Unfortunately, there is still a proportion of patients who do not respond to or lose response to ADA. In patients who do not respond, increasing the dosing frequency to once a week has been shown to be effective in recapturing response in CD29. However, to date, the data regarding dose escalation of ADA have been retrospective. To assess this prospectively, the Study to Evaluate Efficacy and Safety of Two Drug Regimens in Subjects With Moderate to Severe Crohn's Disease (SERENE-CD) trial has randomly assigned patients to receive higher induction and maintenance doses to establish whether primary and secondary loss of response can be avoided by maintaining higher serum drug concentrations from the outset30. If a benefit is observed, this may lead to a change in the approved dosing regimen.Measuring and monitoring. Higher concentrations of serum drug are associated with better outcomes, not only clinical remission but also endoscopic healing and deeper histological remission31. However, for ADA, unlike for IFX, there is evidence to suggest that it may not need to be a trough level that is taken. Wardet al. performed a prospective observational study on 19 patients with CD on maintenance ADA and took serum levels at multiple intervals during the usual 14-day cycle32. From this, the authors concluded that although ideally a trough levels should be taken, if a level of at least 4.9 μg/mL is detected during the first 9 days following a dose, it can reasonably predict an adequate trough level32. A recent retrospective study including 382 patients with IBD (311 of whom had CD) found that proactive TDM may be associated with a lower risk of ADA treatment failure compared with standard of care 33. However, a prospective randomised trial directly comparing these groups  has yet to be conducted.16.For their 2017 guidelines, the AGA reviewed data from four studies that reported the proportion of patients not in remission above ADA trough concentrations of 5 ± 1 or 7.5 ± 1 µg/mL. This proportion progressively decreased from 17% at a threshold of 5 µg/mL to 10% at a threshold of 7.5 µg/mL. They therefore gave a “conditional” recommendation for the use of 7.5 µg/mL as the target trough concentrationCombination with a conventional immunosuppressant. There are varying results from real-world cohort studies in terms of the effect of concomitant immunomodulator therapy on response rates to ADA. Previously, it was felt that the addition of immunomodulators offered no additional benefit in terms of prevention of anti-drug antibodies34. However, recent data from PANTS demonstrated that immunomodulators significantly reduce the immunogenicity of ADA 24.Dosing regimen modification. The induction and maintenance dosing schedule for VDZ involve an intravenous infusion at weeks 0, 2 and 6 and then every 8 weeks. The dose is standardised at 300 mg per infusion and is not weight-based like IFX. There is scope for variation in the maintenance dosing of VDZ to every 4 or 6 weeks, especially in those who are secondary non-responders. A 15% relapse rate was observed in one study that switched patients from every-4-week to every-8-week VDZ (and appears similar between UC and CD). Upon dose intensification back to every 4 weeks, 80% re-entered remission35. This suggests that, in patients with CD, increasing dose frequency of VDZ to every 4 weeks could lead to an improvement. A recent meta-analysis echoed these findings of dose escalation to recapture response in secondary non-responders36. The results revealed that a high proportion of patients with CD were secondary non-responders (47.9 per 100 patient-years) and that 56 (50%) out of 111 of secondary non-responders re-entered remission upon dose escalation36. An observational cohort study of 36 patients with IBD (18 with UC and 18 with CD) with a previous suboptimal response to every-8-week dosing also demonstrated a significant reduction in CRP  after 24 weeks of every-4-week dosing , and only week 6 levels showed an association with mucosal healing43. Drug levels and outcomes vary in the supporting literature and need further validation in larger prospective studies. Until data from larger studies are available, VDZ should be administered with dose optimisation based on objective measures of disease activity.A dose-response relationship for VDZ was observed in the GEMINI I, II and III studies. Drug level quartile analysis demonstrated a significantly higher rate of clinical response and remission, amongst patients the highest drug level quartile, compared to the lowestCombination with a conventional immunosuppressant. In patients who receive VDZ, the development of antibodies appears to be low (<5%). This degree of immunogenicity appears unlikely to have a significant impact on clinical outcomes and may decrease over time45. GEMINI II and III studies reported immunogenicity rates of 4.1% and 1%, respectively. GEMINI II found persistent antibodies in just 0.4%, whereas GEMINI III found no persisting antibodies. Although these measurements were performed by using a drug-sensitive assay (and therefore were unable to detect antibodies in the presence of drug), the results have been broadly corroborated in other studies using drug-tolerant assays45. Studies testing samples with drug-tolerant assays found low immunogenicity rates of 17% (7 of 41 patients) during the induction phase, 3% during the maintenance phase and 2.2% (4 of 179) after the first infusion45. When antibody development was demonstrated in the induction phase, 3 of the 7 responded to induction therapy45. The transient phenomenon of immunogenicity using VDZ is supported by undetectable antibody burden by week 40 when detected after first infusion44. The literature therefore supports low immunogenicity rates in treatment with VDZ and shows that antibody development is unlikely to have a significant impact on clinical outcomes.46. An integrated summary of VDZ suggests that whilst its low rates of immunogenicity could be further reduced by concomitant use of immunomodulators, the long-term risk-benefit should be evaluated and may not be in favour of their use47. This risk-benefit analysis is clearly different from that of IFX, which has much higher rates of immunogenicity. The only note of caution in this regard is that there has been no dedicated randomised study comparing combination therapy with VDZ alone to provide a more definitive answer to this question.Based on the majority of current evidence, combination therapy with immunomodulators to solely prevent immunogenicity does not appear to be a necessary strategy with VDZ. This is highlighted in a study that analysed pharmacokinetics and pharmacodynamics which demonstrated that the clearance and concentration of VDZ were not affected when co-administered with other immunomodulators48. They can also aid in maintaining remission up to 52 weeks. In a study where calcineurin inhibitors were used alongside VDZ, 44% of patients with CD had achieved steroid-free remission at week 1448. This study had a small cohort and further studies are warranted.VDZ appears to have a more gradual onset of action than other biologic agents, so initiation of treatment often requires bridging with other agents, at least until the end of induction. Corticosteroids are ideally placed to fulfil this role, but in those who are refractory, bridging with calcineurin inhibitors has been shown to be safe and effectiveReal-world effectiveness data. In addition to the randomised controlled trial data of ustekinumab’s efficacy demonstrated by the UNITI program, there exists a growing body of observational data of its effectiveness in clinical practice. One such study, carried out as a collaboration between three IBD centres in London, included a cohort of 149 patients and reported week 32 response and remission rates of 63% and 39%, respectively49. These are broadly consistent with another large cohort (n = 167) from Canada which described corresponding week 24 rates of 60% and 25%50. The London cohort also reported a range of other endpoints, including biological response (50% reduction in CRP) and biological remission (CRP of less than 5 mg/L in patients with a baseline CRP of more than 5 mg/L) (49.5 mg/L) 49.Dosing regimen modification. The induction dosing schedule for UST is fixed and involves an intravenous infusion of 6 mg/kg at week 0 followed by a subcutaneous 90-mg dose at week 8. There is scope for variation in the frequency of maintenance dosing. Depending on response, subsequent 90-mg subcutaneous maintenance doses are given every 8 or 12 weeks. The maintenance dosing regimen of UST, unlike that of other biologics, is guided by an assessment of response to induction therapy. This requirement encourages good clinical practice. However, it can pose certain difficulties and requires additional investigations.51. In summary, given the feasibility issues at play as well as the desire to use an objective marker in combination with symptoms52, faecal calprotectin appears to be ideally placed53. However, this can be applied only to patients with an elevated measurement at baseline.To establish adequacy of response to UST induction, a review at or just prior to week 16 is advised. Different modalities—including paired Harvey–Bradshaw index (HBI) scores and biochemical markers such as CRP and faecal calprotectin, endoscopic or radiological evaluation—may be used to determine treatment effect. However, the feasibility and acceptability of repeating endoscopic or radiological evaluations within this time frame are likely to limit their utility in this context. Nonetheless, the well-recognised discrepancy between clinical and endoscopic remission in CD should be borne in mind. For example, a recent prospective study of UST induction showed an endoscopic remission rate at week 24 of 7.1%. The concurrent clinical remission rate was 39.5%54. Another factor for consideration is that rates of endoscopic response and healing appear to be more favourable in every-8-week rather than every-12-week dosing55. Therefore, if there is any doubt as to which dosing regimen to use, literature supports a decision to opt for every-8-week dosing.If an adequate response is achieved, every-12-week dosing is considered appropriate, but in cases of partial response, every-8-week dosing is recommended. In circumstances where there is a deterioration or complete non-response, switching to another treatment (or surgery) is appropriate. However, it is recognised that in some patients a late response is seen, and given that patients may have already failed other therapies, it would be reasonable to persist and suggest a further every-8-week dose after week 1657. Dosing frequency can be reduced from every 8 weeks to every 12 weeks if remission is sustained, although there are little data to show the effect of this.There is also scope to adjust the frequency of maintenance dosing, even once already established. Post-hoc analysis of randomised controlled trials and observational evidence support changing from every-12-week to every-8-week dosing if a loss of response is observed58.Another important practical factor for clinical use was addressed by a retrospective Canadian study that examined the rates of peri-operative complications associated with the use of UST (20 patients) compared with anti-TNF therapy (40 patients). Their findings suggested that there was no difference between the two groups with a post-surgical follow-up of 6 months, although the UST cohort of patients were more likely to be on concomitant immunomodulatory therapy and were more often undergoing surgery as an emergency. These findings will be reassuring to clinicians but should be confirmed in prospective studies with a larger cohort of patientsMeasuring and monitoring. Access to UST TDM is currently limited and therefore it has not yet been integrated into routine clinical practice. However, a dose-response relationship has been demonstrated, and measuring serum levels will most likely be a useful tool to guide maintenance dosing in the future59. For example, one recent study showed higher UST levels in endoscopic responders at every study time point 51. In addition, potential therapeutic thresholds have already been postulated. A post-hoc analysis of UNITI suggested that a maintenance serum trough level of UST in the range of 0.8 to 1.4 µg/mL predicted clinical remission at 6 months56. However, for endoscopic remission, higher levels appear to be required. A recent study suggested that a minimum UST drug level of 1.9 µg/mL was necessary to achieve an endoscopic response at 6 months51. Another study suggested that a maintenance trough concentration of more than 4.5 µg/mL at 26 weeks correlated with biomarker and endoscopic response59. However, there exists some discrepancy between these observations; in the former study, only 1.6% of patients had a 26-week trough level over 4.5 µg/mL. This may reflect insufficient dosing even whilst on an every-8-week regimen51. It is possible that, in some cases of non-response, intensification of the dosing regimen (outside of licence) from every 8 weeks to every 4 weeks provides more adequate drug exposure and increased remission rates. However, further research in this regard is needed. The variation in serum levels between studies could be explained by intra-assay variation and other factors such as high baseline albumin, lower baseline faecal calprotectin and female sex (which are shown to be independent predictors of higher levels during induction51). Currently, UST level concentration assays are not commercially available on a wide scale, so despite the growing body of evidence, at present dose adjustments are made empirically.Combination with a conventional immunosuppressant. In patients who receive UST, immunogenicity rates appear to be low. In addition, the use of concomitant immunomodulatory therapy does not seem to affect serum concentrations. Both of these findings were observed in a comprehensive pharmacokinetic and pharmacodynamic analysis of the UNITI trials56. A post-hoc analysis of the UNITI trial revealed anti-UST antibodies in just 2.3% of 1366 patients during a year of treatment. These low rates are mirrored in another study showing antibodies in roughly 2% of patients (1 of 57) at weeks 8 and 1656. The former study used a drug-tolerant assay, the latter a drug-sensitive assay. Although the use of concomitant immunomodulation does not appear to impact UST concentrations56, prior exposure to anti-TNF agents and amount of previous anti-TNF exposure have been shown to negatively influence maintenance serum UST levels51. It may be appropriate to consider discontinuation of immunomodulatory therapies after commencing UST if the sole reason for their use was to reduce immunogenicity.Despite great strides in terms of understanding how best to use biologic agents, there remain many unanswered questions. For example, even though no clear evidence suggests that using UST or VDZ in combination with an immunosuppressant is beneficial, it must be remembered that this did not become apparent for IFX until a dedicated trial, specifically aiming to answer that question, had been conducted. This trial is yet to be conducted for UST or VDZ. We also have a relatively limited understanding of their efficacy for IBD manifestations such as perianal fistulation and pouchitis, and most of the data for these indications come from post-hoc analyses of randomised trials and observational studies. In addition, although there have been no signals of harm, we currently have a relatively limited understanding of their safety in pregnancy, a factor which can play a significant role in guiding treatment decisions.60. Although biologic combination regimens currently appear unfeasible on a cost basis alone, the impact of biosimilar versions is likely to make this type of strategy more achievable in the future. Finally, we have some way to go in terms of predicting which patients would benefit most from biologic therapy. In an attempt to address this question, the currently recruiting Predicting Outcomes for Crohn’s Disease Using a Molecular Biomarker (PROFILE) study uses a biomarker panel to separate CD patients at diagnosis into two cohorts: those likely to develop severe disease and those predicted to have a milder disease course. Patients within each group will be randomly assigned to receive either combination therapy with an immunomodulator and IFX (“top down” treatment) from the point of diagnosis or a “step up” approach of an immunomodulator initially followed by IFX only in the case of refractory disease. The trialists predict that patients in the severe group will benefit more from biologic treatment from the point of diagnosis, and if this is proven correct, it is likely that biologic therapies will appear earlier in treatment algorithms for this group of patients.An ongoing source of debate and controversy in the era of multiple biologic mechanisms is biologic sequencing. We now have to decide not only which mechanism—and, in the case of anti-TNF, which agent—to use first but also which mechanism to switch to in cases of treatment failure. Although it is can be clearly appreciated from their registration trials that VDZ and UST are less efficacious when used after anti-TNF, it is not yet clear whether the efficacy of the anti-TNF agents is diminished but prior exposure to novel biologics. There is also interest in the concept of combining biologic agents with differing and perhaps complementary mechanisms of action. For example, a trial is under way which combines VDZ, ADA and methotrexate for CD patients considered at high risk for complicated diseaseOne thing that appears certain is that the range of biologic  agents available for the treatment of CD will continue to expand—and rapidly. With several novel agents in late-phase trials and many more in earlier phases, the degree of complexity when making treatment choices will increase. Most importantly, with it, so will the range of options we have to offer patients with CD.Biologic therapies have completely changed the landscape of IBD care, and although there is renewed interest in small-molecule therapies, they appear likely to remain central to the management of patients with more severe or refractory disease. Despite this, there remains a great unmet need with many patients failing to respond to induction therapy or losing response after an initial improvement. Only through a broad spectrum of work carried out over many years were we able to fully appreciate the nuances necessary to optimise the effect of the anti-TNF agents. Indeed, we are still learning. Although some of this understanding can be extrapolated to help us maximise the benefit of novel biologics, such as VDZ and UST, an entirely new program of work is necessary. The fact that much of this research is already under way is cause for optimism and means that over time we will hopefully be able to meet the unmet need of patients with IBD.


Since there is a high prevalence of high risk alcohol use in patients with HIV in Africa, with negative health effects, there is a need for short interventions to reduce alcohol use.We studied the efficacy of a short intervention aiming to reduce alcohol use based on the Information-Motivation-Behavioural Skills Model in patients with HIV with high alcohol use (measured by AUDIT). The study was performed in three outpatient clinics in South Africa. The intervention group received in one-session intervention a personalized feedback on AUDIT results trying to make people aware that they are in the medium- or high-risk drinking category. Both the intervention and the control group received a health education leaflet.A total of 560 patients participated in the study with a follow up of 1 year. There was a significant decrease in total AUDIT scores between baseline and follow up points 1 (5 months) and 2 (1 year) in both groups. There was no significant decrease between time points 1 and 2. However, between the intervention and control groups there was no difference in reduction of alcohol use to abstinence or low risk alcohol use over time as there was no difference in absolute decrease in AUDIT-score or percentage of change in AUDIT score. The intervention had no influence on the quality of life outcomes, depression scores, stigma, tobacco use, viral load and therapy adherence at both time points. In all secondary outcomes, there was no significant interaction between intervention and time.The brief intervention was not successful at reducing alcohol use both 5 and 12 months after the intervention. However, there was a beneficial effect on reported hazardous or harmful alcohol use at least over a short term follow up period in both study groups. It might be that only an interview and/or the distribution of a health leaflet can be successful in reducing alcohol use but this needs to be investigated with more objective measures of alcohol use. To sustain an effect, most likely repetitive contacts with hazardous or harmful alcohol drinkers will be needed during a long follow up period. The prevalence of alcohol use disorders among patients with HIV is high in sub Saharan Africa. Previously we showed that of patients with HIV visiting primary health care clinics in South Africa up to 25.1% of the total group, and up to 40.3% of male patients were hazardous drinkers  or harmful drinkers  [Several studies showed the negative associations of alcohol use in patients with HIV. The prevalence of HIV infection is higher in alcohol users –6, most Therefore, there is a need to identify patients with HIV with an unhealthy alcohol use to test interventions aimed at reducing alcohol use. Previously conducted studies in patients with HIV in sub-Saharan Africa mostly focus on lowering sexual risk behaviour by reducing alcohol intake before sexual contacts .Given the lack of human and financial resources in sub-Africa there is a need to evaluate whether a brief and simple intervention is able to identify a real or potential alcohol problem and to motivate an individual to do something about it. Brief interventions have been used for a long time to change behaviour. By definition they are brief, usually conducted in a one-to-one situation and involve raising awareness of the problem, share knowledge and get the person involved to think about making changes to his or her behaviour. It can be delivered in one or multiple sessions. The design of brief interventions can differ in different settings. We conducted a randomized controlled trial to evaluate a brief intervention to reduce alcohol use based on the Information-Motivation-Behavioural Skills Model. This model has proved to be successful in different settings to change behaviour ,19.A systematic review and meta-analysis of brief interventions  for alcohol reduction in primary care patients including 19 trials  with 5639 individuals showed that of 17 trials reporting a quantified outcome measure in alcohol consumption, 8 reported that brief intervention for alcohol reduction in primary care patients was effective in reducing alcohol consumption at 6 and 12 months . Seven tAt the start of our trial there was no reported study on a single-session brief intervention aiming to reduce alcohol use in patients with HIV in Africa. The aim of our study was to evaluate the effectivity of a brief intervention to reduce alcohol use in patients living with HIV with hazardous and harmful drinking patterns attending a primary care setting in South Africa. Taking account of the overcrowded, overwhelmed HIV clinics in this setting, the goal was to develop a short, single-session, easy-to-use intervention to reduce alcohol use among patients with HIV. We hypothesized that patients in the intervention group would reduce their drinking levels more than patients in the control group.A randomized controlled clinical trial was performed in 3 HIV clinics based within primary health care clinics in townships surrounding Pretoria in South Africa comparing a brief intervention to reduce alcohol use with standard of care combined with distributing a health education leaflet on responsible drinking. In these clinics patients with HIV receive basic care and ART free of charge. The trial protocol was described before .Patients with HIV-1 infection who were 18 years or older and who visited the selected outpatient clinics for their HIV care were eligible for this study. Patients with mental impairment, not able to provide informed consent; , or those already undergoing alcohol reduction treatment  and pregnant women  were excluded from this study. All consecutive outpatients who gave informed consent were screened for alcohol use. Details of this population have been published before [Research assistants administered a range of questionnaires at baseline, 3 months and 12 months follow up in English or Tswana, the most spoken local languages.A researcher designed questionnaire was used to collect data on patients’ age, gender, population group, language, educational level, marital status, highest educational qualification, main source of family income and residential status.A researcher designed questionnaire and data extraction sheet were used to collect data on first positive HIV test, start date for ART, ART regimen, co-infections, co-medication, CD4 cell count at initiation of therapy and the latest measured value and HIV viral load.The 10-item Alcohol Use Disorder Identification Test (AUDIT) was used to assess alcohol use levels . A scoreTo reduce the stigma of alcohol use, the WHO suggests integrating the screening of alcohol use with screening for other health-related behaviours. Therefore, we included 2 questions on the use of tobacco products and anthropometric measurements were taken to assess the risk factor of overweight .The WHOQoL-HIVBREF questionnaire was used to evaluate the quality of life, which comprises the physical (domain 1), psychological (domain 2), level of independence (domain 3), social relationships (domain 4), environment (domain 5) and spirituality/religion/personal beliefs (domain 6) domains . Two queThe 10-item version of the Center for Epidemiologic Studies Depression (CES-D) Scale was used to assess symptoms of depression ,31. A cuThe seven items internalized AIDS-related stigma scale for people infected with HIV was used to reflect self-defacing beliefs and negative perceptions of people living with HIV/AIDS .Adherence to ART was assessed only in patients on ART by self-reported adherence by recall of missed doses during the previous 7 days . The secResearch assistants (four nurses), not attached to the clinics, were trained to deliver the interventions to the patients and follow them up. The training consisted of (video recorded) role play, general skills training techniques and training on alcohol-, HIV- and sexual-related issues. The sites were visited twice weekly by one of the investigators to observe whether there was adherence to the protocol and to offer support and supervision to the research assistants. During regular study team meetings occurring problems were discussed and solved.The brief intervention was based on the WHO brief intervention package for hazardous and harmful drinking, which is based on the Information-Motivation-Behavioural Skills Model . PatientPatients in the control group only received a health education leaflet on responsible drinking.The primary outcome of the study was reduction from hazardous (AUDIT between 7 to 15 for women and 8 to 15 for men) or harmful (AUDIT between 16 to 19) alcohol use to alcohol abstinence (AUDIT score 0) or low risk alcohol use (AUDIT score 1–6 for women and 1–7 for men) at time points 1 and 2.Secondary outcomes were reduction in absolute numbers and reduction in percentages in AUDIT score at time points 1 and 2. To measure any other possible effects of the intervention, other secondary outcomes were changes in medically related outcomes , quality of life outcomes (measured with the WHOQoL-HIVBREF tool), depression score, level of experienced stigma and tobacco use.The sample size was calculated using Open Epi (version 3) . Based oDescriptive statistics describing patient characteristics were represented in means, standard deviations (SD), medians and interquartile ranges (IQR), and percentages.The primary outcome was compared between the intervention and control group by using a logistic Generalized estimating equations (GEE) model to account for correlated patient response . A modelSecondary outcomes  were compared between intervention and control group using a linear mixed model , accountAll statistical tests were two-sided and were considered statistically significant at α = 0.05. The GEE, MIXED, and GLIMMIX procedures of SAS version 9.4  were used to fit the logistic GEE and mixed models.Public trial registration: Pan African Clinical trial Registry: PACTR201202000355384.Ethical approval was given by the Medunsa Research and Ethics Committee (Project number: MREC/H/165/ 4152011: IR). The Tshwane Research Committee has provided approval for the study (Project number: 2011/46). Procedures followed were in accordance with the ethical standards of the Medunsa Research & Ethics Committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards . Written informed consent was obtained from all individual participants reincluded in the study.From January until June 2012, 2352 patients were approached to participate; 103 refused (response rate 95.6%) and 2249 were eligible and agreed to participate in the study .The follow up period was up to December 2013. Of these 2249 eligible patients, 560 (24.9%) fulfilled the criteria for hazardous or harmful alcohol use and 267 patients (47.7%) were randomized into the intervention group and 293 (52.3%) in the control group . Table 1The median duration of the interview was 40 minutes (IQR 30–47 minutes) in the intervention group (including the intervention) and 35 minutes (IQR 25–40 minutes) in the control group. Almost 91% of the patients in the control group were in the hazardous alcohol use group and 9.2% in the harmful alcohol use group, compared to 77.9% and 22.1% respectively of patients in the intervention group. Of patients receiving ART , the duration being on ART was longer in the control group than in the intervention group. The last measured CD4 count was higher in the control group . The medFor the whole study group there was a significant decrease of around 6 points (SD = 0.27) in the absolute AUDIT score from baseline at both time points 1 (p<0.0001) and 2 (p<0.0001). The difference in AUDIT-score between time points 1 and 2 was not significant .In both the intervention and control group there was a significant decrease in AUDIT scores at follow up time points 1 and 2 compared to baseline , Table 2Overall, around 58% and 61% of the patients had a reduction of alcohol use from hazardous or harmful at baseline to abstinence or low risk alcohol use at time points 1 and 2, respectively. Around half of the patients both in the control as the intervention groups reached the group of abstinence or low alcohol use at both time points . Using tThe difference in AUDIT score from baseline to first and to second follow up did not differ between the intervention and control groups . Being mThe percentage change in AUDIT score was not significantly different between the intervention and control group. Men (compared to women) and patients on ART (compared to patients not on ART) had a significantly higher percentage of change in AUDIT score. In patients with higher stigma scores at baseline, the percentage of change in AUDIT score was less than in patients with lower stigma scores.The mean last measured CD4 count was significantly lower in the intervention group than in the control group at time point 1 (p = 0.036), but not at time point 2 . The domSince there is a high prevalence of high level alcohol use with risk for morbidity among patients with HIV there is a need for interventions to reduce alcohol use. In sub-Saharan Africa the health systems providing care to patients with HIV are overwhelmed by the large numbers of patients using the system. Possible interventions should be short and easy to implement in daily care provision. The brief intervention evaluated in this trial was based on an Information-Motivation-Behavioural Skills Model and was delivered once during a standard clinic visit. There was no difference in alcohol reduction in the intervention compared to the control group both 5 as 12 months after the counselling session. However, the alcohol use in both groups reduced significantly between baseline visit and visit 1 (after a median time of 5 months). There might be different explanations for this. Both the intervention as the control group received a health education leaflet on responsible drinking, emphasizing the idea of sensible limits. With both the baseline interview, covering questions about alcohol use and health, and the health education leaflet, the patients in the control group might also have been triggered to reduce their alcohol intake. This effect of alteration of study participants’ way of thinking about their alcohol use, only after assessment of it by interview or questionnaire, was recognized previously ,42,43,44There was no further reduction of alcohol use between time point 1 and time point 2; the reported alcohol use remained stable. These results are comparable to a study in Kampala in Uganda which also showed a significant decline in AUDIT-C score in both intervention as control arms in the 6 months follow up after a similar single brief alcohol reduction intervention session. In the latter study there also was no difference in both arms regarding alcohol reduction  but the In our study there was a significant higher reduction in percentage and absolute AUDIT score in men compared to women. This is in contrast with the study in Uganda that showed the opposite effect . In our The reduction in alcohol use in both groups still remains significant and important for health outcomes. The risk of all-cause mortality, cancers and high blood pressure rises in the general population with increasing levels of alcohol consumption,54. MoreThere are several possible limitations in this study. There was a difference in AUDIT-score between the intervention and control group at baseline, with a higher mean score in the intervention compared to the control arm. However, because randomisation was done, any observed differences at baseline is attributed purely by chance and no conclusion from this can be derived . A limitPatients in the intervention group had a lower last measured CD4 cell count at baseline and at time point 1. Data on influence of (heavy) alcohol use on CD4 cell count is contrasting, in our previous report of the whole study group (n = 2230) there was a significant lower last measured CD4-cell count in high risk drinkers versus low risk drinkers  .Finally, the study was conducted in three primary HIV clinics in one region in South Africa and therefore results might not be generalizable to other settings.A brief alcohol intervention, based on an Information-Motivation-Behavioural Skills Model and delivered once during a standard clinic visit, was not more successful at reducing alcohol use both at 5 and 12 months after the intervention, compared to only the administration of a questionnaire on alcohol use and the distribution of a health educational leaflet on responsible drinking. However, there was a beneficial effect on reported hazardous or harmful alcohol use at least over a short term follow up period with both interventions. It might be that only an interview and/or a health leaflet can be successful in reducing alcohol use but this needs to be investigated with more objective measures of alcohol use. To sustain an effect most likely repetitive contacts with harmful alcohol drinkers will be needed during a long follow up period.S1 FileChecklist of information to include when reporting a randomised trial.(PDF)Click here for additional data file.S2 FileScreening and brief intervention for alcohol problems in HIV outpatients in the clinics associated with Dr. George Mukhari Hospital, Ga-Rankuwa, South Africa: a single-blinded randomized controlled trial.(DOCX)Click here for additional data file.


The majority of the world’s children live in low- and middle-income countries and face multiple obstacles to optimal wellbeing. The mechanisms by which adversities – social, cultural, psychological, environmental, economic – get ‘under the skin’ in the early days of life and become biologically embedded remain an important line of enquiry. We therefore examined the contribution of childhood adversity through pregnancy and the first year of life to hair and salivary cortisol measures of early life stress in the India SPRING home visits cluster RCT which aims to improve early childhood development.We assessed 22 adversities across four domains: socioeconomic, maternal stress, family-child relationship, and child and summed them to make a cumulative adversity score & quintiles, and four subscale scores. We cut 3 cm of hair from the posterior vertex and took three saliva samples from morning till late afternoon on each of two days . We analysed both for cortisol concentration using ELISA techniques. We used multiple linear regression techniques to assess the relationship between cumulative adversity and log hair cortisol concentration and saliva diurnal slope and area under the curve.We assessed 712 children for hair, and 752 children for saliva cortisol at 12 months of age. We found a strong positive relationship between adversity and hair cortisol; each additional adversity factor was associated with hair cortisol increases of 6.1%  and the increase from adversity quintile one to five was 59.4%. Socioeconomic, relationship and child scales were independent predictors of hair cortisol ; relationship 11.8% ; child 7.9% . We did not find any association between any measures of adversity and either of the saliva cortisol outcomes.This is the largest study of hair cortisol in young children, and the first in a low- and middle-income country setting. Whilst the short-term diurnal measures of cortisol did not appear to be linked with adversity, chronic exposure over several months appears to be strongly associated with cumulative adversity. These findings should spur further work to understand the specific ways in which adversity becomes biologically embedded, and how this can be tackled. They also lend support to ongoing action to tackle childhood adversity in communities around the world. Childhood adversity is associated with negative effects across the lifecourse including on growth, development, behaviour and academic ability in childhood and conditions including depression, cardiovascular disease and healthy ageing later in life . The queCortisol is one of the most studied of these mechanisms, and we examined the relationship between this hormone and childhood adversity in young infants aged 12 months in India. Cortisol is the end product of the hypothalamic-pituitary-adrenal (HPA) axis and has been widely used as a stress biomarker, but rarely in such young infants. It can be measured in multiple ways. The concentration of cortisol in hair gives a measure of chronic exposure to stress over a period of months . MeasuriChanges in cortisol are well documented in older children exposed to adversities including those exposed to deprived care in Romanian orphanages  where flIn the work described in this paper we therefore set out to assess the relationship between early life adversity and these chronic & diurnal measures of cortisol in hair & saliva in children enrolled in the Early Life Stress sub-study of the SPRING cluster randomised controlled trial in rural Haryana, India. Our hypothesis was that adversity would be associated with increased hair cortisol, and in saliva with increased daily exposure to cortisol and flattened diurnal slope. This is the first time hair cortisol has been reported in children from a low/middle-income country (LMIC), and few studies examine both saliva & hair simultaneously.22.1SPRING-ELS was a sub-study of the Wellcome Trust funded SPRING cluster randomised controlled trial in India. It focussed on cortisol measures of early life stress, and on early childhood adversity in children enrolled in SPRING. Details on SPRING are available elsewhere  but in brief SPRING developed an innovative, feasible, affordable & sustainable community-based approach to delivering a home visiting programme aiming to improve child growth & development at-scale in India & Pakistan, two countries with high burden of disadvantage. Implementation was evaluated by parallel cluster randomised controlled trials where clusters represent geographical areas served by a health sub-centre with a functioning auxillary nurse midwife, covering a population of at least 8000. The primary outcomes were height-for-age, the best early childhood predictor of human capital , and Bay2.2Children were identified by a surveillance system whereby trained resident fieldworkers visited each household in the study area every 8 weeks to identify pregnancies and births, and follow-up pregnant women & children already identified. Socioeconomic data was collected at enrolment and assessors were trained to take saliva and hair samples from children, and to do adversity assessments with their mothers when infants turned 12 months of age. Full implementation of the SPRING intervention was achieved in May 2015 and these children were born from 18 June 2015. Assessments were therefore done when SPRING had been running for at least one year in intervention clusters.2.2.1These were selected to be contextually important based on formative research with local mothers and grandmothers, advice from child development experts and reviews of the literature on existing tools. The adversities covered four domains as follows: 1) household-level socio-economic factors, 2) maternal stressors, 3) child-carer relationships and 4) child-related factors. The focus was on these groups because young children in this setting spend most of their time and interact most closely with family members inside the home. The aim was to focus on a broad range of potential impediments to wellbeing because children can be resilient to single adversities, but combinations of these may be more harmful and overwhelm protective factors in a child’s life .Data on 22 adversities were chosen. These are summarised in Socioeconomic:1) Asset index - being in the lowest quintile for the population at enrolment  2) Low parental education – no education or primary-schooling only (asked at enrolment) 3) Father occupation - father did not work, was seasonably employed or was a casual labourer at 12-month assessment 4) Mother married under the legal age of 18 years 5) Family debt - mother reported family debt or being unable to afford to buy food for herself or her child at any point between becoming pregnant and the 12 month assessment.Maternal stress:1) Death of one or more of mother’s close family members since becoming pregnant 2) Mother seriously injured or ill since pregnancy 3) Any violence towards mother from husband  or any Relationship:1) Any family member was unhappy when they found out that the child was a girl 2) Moderate or high concern level on Mother Object Relations Scale – short form (MORS-SF) at 12-month assessment. MORS-SF is a screening tool consisting of 14 short statements which a mother is asked to rate on a Likert-type scale to identify potential problems in early mother-infant relationship  3) Very Child:1) Child born prematurely (asked at 12-month assessment) 2) Child hospitalised in first year of life 3) Separation of mother & child for more than a week in the first year of life 4) Inadequate care – child left alone or with a child under 10 years for more than one hour in the past week (assessed at 12-month assessment) (From ) 5) OldeAdversity questionnaires were double-entered and verified using a computer program written in C Sharp with an SQL Server 2008 database.2.2.2Trained assessors cut hair samples from the posterior vertex  as clos2.2.3The same fieldworkers took saliva samples three times  on each of two consecutive days . They used Salimetrics SalivaBio Children’s Swabs  which are designed for young infants to avoid choking risk and to be palatable. These swabs have been used in many settings worldwide and are the gold-standard for collection of saliva for cortisol analysis . The firSampling was done as follows. The child was positioned on their mother or grandmother’s lap and the fieldworker gently introduced the swab into the child’s mouth for 30–60 s. When it was observed to be at least 1/3 saturated  it was removed, placed into a storage tube  and labelled with a pre-printed freezer-proof label containing only a sample identifier and anonymised child identifier. Time of sampling, time of last food, drink & last waking were then recorded.g for 15 min and refrozen at −20 °C. Samples were analysed later in batches using a Salimetrics USA high-sensitivity salivary cortisol enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. A randomly selected 10% of samples per batch were analysed in duplicate. The intra-assay coefficient of variation of 5.6% and inter-assay coefficient of variation of 9.2% was within acceptable limits . The aim was to assess more children than this - at least 30 children per cluster for saliva and 35 per cluster for hair - to ensure that the minimum sample size was met even if samples were of insufficient volume  or weight (hair) for analysis.2.4Stata 15 was used for all analyses .2.4.1we used multiple imputation by chained equations (MICE) to account for the missing values in adversity data described in We categorised adversities in three ways as follows: 1) we summed the adversities to create a total adversity score of 0–22 following a cumulative-adversity model  2) we su2.4.2We log-transformed hair cortisol concentrations for each child because of left-skew and then winsorized four remaining outliers to 3SD above the mean. This was the first outcome variable.2.4.3We calculated two outcomes from saliva cortisol measures for each child. The first was saliva cortisol slope which is a measure of the change in saliva cortisol concentration per hour across the day’s sampling. The second was saliva cortisol area under the curve which is a measure of the total hourly exposure of a child to cortisol over the sampling period. For each saliva cortisol result we first winsorized outlying high values to 3SD above the mean. We then used the rise-over-run formula  for children with results at 8am and 4 pm to calculate the saliva slope for each child. Similarly, we used the trapezoid formula to estimate the total cortisol a child was exposed to which was represented by the total area bounded by two parallel lines at each of two time points on the x-axis, the base on the y axis (where cortisol is zero) and the line connecting the two cortisol values on the y-axis. This was done for children with all three samples on at least one day to calculate saliva cortisol area under the curve with respect to the ground.2.5We assessed the relationship between cumulative adversity and hair cortisol using multi-level modelling, accounting for clustering as a random-effect and allocation to the SPRING intervention or control arm allocation as a binary fixed-effect in the model. We first treated adversity as categorical to examine mean hair cortisol for each of the observed cumulative adversity scores, and then as continuous to assess the linear trend in this relationship. We ran the same model with the adversity quintiles replacing cumulative adversity. We ran all of these models including Sex and Body Mass Index (BMI) to assess for possible confounding.2.6We also used multi-level modelling to assess the association of adversity and saliva cortisol slope in the whole sample using a three-level model which accounted for saliva results nested hierarchically within the random effects day, child & cluster, accounting for time of sample collection as a fixed-effect interaction term. Child wake-up time was a fixed-effect to control for any effect of waking time on saliva cortisol values. The difference in saliva cortisol slope between cumulative adversity scores was represented by the interaction term between adversity and time of sample with cortisol result at that time as the outcome.We used a similar approach to model AUC. The difference in AUC between cumulative adversity scores for the whole sample was modelled using predicted margins for cortisol concentration at 8am, noon & 4 pm. The model first used the trapezoid formula described earlier to calculate the AUC at each adversity level, and then subtracted one from the other to calculate the difference. We ran these models including Sex and Body Mass Index (BMI) to assess for possible confounding.2.7We examined the association between the four adversity domains and the three cortisol outcomes using similar models to the analyses with total cumulative adversity. We used a three-step process as follows:We first ran models to explore the association between each domain and cortisol, adjusted only for clustering and trial arm allocation. We then added socioeconomic score to the models for Maternal Stress, Relationship & Child domains to adjust for potential confounding in these associations by socioeconomic status. We finally ran a model including all scales showing either a strong relationship or p-value less than 0.1 to create a mutually adjusted model.2.8Ethics approval was obtained from the London School of Hygiene & Tropical Medicine research ethics committee  and the Sangath Institutional Review board (IRB) . Approval was also granted by the Indian Council of Medical Research’s Health Ministry Screening Committee (HMSC) . The SPRING trial is registered with clinicaltrials.gov, number NCT02059863. Informed written consent was obtained from mothers at identification by the surveillance system and again before a child’s first birthday.2.9The funder (Wellcome Trust) had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript. SB & BK have complete access to the study data and are responsible for the reported study findings, and made the decision to submit for publication.33.11693 children were enrolled for hair assessment, and 1350 children were similarly enrolled for saliva assessment. The flowcharts for both of these sub-samples are shown in 3.23.2.1Most children had a cumulative adversity score of at least one. The maximum score was 12. Cumulative adversity was strongly positively associated with hair cortisol on the log scale as illustrated in 3.2.2The strongest association was with the Relationship scale where each increase in score was associated with an increase of hair cortisol of 15.8% on the log scale. This scale was observed across a range of 0–3 adversities and so is not directly comparable to other scales which were observed over greater ranges as illustrated in 3.3Analyses presented in 4We found that childhood adversity was clearly positively associated with the concentration of cortisol in hair samples taken at one year of age. This assessed cortisol exposure over several months. This relationship was not confounded by BMI or sex. We did not find the same relationship with saliva measures which focus on the cortisol rhythm over two days. This increased chronic exposure to cortisol is likely to be detrimental to child wellbeing and our findings should serve as a wake-up call that children are never “too-young” to be affected by adverse circumstances, that children need protection & support to avoid adversity and that programmers & policy-makers should reiterate efforts to ameliorate these effects.Most studies of hair cortisol in young children are relatively small. We identified 11 studies done in under 5 year olds, a similar number to those identified in a recent systematic review . Most ofThe finding that maternal-stress factors were less critical to cortisol than other factors in our analysis should give pause for thought, as these are often some of key issues considered when attempting to address problems in early childhood. Similar to our findings, Liu et al examined children at 9 months & 1 year and found no difference in hair cortisol by maternal stress, affect or mood  . HoweverThat saliva cortisol was not associated with childhood adversity is noteworthy, and contrary to our hypothesis. We found very flat slopes in the majority of the sample and it is possible that this contributes to this finding. For example, St John et al describe slopes which are 10 times greater than the decline we describe in under one year olds . Having Strengths of our study include the measurement of adversities at the time they are occurring, in contrast with the common approach to Adverse Childhood Experiences focussing on adult recall of childhood events . We wereLimitations are that biological measures were restricted to cortisol whilst biological embedding of adversity is likely to occur through multiple factors acting simultaneously. Assessing multiple biological markers simultaneously  can provide more detailed estimation of the biological toll of adversity . A speciThat hair cortisol was seen to rise with each increase in a wide-range of adversities suggests that efforts to improve this will require a multisectoral approach to both reduce adversities and to design interventions that can protect children who face these – the care for child development curriculum being promoted by WHO/UNICEF is one such example of an intervention promoting stimulation and nurturing care within the household.In attempting to understand the effects of adversity in early childhood on a broad range of outcomes, a key question relates to ways in which these become biologically embedded, causing suboptimal lifelong health & wellbeing. Simultaneously, programmers implementing early-life interventions require tools that can provide insight into the biological impact of these. Hair cortisol has the potential to be of use in both regards.In summary, we present for the first time the finding that early life adversity is related to chronic childhood exposure to cortisol in a low/middle-income country. Most children live in LMICs and these countries are where the burden of adversity and suboptimal health & wellbeing is greatest. Action is ongoing worldwide to tackle childhood adversity at societal, community & household levels and our work reiterates its importance in very early childhood, in a low/middle-income country. Further work is needed to develop deeper understanding into ways in which adversity becomes biologically embedded to help refine this action.Sunil Bhopal: Conceptualisation, methodology, software, formal analysis, investigation, writing – original draft, writing – reviewing & editing, project administration, funding acquisition. Deepali Verma: Methodology, formal analysis, investigation, writing – reviewing & editing, project administration. Reetabrata Roy: methodology, formal analysis, investigation, writing – reviewing & editing, project administration. Seyi Soremekun – formal analysis, writing – reviewing & editing. Divya Kumar – investigation, writing – reviewing & editing, project administration. Matt Bristow – Methodology, supervision, writing – review & editing. Aparna Bhanushali – validation, investigation, writing – review & editing. Gauri Divan - methodology, investigation, writing – reviewing & editing, supervision, project administration. Betty Kirkwood – Conceptualisation, methodology, formal analysis, investigation, writing – reviewing & editing, supervision, project administration, funding acquisition.None