Handwashing with soap has the potential to curb cholera transmission. This research explores how populations experienced and responded to the 2017 cholera outbreak in the Democratic Republic of the Congo and how this affected their handwashing behaviour.Cholera cases were identified through local cholera treatment centre records. Comparison individuals were recruited from the same neighbourhoods by identifying households with no recent confirmed or suspected cholera cases. Multiple qualitative methods were employed to understand hand hygiene practices and their determinants, including unstructured observations, interviews and focus group discussions. The data collection tools and analysis were informed by the Behaviour Centred Design Framework. Comparisons were made between the experiences and practices of people from case households and participants from comparison households.Cholera was well understood by the population and viewed as a persistent and common health challenge. Handwashing with soap was generally observed to be rare during the outbreak despite self-reported increases in behaviour. Across case and comparison groups, individuals were unable to prioritise handwashing due to competing food-scarcity and livelihood challenges and there was little in the physical or social environments to cue handwashing or make it a convenient, rewarding or desirable to practice. The ability of people from case households to practice handwashing was further constrained by their exposure to cholera which in addition to illness, caused profound non-health impacts to household income, productivity, social status, and their sense of control.Even though cholera outbreaks can cause disruptions to many determinants of behaviour, these shifts do not automatically facilitate an increase in preventative behaviours like handwashing with soap. Hygiene programmes targeting outbreaks within complex crises could be strengthened by acknowledging the emic experiences of the disease and adopting sustainable solutions which build upon local disease coping mechanisms. For centuries cholera has been a marker of social inequalities, affecting the most vulnerable members of society and commonly occurring amid social and economic upheaval or disaster . Choleravibrio cholera believed to be responsible for the majority of transmission  is not a soap we want to use because it can damage hands, but we just use it because of poverty.” As with water, soap use within the household involved conscious trade-offs and decision-making between family members:“Getting soap is not easy…In a house of nine children, you understand that a piece of soap will not be prioritized for hand washing. If we manage to afford the piece of soap for 100 Congolese Francs only once a week… How, and on what, can you use just a small piece of soap? Will you use for laundry? For bathing? Or for hand washing? Things become complicated.” Participants explained that it was relatively common to ask neighbours for soap and water if needed but said people would laugh at you if you asked for these items for handwashing.The social environment—norms, routines and social influenceDaily routines were unpredictable for most of the participants, with individuals searching for employment in the fields of others on a day-to-day basis. Daily routines were further complicated by intermittent water supplies and individuals could spend several hours per day searching for water they considered to be safe. Combined, these factors created time and financial pressure. The irregularity of routines, and the daily stressors that accompanied this, decreased the likelihood of handwashing habits forming. This was because there were few routine sequences of behaviour to cue handwashing and there was limited ability to make plans related to handwashing (e.g. to budget to have enough soap in the house).Handwashing was seen as socially desirable and an injunctive norm. Hand hygiene norms were heightened by the outbreak, with participants estimating 65% of their community had increased their frequency of handwashing due to cholera concerns. Despite this, social sanctions or judgment related to not washing hands was low:“I cannot judge people around me for not being clean because sometimes I am not clean too… I think only about 30% of people would judge me negatively [if they saw me not washing my hands] because people are not focused on hand washing behaviour; they can take it as normal that people sometimes forget to wash their hands.” Participants explained that people easily forgave each other for not washing hands because of their difficult circumstances. Social support for handwashing was limited. Participants felt handwashing could be facilitated by family members reminding each other to wash hands at key times, but this rarely happened in practice. The importance of handwashing behaviour was consistently reinforced by NGOs and while some people felt the repetition of handwashing messages helped to remind people, others found it frustrating that these organisations were unable to realise changes to their broader circumstances which would allow them to practice handwashing more regularly.MotivesIn FGDs participants were asked about which motives were associated with handwashing. Motives of love, attractiveness and status  were thought to be strongly associated with handwashing:“No one can fall in love with someone with dirty hands!” “An attractive person is likely to remember to wash hands because she is used to looking nice, so, her hands have to look nice as well.” “Highly educated people are always clean because they do work with white papers and with clean things, and so they also have to have clean hands. And again, these educated people teach others; they cannot go in front, teaching others when they are unclean. They have to have clean hands so that people consider and respect them.” Handwashing was less strongly associated with nurture and was not seen to be associated with affiliation (fitting in with a group). Participants explained that even ‘good parents’ are unable to mind their children and encourage handwashing behaviour because everyone has to work long hours outside the home. Others explained that relationships were typically built on shared interests and needs and so handwashing didn’t necessarily help a person to fit in. Participants in IDIs and FGDs agreed that if a person was hungry, poor or upset they would be unable to prioritise handwashing:“Hygiene and good nutrition work together. Cleanliness cannot be visible in a house where food is absent. I can’t think about handwashing when I am so hungry. Another thing is the kind of life I live, since I am IDP, it has brought me to trauma or psychological problems. Hygiene has become difficult because of the many thoughts crossing my mind like: How my children are going to eat? How am I going to get money? It’s difficult.” Hunger emerged as a particularly prominent barrier to hand hygiene in this setting. At critical moments for handwashing, such as preparing or eating food, people’s hunger would override all else, causing people to forget handwashing. Hunger also caused prevented people from making plans that could facilitate handwashing. For example, participants explained that their limited daily earnings are entirely consumed by purchasing food:“If I do not go to work in the farms all day, we shall not eat… But if you are only able to earn 2500 Congolese Francs, then all of this money will have to go on food. We ask ourselves whether to buy soap… but it is difficult to choose this rather than prioritizing food for our children.” Demographic characteristics Certain personal characteristics influenced handwashing behaviour. Older adults and men who lived alone often were unable to collect sufficient water to meet their needs and therefore reported making handwashing compromises. Their reduced water availability was due to accessibility or cultural barriers associated with gender norms . IDPs typically faced more challenging living conditions than host community members and often described feeling that they were living “like animal”‘ or “living a life that was not our own”. Consequently, many IDP participants reported that the ‘problem of handwashing’ was new for them and if their old lives could be restored their behaviour would also improve:“I can tell you that I had a good life, I was a rich person… but my life changed with displacement…the situation changed to bad and today I am as you see me. take me back to my previous life and you will see my feelings and emotions will change and then I will have a high chance of washing his hands with soap.” The determinants of handwashing behaviour among case households were consistent with those in comparison households across the determinant categories described above. However, there were substantial differences between the two groups on cholera-related risk perceptions and the perceived or actual consequences of cholera on people’s livelihoods and routines.Participants from comparison households. Cholera was the main health concern of almost all participants, but among those who had not had a cholera case in their household, it was seen as a common, chronic challenge that was inseparable from other adversities they faced:“Cholera is a major health problem here … because we work hard, earn less, eat less and rest less. As consequence, we lose weight and look pale and have poor nutrition… Then it is easy for the cholera disease to attack people.” “It has been a long time since we do not have drinking water in this area and that is the reason why cholera disease attacked people… if you have bad food, unhealthy water and your hands are dirty then of course you will suffer from cholera.” The high number of cases in the 2017 outbreak did heighten perceptions of risk and participants from comparison households reported realising cholera was serious within recent months. Participants from comparison households thought cholera generally affected children or people who were already ‘sickly’, ‘unclean’ or ‘pale’. Host community members thought that cholera was primarily a problem that affected IDPs. In terms of the social and economic impacts of cholera, participants from comparison households said they feared people who had cholera and would avoid them while infectious. Those without direct personal experiences of cholera thought its impacts on the lives of cases or their families would be relatively temporary, given that disease was seen to be easy to treat and participants felt that people tended to recover quickly. Participants were divided about whether getting cholera would have a longer-term impact on a person’s reputation:“Someone who has had it [cholera] would not like people to know that he had it because cholera disease really affects a person’s dignity, nobody can wish to get it. It leaves you with no reputation at all.” “Everyone understands…I mean most people know someone who has experienced cholera at some point… So the person who gets infected, he can still recover and get back to his normalcy.” In many ways local explanatory disease models were aligned with ‘Western’ biomedical messaging about the cholera . However, misperceptions about cholera persisted within the community despite familiarity with the disease. Several participants said others in their community do not take cholera seriously because they believe “black people don’t die of germs”. Similarly, some people believed that a certain degree of exposure to “dirtiness” helps to protect you because being too clean may leave you vulnerable to infection. Others felt the continued presence of cholera in Eastern DRC played to the interests of humanitarian organisations:“We know that when [organisations] come just sensitizing about hygiene and cholera, they are paid on this—it’s no use coming all the time disturbing us. They come for their own interest.” Over decades people had realised that most humanitarian aid was provided during cholera outbreaks. These were short term projects which subsided as cholera cases decreased. People had also learned how to make the most of a system that didn’t always appear to have their ongoing interests at heart. For example, on several occasions during our research people from the community who were not participants, approached us with lists of ‘cholera cases’ who needed help.Participants from case households. Participants from case households reported that they were easily able to recognise cholera symptoms. All of these households explained that cholera had affected them suddenly and unexpectedly. Some participants said this in a literal sense, reflecting that people commonly went from feeling healthy to suddenly experiencing vomiting and diarrhoea which lead them to become so weak that they were unable to do anything within hours. More commonly, when participants said cholera was “unexpected” they were expressing that they struggled comprehend how the disease had been able to launch a “surprise attack” on a family like them. Participants therefore attributed their illness to a one-off “mistake” in their behaviour.When the disease initially “attacks”, family members reported being worried. Patients described feeling “empty”, “not of this world” and “seeing only death before them”. When patients returned home from the CTC, their cholera experience was not over. Cholera cases described feeling “stuck” and “destabilized” in multiple aspects of their life. Patients were unable to do agricultural work or household tasks for about a month because they felt “weak like paper”. Given the majority of households in this region survived day-to-day, earning less than 3000 Congolese Francs per day (1.5 USD), this inability to work rapidly put families in a state of economic crisis:“I cannot say that my economy decreased—it was totally blocked!” “A huge economic impact was observed…during that period of our child’s sickness, things really got harder. Although we generally eat badly, that particular time my husband was staying with the child at the hospital, we then ate more badly than usual because of the little amount of money—everything went really bad.” People also reported that cholera affected their roles, responsibilities, and sense of self. Participants felt cholera caused their attractiveness to “fade” and that they now “hated their outlook” due to the amount of weight they had lost. Parents who had been cholera cases felt worried that in the months following their discharge they had become unable to care and provide for their children:“I became like a baby…I had to wait for somebody else to take care of me, like the neighbours, it is like I have lost my role of mother to all these children.” “Suffering from cholera reduced considerably my responsibility as a father, I could not feel respected, and I could not feel myself as a father of the family because I was half-dead.” Another woman explained that she relied on her neighbour to breastfeed her new born baby for several months while she was sick with cholera. Children and older members of the household often had to stand in for parents to do the household chores. Relationships with neighbours and friends also changed. Some participants explained neighbours were integral in helping them through their recovery and that they often gave them food, money and water :“I am still feeling weak and have no money to buy food because I am not working, so some neighbours give me food to eat, but you understand how difficult it is to depend on someone else’s kitchen.” Others explained that neighbours and friends stayed away following their illness and that they felt isolated and stigmatised:“The relationship with neighbours does change because of gossip, they start saying that it is because of your uncleanness…they end up avoiding you” (Man IDI participant who’s elderly brother had cholera)“I lived an isolated life during those hard periods of cholera cases.” However, participants from case households reported being more motivated to wash their hands, explaining this was because they recognised their vulnerability to disease. They also used handwashing as a way of countering any misperceptions from neighbours about their cleanliness. The latter concern seemed to prompt a range of demonstrative action around handwashing. For example, several participants said they have actively encouraged others to practice handwashing:“Though we got infected, we cannot feel discouraged from doing hygiene practices…we even try to improve it and we are telling our neighbours that they should keep on practicing hygienic behaviour because that is the only way to prevent cholera…And other people around here, when they see how clean you are, even if you got sick with cholera, they can decide to take you as their good example of cleanliness” One participant built a dedicated place for handwashing near the toilet after his daughter was admitted to hospital with cholera:“I realise that fighting this cholera disease is very serious these days and so I thought that once we are practicing hand washing, we will be able to prevent our family from this disease…So I decided I had to make the place for handwashing, a place that would be respected… Now the neighbours are just appreciating [the facility] and I am telling them to do the same as I did, but whether they agree or not, I will never give up with the practice.” However, households with cholera cases also felt their circumstances following their exposure to the disease made it more challenging to practice handwashing. This was due to their reduced physical health, their inability to collect sufficient water , increased hunger and malnutrition (due to loss of income), and difficulties affording other basic daily necessities, such as soap. A minority of participants were given a small bar of soap and six water purification tables upon discharge from the CTC. Participants who received these distributions, reported trying to use these sparingly to make them last as long as possible.Our research found that in the Eastern region of DRC, cholera is generally conceptualised as a persistent and commonplace health challenge but also one that is easily treatable. Frequent hygiene promotion sessions in this region have led to high levels of knowledge about the health impacts of cholera, its symptoms and recommended preventative behaviours. However, handwashing with soap was observed to be rare in this setting. By using theory-driven qualitative methods we were able to identify that this was because the psychological, social, and environmental behavioural determinants affecting handwashing in this context combined to limit the ability of individuals to improve their handwashing behaviour. Major barriers to handwashing related to the physical environment or behavioural setting included the absence of handwashing facilities, water scarcity, the unaffordability of soap, the small make-shift houses where displaced populations lived, the use of shared sanitation facilities, and the extended periods people spent working outside of the home. Handwashing behaviour was also hampered by broader experiences of living in poverty and within in a dynamic conflict-prone region with high rates of displacement and livelihood fragility. This was because handwashing was often deprioritised because of hunger, mental health challenges, the unpredictability of routines, and the lack of social support and sanctions around handwashing. The experiences of participants from case households indicated that in complex crises, cholera can have profound non-health impacts on a household’s income, productivity, social status, and sense of control–factors which in turn create additional barriers to handwashing.Despite low rates of actual handwashing practice, our research participants reported handwashing had increased as concerns about cholera were heightened. Many participants felt these changes in behaviour might be sustained beyond the outbreak. Prior literature has indicated self-reported handwashing behaviour tends to increase during outbreaks , 27–31, v. cholerae continues to be shed in their faeces. There is also some evidence that pre-existing and continued malnutrition during this period may prolong shedding Reviewers' comments:Reviewer's Responses to Questions <font color="black"> Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions? </font> The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes********** <font color="black"> 2. Has the statistical analysis been performed appropriately and rigorously?  </font> Reviewer #1: N/AReviewer #2: N/A********** <font color="black"> 3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. </font> The Reviewer #1: NoReviewer #2: Yes********** <font color="black"> 4. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes********** <font color="black"> 5. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Overall, this was well designed matched case-control study and the matching criteria were clearly laid out. However, there following would need some clarification.Methods1. Line 40-42 states the objective was to find out how the people responded to the cholera outbreak and how the outbreak affected their handwashing behavior. Is it right to assume that you wanted to find out if they washed their hands more often after the outbreak? How was this done/measured? was there a measurement of handwashing behavior before the outbreak to compare with that after the outbreak?ResultsFrom table 1, 21% lived in camps while the rest were lived in the community, was there a difference in water supply between the camps and community? Did this difference have any impact on the handwashing behavior?Summary and general commentsThank you for the privilege to review this well written paper. The authors carried out a matched case-control study to examine the hygienic behavior of the people of Kivu during the 2017 cholera outbreak. These results will be very useful for the cholera outbreak literature. I suggest revising for key elements in the methods and results which need a bit more clarification. Again I commend the authors for clarity of thoughts which allowed me to only focus on the science.Reviewer #2: Referee review for: "How does handwashing behavior change in response to a cholera outbreak? A qualitative case study in the Democratic Republic of Congo”This study uses a mixed methods approach to examine perceptions and behaviors associated with handwashing with soap among households that had experienced cholera and households that had not experienced cholera during a cholera outbreak. The outbreak took place in 2017 in a resource poor community in the eastern DR Congo where endemic and epidemic cholera exists. Health education messages involving biomedical information about the health benefits of handwashing had been disseminated over time in the study community. Study results suggest the handwashing with soap was rare during the outbreak among both case and control households despite self-reports of handwashing. The findings highlight that competing demands related to food consumption and livelihoods constrained people living in both case and comparison households from practicing handwashing. The authors conclude that severe psychological, social, and economic impacts that occurred in case households increased barriers to handwashing. The authors underline that handwashing strategies in contexts with endemic and outbreaks of cholera should consider emic understandings of disease and promote sustainable strategies that take into account social and physical environmental factors, including access to handwashing materials, that influence handwashing behavior.Comments1. Little information is provided on the cholera outbreak. It would be helpful to include a description of the outbreak including how long it lasted and the number of cases and deaths.2. The manuscript would benefit from a description of the messages provided to community members, including how messages were disseminated, the content of messages and how this changed during the epidemic, the frequency of dissemination, and the target audience. Were the messages focused on diarrhea prevention or were cholera-specific messages disseminated?3. It is not clear how focus group discussion participants were selected. Also, a description of the composition of the focus groups is needed.4. While the authors mention that some households participated in more than one method, there is no indication regarding the extent to which IDIs were carried out in households where structured observations took place. The methods section should provide a description indicating when more than one method was carried out in one household.5. Little is said about the differences between the IDP and local populations and how their living environments and daily routines affected exposure to interventions, perceptions of cholera, and handwashing practices. It would be helpful to include a description of the different social and physical environments, as well as exposure to messaging, to understand how this may have improved or constrained handwashing. Also, a description of differences in the way the outbreak impacted IDPs and the local population would be helpful.6. The study results suggest the cholera is a general term used to describe a range of diarrheal illness. However, there is no mention of how this may have influenced emic views and the cholera explanatory disease model related to disease severity, causal explanations, and transmission, as well as the perceived need to wash hands. In other words, to what extent were informants able to distinguish disease signs, symptoms, severity, causation, and transmission from other types of diarrheal illness.7. The authors rightly advocate for incorporating emic views and local constructions of disease into messaging and intervention strategies. However, the manuscript lacks some key information on the local explanatory disease models that could potentially be useful when informing strategies. I wonder whether some of this information was collected during the participatory methods that were not presented in the results. If so, it would be useful to include more details on the local explanatory model of cholera and how this may have affected handwashing behaviors. In that regard, what were the results of the participatory methods and how were the findings used.8. In the discussion, the authors cite literature suggesting that behaviors may change over the course of an outbreak. It would be helpful to know whether this research detected any behavioral changes over time as perceptions related to the disease and risk of exposure may have changed.Other commentsRecent research carried out in an IDP camp in North Kivu, which highlights similar findings related to the prioritization of competing needs over handwashing, should be mentioned.Some statements made in the first paragraph of the discussion section do not necessarily reflect the study findings. Examples include:- Cholera is conceptualized as a health challenge that is easily treatable- Handwashing was hampered due to the fact that the work took place in a conflict zone- Health promotion has led to high levels of knowledge related to the health impacts of cholera********** <font color="black"> 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. </font> Yes: Afoumbom Mildred TitaReviewer #1: Reviewer #2: Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. </div> While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, Attachmentreview of DRC handwashing mansucript.docxSubmitted filename: Click here for additional data file. 25 Feb 2022We thank both reviewers for their encouraging words of support and their constructive ideas for strengthening this manuscript. We have explained how we have taken on their feedback below: Reviewer 1 Comments: 7. Methods: Line 40-42 states the objective was to find out how the people responded to the cholera outbreak and how the outbreak affected their handwashing behavior. Is it right to assume that you wanted to find out if they washed their hands more often after the outbreak? How was this done/measured? was there a measurement of handwashing behavior before the outbreak to compare with that after the outbreak?Response: No this is an incorrect assumption. The objective is broader than what reviewer 1 assumes. Given that this was an exploratory study we did not start with a priori assumptions about how behaviour may change during an outbreak. The study focuses primarily on the determinants of behaviour (rather than behaviour itself) and how these may have been shifted by the outbreak. While observational measures of handwashing are included, they were only on a small sample and were designed to be qualitatively analysed rather than to indicate handwashing prevalence or frequency. Similarly participants self-reported their behaviour during many of the methods but we know that self-reported handwashing behaviour tends to overestimate practice. We have not adjusted the objective but rather have added some text in the limitations to better explain this. 8. Results: From table 1, 21% lived in camps while the rest were lived in the community, was there a difference in water supply between the camps and community? Did this difference have any impact on the handwashing behavior?Response: This information has been added to the text. In summary there were no substantial differences. 9: Summary and general comments: Thank you for the privilege to review this well written paper. The authors carried out a matched case-control study to examine the hygienic behavior of the people of Kivu during the 2017 cholera outbreak. These results will be very useful for the cholera outbreak literature. I suggest revising for key elements in the methods and results which need a bit more clarification. Again I commend the authors for clarity of thoughts which allowed me to only focus on the science.Response: We thank Reviewer 1 for their kind words of encouragement. Reviewer 2 Comments10. Little information is provided on the cholera outbreak. It would be helpful to include a description of the outbreak including how long it lasted and the number of cases and deaths. Response: While in DRC data on cases and deaths were shared with us by individual health centers, humanitarian organizations and the district government. However this data was inconsistent and hard to verify. Total case numbers and mortality from the national outbreak are available and have been included in the text but unfortunately due to these data issues we have not been able to include anything more specific for the region. ‘Outbreaks’ of cholera in DRC are, sadly unending, with this region reporting endemic cases every week of the year. Therefore and outbreak within the DRC context refers to a dramatic rise in cases as is typically experienced most years between about August and January. We have not given this detail in the text as we have already mentioned that our study took place during the peak of the outbreak during October and November. 11. The manuscript would benefit from a description of the messages provided to community members, including how messages were disseminated, the content of messages and how this changed during the epidemic, the frequency of dissemination, and the target audience. Were the messages focused on diarrhea prevention or were cholera-specific messages disseminated?Response: This was not assessed formally as part of this study. We have provided some additional description of this based on what participants told us. However we did conduct some parallel research with humanitarians about their programming throughout the Eastern region of DRC. This work has been submitted separately to Conflict and Health. 12. It is not clear how focus group discussion participants were selected. Also, a description of the composition of the focus groups is needed.Response: Information on sampling is provided under the heading ‘sampling’ in the methods section. We have expanded on this information about the composition of the FGDs under this section of the methods. 13. While the authors mention that some households participated in more than one method, there is no indication regarding the extent to which IDIs were carried out in households where structured observations took place. The methods section should provide a description indicating when more than one method was carried out in one household.Response: We have added this information under the IDI section. 14. Little is said about the differences between the IDP and local populations and how their living environments and daily routines affected exposure to interventions, perceptions of cholera, and handwashing practices. It would be helpful to include a description of the different social and physical environments, as well as exposure to messaging, to understand how this may have improved or constrained handwashing. Also, a description of differences in the way the outbreak impacted IDPs and the local population would be helpful. Response: As noted above we have added some information on the similarity of WASH circumstances between IDPs and host communities. In the text we had tried to describe the different housing of both populations. Since these camps were informal and located within the heart of the communities there was little difference in exposure to hygiene promotion, however those outside the center of town were slightly less exposed as is now noted in the text. We have also included more detail on employment. 15. The study results suggest the cholera is a general term used to describe a range of diarrheal illness. However, there is no mention of how this may have influenced emic views and the cholera explanatory disease model related to disease severity, causal explanations, and transmission, as well as the perceived need to wash hands. In other words, to what extent were informants able to distinguish disease signs, symptoms, severity, causation, and transmission from other types of diarrheal illness. Response: We have added content to the limitations section of the paper to address this point. We agree that this may have biased or distorted their reporting of experiences and perceptions towards the disease, however we don’t feel that this would have had a substantial bearing on understandings of disease causation and transmission given that this would be similar to many other diarrheal diseases (at least at a lay population level). Under the knowledge heading in the text I have added that while people viewed it as a catch all term people were able to easily identify cholera-specific symptoms such as rice-water stools. 16. The authors rightly advocate for incorporating emic views and local constructions of disease into messaging and intervention strategies. However, the manuscript lacks some key information on the local explanatory disease models that could potentially be useful when informing strategies. I wonder whether some of this information was collected during the participatory methods that were not presented in the results. If so, it would be useful to include more details on the local explanatory model of cholera and how this may have affected handwashing behaviors. In that regard, what were the results of the participatory methods and how were the findings used. Response: As Reviewer 2 suspected the research aims and methods did not specifically seek to comprehensively understand local explanatory disease models. However quite a lot was learned about this during some of the participatory methods used in the IDIs and FGDs . Most of these findings we have tried to capture in our results already. Specifically the following points are linked to this: - Cholera is seen as a challenge closely associated with poverty and hunger- Cholera is associated with people who are thin, pale and sickly .- Understandings of cholera were aligned with western biomedical models of understanding disease (this point has been added in text) given the history of health promotion in the region. - A minority of people felt that Congolese people are resilient to cholera or that if they got infected it wouldn’t cause them serious illness. - A minority of people felt that if you are too clean you would actually be more vulnerable to cholera. - A minority of people felt that it was in humanitarian and government interests for cholera outbreaks to continue.- People who had had cholera tended to be more likely to see cholera as being random. In terms of the second part of your question, participatory methods were used in throughout each of the IDIs and FGDs . Therefore all the content included in the results relates to these methods. 17. In the discussion, the authors cite literature suggesting that behaviors may change over the course of an outbreak. It would be helpful to know whether this research detected any behavioral changes over time as perceptions related to the disease and risk of exposure may have changed. Response: It is perhaps worth noting that the literature cited in relation to this point is limited by its quality. As noted in text that is because the majority of these studies rely on self-reported behaviour. As noted in the text the consensus across such texts is that behaviour seems to fluctuate over the course of many outbreaks and that one of the many factors driving this appears to be perceptions about the disease and the fear associated with it. This indeed is the premise behind Terror Management Theory which is referenced in the text. Given that we feel we have already explained this literature and its implications we have not added anything to this section. 18. Recent research carried out in an IDP camp in North Kivu, which highlights similar findings related to the prioritization of competing needs over handwashing, should be mentioned. Response: We have added in some additional content into the discussion section based on the work of Blum et al 2019 and Claude et al 2020.19. Some statements made in the first paragraph of the discussion section do not necessarily reflect the study findings. Examples include: a) Cholera is conceptualized as a health challenge that is easily treatable b) Handwashing was hampered due to the fact that the work took place in a conflict zone, c) Health promotion has led to high levels of knowledge related to the health impacts of cholera. Response: In relation to point a) this was mentioned in our results but has now been amended so this is a bit more explicit. In relation to point b) our discussion doesn’t quite claim that handwashing is hampered because of the conflict but rather the social and environmental factors that are associated with poverty, and the circumstances crated by conflict and displacement. We have tried to further clarify this nuance in the text. In relation to point c) We realize that while mentioned the high levels of knowledge in the results section we left out information on where participants felt this knowledge came from. This has now been added to the results section so this link is clearer.AttachmentResponses to reviewers.docxSubmitted filename: Click here for additional data file. 29 Mar 2022How does handwashing behaviour change in response to a cholera outbreak? 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Early studies by Boche et al. [Donanemab, a humanized antibody against the N-truncated pyroglutamate amyloid-β peptide at position 3 (AβpE3), was recently assessed in a phase 2 trial for safety, tolerability and efficacy after passive immunization of patients with early Alzheimer’s disease (AD) . The tree et al.  demonstre et al. .Antibodies against AβpE3 differ in their binding properties against soluble and aggregated conformations of AβpE3-42  react wit test p < 0.001). In Down syndrome, the situation was similar. The overall plaque load was higher than in AD cases and all plaques were positive for pan-Aβ. The pan-AβpE3 positive plaques accounted for 49%, and for donanemab, only 34% . The staining in control cases did not differ. Regarding vascular staining (CAA), donanemab and pan-AβpE3 staining appeared similar to pan-Aβ positive CAA . Although donanemab reacted only with a fraction of amyloid plaques, it strongly detected the central core of plaques . Semi-quantitative analysis of plaques  further supported the quantitative analysis   is a maj.  demonstr. , 6, 11. .  as well . , clearlySupplementary file1 (DOCX 2203 KB)Below is the link to the electronic supplementary material.


Severe neonatal jaundice resulting from elevated levels of unconjugated bilirubin in the blood induces dramatic neurological impairment. Central oxidative stress and an inflammatory response have been associated with the pathophysiological mechanism. Cells forming the blood–brain barrier and the choroidal blood–CSF barrier are the first CNS cells exposed to increased plasma levels of unconjugated bilirubin. These barriers are key regulators of brain homeostasis and require active oxidative metabolism to fulfill their protective functions. The choroid plexus-CSF system is involved in neuroinflammatory processes. In this paper, we address the impact of neonatal hyperbilirubinemia on some aspects of brain barriers. We describe physiological changes in the neurovascular network, blood–brain/CSF barriers integrities, and CSF cytokine levels during the postnatal period in normobilirubinemic animals, and analyze these parameters in parallel in Gunn rats that are deficient in bilirubin catabolism and develop postnatal hyperbilirubinemia.14C]-sucrose permeability measurement. CSF cytokine levels were measured by multiplex immunoassay. The choroid plexus-CSF system response to an inflammatory challenge was assessed by enumerating CSF leukocytes.Gunn rats bearing a mutation in UGT1a genes were used. The neurovascular network was analyzed by immunofluorescence stereomicroscopy. The integrity of the barriers was evaluated by [In normobilirubinemic animals, the neurovascular network expands postnatally and displays stage-specific regional variations in its complexity. Network expansion is not affected by hyperbilirubinemia. Permeability of the blood–brain and blood–CSF barriers to sucrose decreases between one- and 9-day-old animals, and does not differ between normobilirubinemic and hyperbilirubinemic rats. Cytokine profiles differ between CSF and plasma in all 1-, 9-, and 18-day-old animals. The CSF cytokine profile in 1-day-old animals is markedly different from that established in older animals. Hyperbilirubinemia perturbs these cytokine profiles only to a very limited extent, and reduces CSF immune cell infiltration triggered by systemic exposure to a bacterial lipopeptide.The data highlight developmental specificities of the blood–brain barrier organization and of CSF cytokine content. They also indicate that a direct effect of bilirubin on the vascular system organization, brain barriers morphological integrity, and inflammatory response of the choroid plexus-CSF system is not involved in the alteration of brain functions induced by severe neonatal jaundice.The online version contains supplementary material available at 10.1186/s12987-022-00332-0. The brain develops in a controlled environment secured by the blood–brain barrier located at the endothelium of the cerebral microvessels and by the blood–CSF barrier located at the epithelium of the choroid plexuses , 2. The We therefore questioned whether pathologic neonatal hyperbilirubinemia alters the postnatal neurovascular network development, and blood–brain and blood–CSF barrier integrity, which would in turn impact brain maturation and functions. Because the choroid-plexus–CSF system is central to neuroimmune interactions and to the initiation of neuroinflammation, we further hypothesized that pathological hyperbilirubinemia would elevate CSF cytokine levels, sustaining neuroinflammation and impacting brain development. To test these hypotheses, we used the Gunn rat characterized by a deficiency in bilirubin conjugation induced by a spontaneous mutation in the 4th common exon of the UDP-glucuronosyltransferase UGT1a enzymes . HomozygIn this paper we evaluated the complexity of the vascular network, the integrity of blood–brain interfaces using sucrose as a polar, non-metabolized tracer, and the cytokine content in plasma and CSF during postnatal development in normobilirubinemic rats in order to increase our knowledge on these parameters which remain little characterized during development. In particular, developmental concentration profiles of most cytokines in CSF are currently unknown. In parallel, we examined whether these three parameters are modified in hyperbilirubinemic Gunn animals. We also assessed whether the neuroinflammatory response of the choroid plexus-CSF system to a bacterial stimulus was exacerbated in hyperbilirubinemic newborn rats. Altogether, the data point out important early postnatal functional changes in the blood–brain interfaces/CSF system, and show that among brain cells, those forming the blood–brain interfaces do not appear to be primary targets of bilirubin toxicity.Gunn rats were obtained from the SPF animal facility of the University of Trieste and the colony was maintained at the Lyon Neurosciences Research Center animal facility. Animal homozygous for the mutation (jj genotype) were characterized by a yellow skin color clearly visualized shortly after birth, in contrast to wildtype animals (NN genotype) and animals bearing the heterozygous mutation (Nj genotype). They also developed a mild cerebellar hypoplasia , 0.2 (9-day-old), 0.1 (17-day-old) mg of pentobarbital/g as an 1/8 dilution in saline), blood was withdrawn on heparin by intracardiac puncture, and the cisterna magna was exposed. CSF was sampled using a glass micropipette, collected in a low binding tube, and its volume measured. Heparinized blood samples were centrifuged at 5000 rpm and plasma were collected. Fluids were frozen at − 80 °C until use. Following fluid sampling, the brains were dissected out of the cranial box, quickly frozen in isopentane at − 45 °C, and stored at − 80 °C until used for immunohistochemistry.14C]-sucrose used as a polar marker of the barrier cell integrity. The influx constants, apparent brain Kin (App Kin) and true CSF Kin for [14C]-sucrose were determined as described previously [14C]-sucrose was injected intraperitoneally to animals under slight isoflurane anesthesia. Plasma and CSF were sampled after 20 min as described above. Their radioactive content was measured by liquid scintillation counting in a TRICARB 4910 TR (Packard). The brain was collected and frozen on dry ice. Pieces of cortex, pons, and cerebellum, free of meninges, were dissected from the frozen brain in a − 20 °C chest freezer, weighted, and digested in 1 M sodium hydroxide overnight at 4 °C. The digested tissues were further homogenized with a micro-pestle before being analyzed for radioactive content. [14C]-Sucrose plasma concentration x time area-under-the-curves (AUCs) from 0 to 20 min (AUC 0→20 min) were calculated for each individual [14C]-sucrose plasma concentration at 20 min, using the shape of a generic time x sucrose plasma concentration curve obtained by the litter-based method on animals sacrificed at various time points between 2 and 30 min. As no differences were found in sucrose plasma concentrations over time between males and females, the values were pooled to build up the generic sucrose plasma concentration x time curves for 1-day-old and 9-day-old animals. The curves were built separately for NN/Nj and for jj animals, as the jj genotype slightly affected the shape of the curves at both ages −1. min−1, i.e. 10–3 min−1.20min is [14C]-sucrose concentration in discrete brain regions at 20 min, expressed as µl . g−1 . min−1.The permeability of the blood–brain barrier  and of the blood–CSF barrier was measured towards [eviously , 25. Bribrainin  values were corrected for the amount of sucrose associated to the brain vascular space to generate true Kbrainin , using the following plasma volume determined as described infra: Cerebral cortex: 5.0 ± 1.4, Pons: 5.7 ± 0.7, Cerebellum 12.6 ± 0.6 µl/g, .For 9-day-old animals, App K2HPO4 12; KH2PO4 2; pH 7.4) . Rat IgG concentrations were measured in plasma and brain homogenate samples by ELISA . The vascular volume, expressed as µl/g, was calculated as being the difference of the IgG amount per gram of tissue between non-perfused and perfused animals, divided by the IgG plasma concentration in the non-perfused animals.After pentobarbital anesthesia, a blood sample was collected by intracardiac puncture and plasma was frozen. Half of the animals from one litter underwent a transcardiac perfusion with a physiological solution . Areas with large vessels were omitted from the study by introducing a filter in the analysis that takes into account only vessel profiles whose surface area were comprised between 10 and 25,000 µm2. For each animal and each area of analysis, data from the 3 serial sections were averaged. The number of microvessel segments per field of observation and the vessel area per field of observation were used as proxies for the quantification of microvascular network development and vascular volume, respectively.Rat brains were cut at − 20 °C using a NX50 Microm Microtech cryostat. Ten-µm thick sagittal sections were collected on glass slides and stored at − 20 °C until used for immunostaining. Sections were fixed at room temperature in 4% paraformaldehyde in PBS for 10 min, then blocked for 1 h in 1% BSA, 8% normal goat serum, 0.3% Triton in PBS. After overnight incubation at 4 °C with the anti- RECA-1 antibody  in PBS supplemented with 1% BSA, 1% normal goat serum, and 0.3% Triton, sections were washed 3 times with PBS supplemented with 0.3% Triton, and incubated with secondary Alexa Fluor 488-conjugated goat antirabbit antibodies  for 1 h at room temperature. Sections were counterstained with DAPI for 10 min and then mounted in aqueous medium. Negative controls were performed by omitting the first antibody. For each area of analysis, three serial sections were examined at X160 with a fluorescence stereo zoom microscope (Zeiss Axio Zoom.V16) equipped with an AxioCam 503 camera, and image acquired through zen 2.3 Pro software (Zeiss Microscopy). Quantitative analysis was done using ImageJ software to determine the number of vessel segments per field as well as the vessel surface area  according to the manufacturer’s protocol. Sample volume needed for plasma and CSF analyses, in singlicate, at the minimal required dilution recommended by the manufacturer (ie 2), was 15 µl. When CSF sample withdrawn from one animal was smaller than 15 µl, samples from 2 animals were pooled, and plasma of these two animals were pooled in the same proportions. Analyte quantification was performed on the Bioplex-200 platform.Eight-day-old rats were injected intraperitoneally with 1 mg/kg PAM3CSK4 , or 0.9% saline. CSF was sampled 14 h later , and totStatistical tests and significance are described in figure legends when appropriate.Cerebral endothelial cell proliferation leads to prominent extension and growing complexity of the vascular network postnatally . To testin values were obtained by correcting apparent Kin values for the amount of sucrose remaining in the brain vascular space (see methods). This correction could not be made for 8-h-old animals, as the low difference in cerebral IgG levels between perfused and non-perfused animals prevented an accurate determination of the vascular volume at that stage. The blood–brain barrier permeability toward sucrose was not different between male and female normobilirubinemic rats  decreased from 2.34 to 1.14 in the cortex, from 3.26 to 1.77 in cerebellum, and from 2.63 to 1.43 in the pons between 8-h-old and 9-day old animals , despite the concurrent growth of the vascular network in all these structures. Similarly, the blood–CSF barrier permeability to sucrose did not differ between male and female  and sustained (9 days) barrier cell exposure to bilirubin, respectively. For 9-day-old animals, true brain tissue Kats Fig. A, B. As 27 Cytokines including 5 chemokines were investigated by the multiplexed antibody-based technology in the CSF and plasma of NN, Nj and jj animals.There was no difference between males and females in the levels of immune mediators in either the plasma or the CSF at all stages of development investigated and in any of the genotype groups. Data from both sexes were therefore pooled in the following analyses.). In plasma, the interindividual variability was higher than in CSF. Developmental variations were restricted to IL-1β present at a low level in 1-day-old animals as compared to levels measured at later stages of development, and to IL-18, MCP-1, IP-10, VEGF whose levels were higher in 1-day-old animals than in 9- or 17-day-old animals.  animals in 1-, 9- and 17-day-old animals is shown in Additional file 4The comparative analysis of normo- and hyperbilirubinemic animals revealed no difference in the CSF or plasma levels of cytokines between NN and jj animals in 1-day-old rats (i.e. shortly after the free bilirubin plasma level increases in jj rat pups), (not shown). The differences closest to statistical significance were observed for CSF levels of IL-1β (p < 0.077) and RANTES p < 0.053), both being lower in jj than in NN animals. In 9-day-old rats that underwent (when carrying the jj genotype) sustained exposure to bilirubin, no difference in cytokine concentration was observed in CSF between NN, Nj and jj animals (not shown). In the plasma of these animals, only the level of MCP-1 was significantly but slightly (1.3 fold) increased in Nj animals as compared to NN animals. This increase was not observed in hyperbilirubinemic jj animals (Anova followed by Tukey multiple comparisons test). In 17-day-old animals, changes in cytokine levels between animal groups were also limited, both in CSF and in plasma. In CSF, Nj animals had overall decreased levels of cytokines by comparison to NN animals, as exemplified in Fig. 3, both bSystemic exposure of developing animals to the gram negative bacterial lipopeptide P3C leads to an infiltration of white blood cells into the CSF through the choroid plexuses . To asseThis work first brings forwards developmental specificities of the brain vasculature and shows that the latter is not impacted by hyperbilirubinemia. The analysis of the number of vessel segments and of the vessel surface per surface area of thin brain sections provides an index of the microvascular network complexity and volume, respectively. It shows that the parenchymal microvascular network undergoes extensive growing complexity between 9 and 18 days after birth in the cerebral cortex of rodents as previously described in pioneer studies , 28 and The cerebral capillaries have both scaffold and paracrine signaling functions that modulate the development and differentiation of multiple neural cell populations, including those migrating radially from neurogenic niches to cortical areas , 33. Conin constants which represent the fraction of the blood-borne molecule that crosses barriers to reach the CSF and neuropil. The distribution of polar tracers such as sucrose into the CSF and brain depends on the efficacy of tight junctions to seal the paracellular pathway between blood and brain/CSF compartments, the formation of transcytotic pathways across barriers cells, the surface area available for exchange, the rate of CSF-extracellular fluid exchange, the brain-to-blood backflux, and the CSF turnover rate. The impact of the last two parameters is minimized in our 20-min experimental setting. Endothelial and choroidal junctions are already tight at birth [in values towards sucrose have been measured in 1-day-old rats as compared to 9-day-old animals, not only in CSF but also in tissue despite the less developed vascular network in 1-day-old animals. The reasons for this age-related decrease in apparent permeability are not fully elucidated and have been discussed elsewhere [The blood–brain permeability to sucrose measured in different brain regions is not altered by bilirubin either, whether sucrose transfer across the blood–brain barrier is measured a few hours after bilirubin concentration rises in the blood, or following a chronic exposure of endothelial cells to bilirubin, such as in 9-day-old rats. Similarly, no change in the blood–CSF permeability to sucrose is observed following choroidal exposure to high plasma concentrations of bilirubin. Barrier permeabilities were evaluated by measuring sucrose kat birth , 36, butat birth , 38. Ovelsewhere , 36, 39.lsewhere . As the lsewhere .Alteration of the blood–brain barrier involving tight junction disorganization has been reported at the adult stage in animal models of hepatic encephalopathy in which serum bilirubin levels are abnormally high –44. The The choroid plexuses are rapidly activated following systemic inflammation and the CSF supports pro-inflammatory mediator circulation . We deteTo our knowledge our study is the first to report specific CSF developmental profiles for these immune cytokines in the healthy rat during the postnatal period. The physiological meaning of the changes in CSF concentration for each individual cytokine needs to be investigated, as is their cellular origin. The choroid plexus epithelial cells, ependymal cells including tanycytes and immune and non-immune cells harbored in the ventricular, subarachnoid, and cisternal spaces are potential sources, in line with the role attributed to the choroid plexus-CSF system in securing brain development , 63. AstConsidering the proximity of CSF compartments with periventricular and hippocampal stem cell niches which harbor the main cellular targets of cytokines during development, CSF-borne cytokines are likely to fulfill important endocrine signaling. An alteration of the developmental profile of these bioactive molecules in CSF, induced by perinatal injuries such as sepsis, may not only participate in spreading systemic inflammation to the brain, but also directly lead to unbalanced neuronal signaling. This would ultimately alter normal neural network organization and lead to neurodevelopmental diseases , 66, 67.In conclusion we describe postnatal developmental changes in the vascular network formation and in blood–brain interfaces permeability, and show that none of these parameters is impacted by acute or chronic hyperbilirubinemia mimicking the human postnatal pathologic jaundice. Our study also provides evidence for a central and a peripheral pool of cytokines that are of different origins and a developmental regulation of several CSF-borne cytokines, that suggest a specific role of selected cytokines in neurodevelopment. The influence of hyperbilirubinemia on both CSF and plasma cytokines is marginal, and bilirubin reduces innate immune cell infiltration in the brain of inflamed animals. Altogether our data indicate that a direct effect of bilirubin on the vascular system organization and brain barriers integrity, and the inflammatory response of the choroid plexus-CSF system to bilirubin, are not involved in the alteration of brain maturation induced by neonatal jaundice.Additional file 1. Additional figures S1, S2 and S3.Additional file 2. Additional figures S4, S5 and S6.


Cryptococcus neoformans infection, IL-1ra, IL-9, and VEGF were significantly elevated in immunocompetent cases. Cytokine levels usually reached peaks within the first 2 weeks of antifungal treatment and gradually decreased over time. PCA demonstrated a co-correlated CSF cytokine and chemokine response consisting of Th1, Th2, and Th17 type cytokines. Prognostic analysis showed that higher scores for the PCs loading pro-inflammatory cytokines, IFN-γ, TNF-α, and IL-12; and anti-inflammatory cytokine, IL-4; and chemokines, Eotaxin, FGF-basis, and PDGF-bb; as well as lower scores for the PCs loading RANTES were associated with disease severity, as defined by a Glasgow Coma Scale of <15 or death. In conclusion, combined inflammatory responses in CSF involving both pro- and anti-inflammatory cytokines and chemokines are upregulated in HIV-uninfected CM, and associated with disease severity.The cerebrospinal fluid (CSF) immune responses in HIV-uninfected cryptococcal meningitis (CM) have not been well studied. In this study, we aimed to explore the phenotype of CSF immune response during the course of disease and to examine relationships between phenotypes and disease severity. We profiled the CSF immune response in 128 HIV-uninfected CM and 30 pulmonary cryptococcosis patients using a 27-plex Luminex cytokine kit. Principal component analyses (PCA) and logistic regression model were performed. Concentrations of 23 out of 27 cytokines and chemokines in baseline CSF were significantly elevated in CM patients compared with pulmonary cryptococcosis cases. In CM patients with  Cryptococcal meningitis (CM) with high mortality and morbidity poses a great public health challenge worldwide. It occurs not only in those with HIV/AIDS and natural or iatrogenic immunosuppression but also in apparently immunocompetent individuals. CM causes 15% of AIDS-related deaths and results in approximately 181,100 deaths annually –5. The mCryptococcus are crucial for infection control. T helper 1 (Th1) cell responses involving the release of pro-inflammatory cytokines interferon (IFN)-γ, tumor necrosis factor α (TNF-α), interleukin (IL)-12, lead to classical activation of macrophages to kill Cryptococcus  test   or died within 2 weeks after initial antifungal treatment (AFT) were considered as severe cases, otherwise as non-severe cases. Lumber puncture was done at physician discretion, according to clinical need. Surplus CSF samples were collected at baseline, before or within 7 days after AFT, and during the follow-ups. A total of 505 CSF samples were obtained.A total of 128 HIV-negative adults age ≥19 years) with a first episode of CM were consecutively enrolled from January 2014 to December 2017 in Huashan Hospital. All patients met ≥1 of the following: positive CSF smear for  years wiThirty cases of proven or probable HIV-uninfected pulmonary cryptococcosis were included as control group. Proven cases were diagnosed through histopathology or tissue culture . A probaCSF samples were centrifuged at 600 g for 10 minutes within 1 hour of collection. The supernatants were frozen at -80°C until analysis using Bio-Plex Pro™ Human Cytokine 27-plex Assay kit and Bio-Plex® 200 system . All samples were tested at first thaw and being run at 1:2 dilution.Demographic and clinical data were recorded on standardized forms. All participants provided written informed consent. This study was approved by the Ethic Committee/Institutional Review Board (HIRB) of Huashan Hospital, Fudan University.Continuous variables were compared with Mann-Whitney U test and categorical variables by χ2 analysis or Fisher’s exact test. Concentrations of baseline CSF cytokine and chemokine were analyzed by principal component analysis (PCA), resulting in linearly uncorrelated principal components (PCs) by reducing dimensionality of such datasets. Associations between PC scores and clinical variables were examined by Pearson’s correlation test. Logistic regression model was constructed using stepwise regression with the objective of determining the independent factors for disease severity. All variables with a value of P < 0.05 in the univariate analysis were included. The results of the multivariate analysis were expressed as odds ratio (OR) and the corresponding 95% confidence intervals (CIs). Data were analyzed with the use of SPSS, version 17.0 (SPSS Inc.); and Prism, version 8 (GraphPad Software). All tests were two-sided, and significance was defined as P < 0.05.Cryptococcus strains were isolated, of which 83 (79.8%) were identified as C. neoformans and 5 (4.8%) were C. gattii. Forty-four (42.3%) patients had extracranial lung  or lymph node  infection. Mortality at 2-weeks was 7.7% and 34 (32.7%) developed abnormal mental status. Of 30 pulmonary cryptococcosis cases, 13 (43.3%) were diagnosed by surgical pathology or percutaneous biopsy. Five out of 10 immunocompromised patients had solid tumor. No significant difference was identified in demographics between 2 groups.Baseline CSF samples were available from 104 out of 128 CM cases enrolled, and from 30 pulmonary cryptococcosis cases, whose demographic and clinical characteristics are compared in C. neoformans, IL-1ra (P = 0.036), IL-9 (P = 0.027), and VEGF (P = 0.017) were significantly elevated in immunocompetent cases (Concentrations of baseline CSF cytokines are shown in PCA was used to determine the variance of co-correlated cytokine and chemokine measurements among 104 CM patients. The majority of variance (77.3%) in dataset could be explained by the first 4 PCs exceeding an eigenvalue of 1  from 128 CM cases with an average follow-up of 92 days. Most cytokines reached peaks within 2 weeks of AFT and gradually decreased over time. The trends were more obvious when cytokines were categorized and compared by disease courses, briefly described as CM1 (within 2 weeks of AFT), CM2 (2–10 weeks after AFT), and CM3 (after 10 weeks of AFT). One hundred and eighty-six (36.8%) samples were collected in the period of CM1, 175 (34.7%) were in CM2, and 144 (28.5%) were in CM3. The median levels of 24 out of 27 CSF cytokines in CM2 were significantly lower than those in CM1 (P < 0.05). In CM3, cytokine levels declined further, albeit with slower decline rates. Median levels of 10 cytokines in CM3 reduced to baseline control levels while the other 17 cytokines were still significantly higher than control levels. Moreover, GM-CSF and MCP-1 did not significantly change throughout the course of the disease, and the ratio of IFN-γ/IL-10 and IL-12/IL-10 progressively increased during follow-up. Cytokines that increased over 10 times compared to controls in CM1 all fell by over 50% in CM2: IL-6, IL-1ra, IP-10, IL-8, IL-1β, IL-10, and TNF-α. The most significant decrease was found in IL-6, exhibiting a fall by 95%. Levels of G-CSF and IFN-γ, presenting 8.1 to 9.2-fold increase in CM1, also dropped by over half in CM2. Chemokines , increased 6.0 to 9.8 times at baseline, declined relatively slow Figure 3Associations of baseline CSF cytokines and chemokines as well as demographic, clinical and laboratory characteristics with disease severity were explored Table 2.Cryptococcus in HIV-uninfected patients.Our study utilized a well-characterized large cohort of HIV-uninfected individuals with CM to do profiling studies on CSF cytokines and chemokines. Levels of baseline CSF cytokines and chemokines were significantly elevated in CM patients compared with pulmonary cryptococcosis cases and could be resolved into 4 principal components, 2 of which were associated with severe disease, as defined by altered mental status or death. Our data provide important additional insight into the immune response against C. neoformans infection, IL-1ra, IL-9, and VEGF were significantly elevated in immunocompetent cases, indicating the underlying influence of immune status in HIV-uninfected hosts.Our data showed that baseline CSF cytokines were significantly elevated in cryptococcosis with CNS involvement . NotablyCryptococcus infection were also reported to have defective macrophage signaling, suggested by STAT5-blocking antibodies to GM-CSF, with retention of normal T-cell activity  stimulation, and IL-4 levels progressively decreased during treatment, also indicating that a progressive shift favoring a pro-inflammatory pattern is crucial in controlling cryptococcal infection  of Huashan Hospital, Fudan University. The patients/participants provided their written informed consent to participate in this study.Y-KJ, R-YW, TH, and L-PZ conceived, designed, and managed the study. L-PZ, R-YW, J-HC, XW, and H-ZZ supervised patient recruitment. Y-KJ, R-YW, L-HZ, J-HC, YL, R-SZ, and W-JQ contributed to sample collections. Y-KJ, R-YW, L-HZ, and J-HC conducted the experiment for cytokine and chemokine detection. Y-KJ, R-YW, L-HZ, and YL contributed the microbiological data. R-SZ, W-JQ, XW, and H-ZZ contributed to the epidemiological investigations. Y-KJ, R-YW, and L-HZ analyzed the data. Y-KJ, R-YW, L-HZ, TH, and L-PZ drafted the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the National Key Research and Development Program of China (2021YFC2300400), Shanghai Municipal Science and Technology Major Project, and the National Natural Science Foundation of China (Grant No. 81971911).We thank all the patients who participated in this study and all the health workers in Huashan Hospital, Fudan University.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Epstein–Barr virus (EBV), positive in 52.5% of samples, was the most commonly reported pathogen, followed by cytomegalovirus (CMV), John Cunningham virus (JCV), torque teno virus (TTV), cryptococcus neoformans (CN), toxoplasma Gondii (TE), and mycobacterium tuberculosis (MTB). 76.2% of the EBV identification and 54.2% of the CMV identification were not considered clinically important, and relative less sequence reads were reported in the clinical unimportant identifications. The clinical importance of the presence of TTV in CSF was not clear. Detection of JCV, CN, or TE was 100% suggestive of specific CNS infection, however, 60% of the MTB reports were considered contamination. Moreover, of the 44 (55%) mixed infections reported by mNGS, only 4 (5%) were considered clinical important, and mNGS failed to identify one mixed infection. Additionally, except for MTB, CSF mNGS tended to have high sensitivity to identify the above-mentioned pathogens . Even all the diagnostic strategies were evaluated, the cause of neurological symptoms remained undetermined in 6 (7.5%) patients. Overall, our results suggest that mNGS is a very sensitive tool for detecting common opportunistic CNS pathogen in HIV-infected patients, although its performance in CNS tuberculosis is unsatisfactory. EBV and CMV are commonly detected by CSF mNGS, however, the threshold of a clinical important detection remains to be defined.Although considerable interest in metagenomic next-generation sequencing (mNGS) has been attracted in recent years, limited data are available regarding the performance of mNGS in HIV-associated central nervous system (CNS) infection. Here, we conducted a retrospectively analyzing of the cerebrospinal fluid (CSF) mNGS reports and other clinical data from 80 HIV-infected patients admitted to the Second Hospital of Nanjing, China from March, 2018 to March, 2022. In our study, CSF mNGS reported negative result, mono-infection, and mixed infection in 8.8, 36.2, and 55% of the patients, respectively.  Central nervous system (CNS) opportunistic infection remains an important cause of morbidity and mortality in HIV-infected severely immunocompromised patients . Timely Fortunately, the limitations of traditional microbiological tests may be overcome by recently developed metagenomic next-generation sequencing (mNGS). This novel strategy identifies nucleic acids from clinical samples in a target-independent way, and thus could theoretically detect all the pathogens in a single run . The cliA recently study suggested that CSF mNGS may be a useful tool for the diagnosis of CNS infection among HIV-infected patients . The stuThis study included 80 HIV-infected patients hospitalized at the department of infectious diseases in the second hospital of Nanjing, China from March 2018 to March 2022. The setting is a tertiary referral hospital and is a designated hospital that provides HIV care. The inclusion criteria for this study were hospitalized HIV patients: (1) with neurological symptoms; (2) CD4 count less than 200 cells/μl; and (3) having CSF mNGS reports. The patients were given conventional microbiological tests. In addition, CSF samples were sent for PMSeq-DNA, which is a commercial mNGS service targeting pathogens’ DNA sequences offered by BGI-Shenzhen . Fresh CBefore nuclear acid extraction, CSF sample was mixed with enzyme and glass beads, and was vortexed vigorously at 2,800–3,200 rpm for 30 min. Then, total DNA was extracted from 300 μl CSF sample using TIANamp Micro DNA Kit . DNA libraries were constructed by DNA-fragmentation, end-repair, adapter-ligation, and PCR amplification. After quality control with Agilent 2100, libraries were sequenced by BGISEQ-50 platform. Low-quality sequence reads and sequence reads mapped to the human reference genome (hg19) were removed. The remaining data were classified by aligning to microbial genome databases covering bacteria, fungi, viruses, and parasites. The reference databases could be downloaded from NCBIWe collected the demographic, clinical, laboratory, and imaging data of the patients from an electronic health record system. The electronic report of CSF mNGS was provided by BGI-Shenzhen, and pathogen list and the sequence reads were extracted from each CSF mNGS report. The final diagnosis was made by the expert panel through evaluation of the all-available clinical data, including clinical features, conventional microbiological tests, mNGS result, and the response to treatment. The clinical interpretation of the mNGS report focused on: (1) did a positive detection necessitates antimicrobial treatment? and (2) did a negative report guaranteed the excluding of specific CNS infection?p values were two-sided and the significance level was set at 0.05.Categorical variables were described as frequencies and proportions. Continuous variables were expressed by medians with interquartile ranges (IQR). Comparisons among different groups were done using Mann–Whitney U test or Jonckheere-Terpstra test, when appropriate. 95% CIs for sensitivity, specificity, positive predictive value, and negative predictive value were calculated according to the Wilson intervals method described by Lawrence D. Brown in 2001 . StatistThe clinical, laboratory, and imaging characteristics of the patients were summarized in Epstein–Barr virus (EBV) was the most common pathogen detected by CSF mNGS, being positive in 42 (52.5%) of the 80 CSF samples. The next six common pathogens were cytomegalovirus (CMV), John Cunningham virus (JCV), Torque teno virus (TTV), Cryptococcus neoformans (CN), Toxoplasma Gondii (TE), and Mycobacterium tuberculosis (MTB), which were detected in 24 (30.0%), 13 (16.3%), 12 (15.0%), 8 (10.0%), 6 (7.5%), and 5 (6.3%) of the 80 CSF samples, respectively  gave negative report and 73 (91.2%) reported at least one pathogen. The presence of mixed pathogens was identified in 42(52.5%) patients . Overallectively .p = 0.041] or JCV . The number of sequence reads was similar between EBV and TTV . Finally, although VZV was only detected in two patients, high numbers of VZV sequence reads were detected in both patients.Variable numbers of sequence reads were reported by CSF mNGS, and seemed likely to be associated with the type of the pathogen . Althougp = 0.001, Of the 42 patients tested positive for EBV DNA, 5 (11.9%) had CNS lymphoma , 5 (11.9%) may possibly have EBV encephalitis (EBV was the only pathogen reported by mNGS), and 32 (76.2%) were not considered to have CNS diseases related to EBV infection. There was a trend of decreasing number of EBV sequence reads among patients with CNS lymphoma, EBV encephalitis, and clinically unimportant EBV detections  were detected positive. Serum anti-Toxoplasma IgG antibody was positive for the six patients with Toxoplasma Gondii sequence reads. All the eight patients with Cryptococcus neoformans sequence reads were confirmed to have cryptococcal meningitis using CSF cryptococcal antigen lateral flow assay.Molecular detection of MTB in the CSF is an important strategy for diagnosing tuberculosis meningitis. The presence of MTB DNA in CSF samples is suggestive of CNS tuberculosis . In the Treponema pallidum, Nocardia spp., and Chlamydophila psittaci) had ever been considered the etiological agents of CNS infections with 11 positive identifications in total. Among these, only one of the two positive identifications of Nocardia spp. was considered contamination. The other 16 pathogens were either considered as non-CNS pathogens (such as HBV) or environmental pathogens.The remaining 21 pathogens account for 35 positive identifications in 22 patients . Only fiCryptococcus neoformans, Toxoplasma Gondii, and MTB) in the CSF, regardless of the number of the sequence reads, were generally considered as clinically important, if not contaminated by other samples. Interpretation of the identification of EBV, CMV, and TTV was more complicated, since those viruses could persist in healthy individuals  samples. Of the seven most common pathogens detected by CSF mNGS, the presence of 4  patients. The finial composite diagnoses for the patients were shown in HIV-infected patients are vulnerable to CNS opportunistic infections, the most common disease among which are cerebral toxoplasmosis, progressive multifocal leukoencephalopathy, tuberculous meningitis, cryptococcal meningitis, and cytomegalovirus encephalitis . CNS lymAsymptomatic carry of TTV is very common in healthy individuals. Human pathogenicity of TTV has not been fully established . The titTreponema pallidum in two patients with neurological syphilis, the sensitivity for diagnosing neurological syphilis was not satisfactory. Taken together, although mNGS may be able to detect all kinds of pathogens from clinical samples, it has variable sensitivity for pathogen identification depending on the categories of the causative agents and the severity of the diseases.For a clinical perspective, detection of MTB, CN, and TE sequences in the CSF samples is generally regarded as etiological confirmation. In the present study, the sensitivity and specificity of CSF mNGS for diagnosing CNS CN and TE diseases both reach 100%. However, diagnostic performance of CSF mNGS for CNS TB was poor. The false positive detection of MTB sequences in CSF samples implies that mNGS could be also subjected to cross-contamination, a drawback that also has been encountered by traditional molecular diagnostic tests. In HIV-seronegative patients, the sensitivity of CSF mNGS for diagnosing tuberculosis meningitis ranges from 58.8 to 84.44% . Since MSeveral reasons could explain why the detection of JCV, VN, and TE from CSF samples was much reliable for diagnosing the related CNS diseases compared with that of the CMV and EBV. It is well known that CMV and EBV could establish life-long persistence and replicate at low subclinical levels in reservoirs . They maAspergillus spp. from CSF samples in two HIV-infected patients  patients. At present, there is no consensus standard for clinical interpretation of mNGS results. This process is not always objective and would inevitably introduce bias. For example, a recent study unexpectedly identified patients . Althougmportant . The autOur study was limited by retrospective design and relatively low sample size for specific CNS infection. The calculated sensitivity and specificity of CSF mNGS for diagnosing CNS infection should be interpreted with prudence. Due to the shortage of commercial diagnostic kits and the CSF sample volume restriction, it was not possible for us to validate every mNGS report with pathogen-specific PCR or other certified traditional microbiological tests. For those reasons, interpretation of the mNGS results is still challenging. Finally, the unbiased mNGS in our study only targeted the DNA elements mainly due to the high cost of mNGS, and therefore it may miss the identification of some RNA virus in the CSF samples. Whether adding RNA sequencing  to improve the diagnostic performance needs to be further explored. Despite those limitations, the finial diagnoses were clear for the vast majority (> 90%) of the cases when CSF mNGS was integrated into diagnostic panel. This finding suggests that CSF mNGS is a promising microbiological diagnostic test for CNS infections in HIV-infected patients.Metagenomics sequencing analysis is a “finding the needle in a haystack” screening technique and may be the most complex among all known laboratory-developed tests. It is a long road to standardization in a real clinical context, but to achieve better quality and higher reproducibility, efforts could be made in simple and easy wet-lab manipulations, for example, using library prep/PCR in one-tube techniques. Barcoding the library samples with unique dual-index combinations to minimize cross-talking, especially to protect those uncertain samples from reads generated by significant infectious samples. Make sure process controls are always included to further examine “kitome,” and “splashome” contamination batch by batch. It is recommended to monitor the variations of lab environmental microbiomes  in the mid or long-term. Additionally, to improve the test’s predictive value, a well-curated clinical microbe’s database and joint interpretation by an expert panel  are also needed.In conclusion, CSF mNGS is a very sensitive tool for detecting common opportunistic CNS pathogen in HIV-infected patients; however, its performance in CNS tuberculosis is unsatisfactory. CMV and EBV are commonly detected by CSF mNGS from CSF samples in HIV-infected patients, the clinical importance of which is likely to be associated with the burden of the viruses. Clinical interpretation of CSF mNGS results is still challenging because of sample cross-contamination, environmental microbial contamination and lack of consensus standard for distinguishing carrier state/colonization and infection. Those obstacles need to be addressed in future studies.The original contributions presented in the study are included in the article/ZH and ZP conceived and designed the study. YunZ, WZ, XY, and XL collected the data. CC, YC, HW, and ZH were involved in clinical interpretation of mNGS results and the clinical management of the patients. YunZ, WZ, XY, YuaZ, XW, and YW analyzed the data and drafted the manuscript. All authors contributed to the article and approved the submitted version.This study was funded in part by the National Key Research and Development Program of China (2019YFC1200500 and 2019YFC1200501) and Nanjing Medical Science and Technique Development Foundation (ZKX21037). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Coronavirus disease 2019 (COVID-19) results from SARS-CoV-2-induced hyperinflammatory immune response, orchestrated by granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF increases interleukin-6 (IL-6) levels, ultimately leading to increased C-reactive protein (CRP). The LIVE-AIR trial demonstrated that lenzilumab, the GM-CSF neutralizing antibody, improved the likelihood of survival without invasive mechanical ventilation (IMV) in hospitalized COVID-19 patients requiring supplemental oxygen but not IMV. This sub-analysis correlated levels of cytokines before and after lenzilumab treatment.LIVE-AIR was a phase 3, randomized, double-blind, placebo-controlled trial (NCT04351152). Patients hospitalized with COVID-19 pneumonia, requiring only supplemental oxygen, were randomized to receive lenzilumab  or placebo IV infusion, in addition to standard of care which included remdesivir and corticosteroids. Blood taken at baseline (BL) and subsequent to treatment through day 10 (D10) were obtained and analyzed by high sensitivity enzyme immunoassay for GM-CSF, IL-6, and CRP.e-transformed for all cytokines and biomarkers) were linearly correlated with higher baseline GM-CSF levels . Baseline CRP levels were linearly correlated with higher baseline IL-6 levels . GM-CSF levels decreased with lenzilumab treatment on day 1 (D1) which persisted through D10 (Table). In contrast, GM-CSF increased with placebo treatment. IL-6 levels decreased only with lenzilumab treatment. CRP following lenzilumab or placebo treatment decreased on D1 to similar levels and further decreased on D10 only with lenzilumab treatment.Baseline IL-6 levels |Humanigen, Inc: Grant/Research Support|Merck: unrestricted educational grant (to the institution)|ViiV: Advisor/Consultant Christopher Polk, MD, Gilead: Advisor/Consultant Jason Baker, MD, Gilead: Grant/Research Support|Humanigen, Inc: Grant/Research Support Vincent Marconi, MD, Gilead: Grant/Research Support.