The COVID-19 pandemic has likely affected the already high unmet need for family planning in low- and middle-income countries. This qualitative study used Andersen’s Behavioral Model of Health Service Use as a theoretical framework to explore the possible ways in which the COVID-19 pandemic, including the impact of a 3-month government mandated lockdown, might affect family planning outcomes in rural Uganda. A secondary aim was to elicit recommendations to improve family planning service delivery in the context of COVID-19.Between June and October 2020, we conducted four focus group discussions with men and women separately (N = 26) who had an unmet need for family planning, and 15 key-informant interviews with community leaders and family planning stakeholders. Data were analyzed using thematic analysis.We identified a significant disruption to the delivery of family planning services due to COVID-19, with potential negative effects on contraceptive use and risk for unintended pregnancy. COVID-19 had a negative effect on individual enabling factors such as family income, affecting service access, and on community enabling factors, such as transportation barriers and the disruption of community-based family planning delivery through village health teams and mobile clinics. Participants felt COVID-19 lockdown restrictions exacerbated existing contextual predisposing factors related to poverty and gender inequity, such as intimate partner violence and power inequities that diminish women’s ability to refuse sex with their husband and their autonomy to use contraceptives. Recommendations to improve family planning service delivery in the context of COVID-19 centered on emergency preparedness, strengthening community health systems, and creating new ways to safely deliver contractive methods directly to communities during future COVID-19 lockdowns.This study highlights the consequences of COVID-19 lockdown on family planning distribution, as well as the exacerbation of gender inequities that limit women’s autonomy in pregnancy prevention measures. To improve family planning service uptake in the context of COVID-19, there is a need to strengthen emergency preparedness and response, utilize community structures for contraceptive delivery, and address the underlying gender inequities that affect care seeking and service utilization. This study explored the potential impact of the COVID-19 pandemic and a 3-month government mandated lockdown on barriers to accessing family planning services in rural Uganda, and recommendations to improve service delivery in the event of future COVID-19 restrictions. Data were collected from four focus group discussions with men and women separately (N = 26) who had an unmet need for family planning, and 15 interviews with community leaders and family planning stakeholders. The delivery of family planning services was disrupted due to COVID-19, negatively affecting community members’ ability to access services, such as by reducing their income. COVID-19 also disrupted community and health system distribution of services, such as through a transportation ban and the suspension of all community-based family planning delivery through village health teams and mobile clinics. Participants felt that COVID-19 lockdown restrictions worsened intimate partner violence, and with men at home more, limited women’s ability to use contraceptives without their partner’s knowledge and resulted in more sex between partners without women being able to refuse. Taken together, these consequences were thought to increase women’s risk of unintended pregnancy. Recommendations to improve family planning service delivery in the context of COVID-19 centered on measures to improve the health system’s response to emergencies and to safely deliver contraceptive methods directly to communities during future COVID-19 lockdowns. The successful implementation of community-based family planning will depend on efforts to increase men’s acceptance of family planning, while addressing underlying gender inequities that diminish women’s ability to time and space pregnancy. Early in the COVID-19 pandemic, it was projected that the pandemic would result in a 10% reduction in contraceptive use and a significant rise in unintended pregnancies in low and middle-income countries (LMICs) .LMICs such as Uganda are particularly vulnerable to COVID-19 disruptions that affect reproductive health; Uganda has an already high fertility rate (5.45 children per woman), low contraceptive use (29.2%), and a high maternal mortality rate  . While tWhile it is expected that such restrictions have affected reproductive healthcare access globally , 9–11, rThis qualitative study uses Andersen’s Behavioral Model of Health Service Use (ABM) as a theoretical framework to explore the effect of the COVID-19 pandemic on barriers to family planning service delivery and use in rural Uganda immediately following a 3-month government mandated lockdown –25. The Predisposing factors include individual and contextual factors that lead to a predisposition of people to use services. Examples of individual factors associated with greater contraceptive use in Uganda and other LMICs include demographic and social characteristics, such as higher education levels and being in a committed/marital relationship , 29. IndContextual factors that predispose individuals to use health services include the demographic and social composition of communities, collective and organizational values, cultural norms and political perspectives , 27. FerEnabling factors are those that enable or impede use of health services. Individual enabling factors include an individual’s income, access to health insurance to pay for health services, and the price of health care. In Uganda, greater wealth is associated with greater contraceptive use , 36. COVAt the contextual level, community enabling factors include health system barriers, like waiting time for health care, quality of care, and the financial and organizational resources available within the community for health services, such as the rate of health insurance coverage, the relative price of goods and services, the distribution of health facilities and personnel, provider availability, and outreach and education programs , 27. ResPerceived need for health services at the individual level is defined as how people view and experience their own general health, functional state and illness symptoms. In contrast, evaluated need is an objective measurement or professional assessment of one’s health status. The ABM also defines need at the contextual level, including environmental need characteristics or environmental health conditions  and population health indices . Since the current study is a qualitative inquiry with community members, we focus on individual-level perceived need.Taken together, the ABM provides an appropriate framework to explore COVID-19’s effect on family planning barriers through the perspectives of community members and family planning stakeholders who have experienced or witnessed the effects first-hand. As displayed in Fig. This qualitative study aimed to understand the determinants of contraceptive use in a rural area of Butambala district in central Uganda as part of the formative phase of a pilot intervention. Given the onset of the COVID-19 pandemic during the study, we expanded the aims of the study to include the exploration of the impact of COVID-19 on barriers and facilitators to contraceptive use and factors influencing risk of unintended pregnancy, which is the focus of the current paper. Data were collected from June and October 2020 in a rural district of Butambala in central Uganda immediately following a 3-month government-mandated lockdown from March to June 2020. Restrictions during lockdown included stay-at-home orders and a curfew, a ban on public transportation, the closure of nonessential businesses and schools, and social distancing measures such as the ban of group gatherings. The study was carried out with support from the Butambala District Health Team, comprised of technical health officials responsible for the strategic planning and allocation of resources to meet the health needs of the community.Participants were recruited from three villages within the same rural district located approximately two hours from the capital city of Kampala. The population of each selected village is approximately 100 households each. All villages are largely Muslim and Christian, with polygamy commonly practiced. A governmental health center III (HCIII) located in the sub-county served the three villages, offering free contraceptives and individual and couples family planning counseling. HCIIIs offer condoms, oral pills, injectable contraceptives, intrauterine devices (IUDs), and implants. However, the contraceptives offered can vary across HCIIIs based on stock availability and the capacity of providers to offer long-acting reversible methods (LARCs). A General Hospital (HCV) in the district provides all the short-term reversible methods, LARCs, and non-reversible methods . In addition, the Village Health Team (VHT), a cadre of community health workers, serve as a liaison between the community and health facilities, and support community family planning efforts. VHTs distribute short-term methods directly in the community  and provide individual and group community education about family planning. In addition, an international nongovernmental organization, Marie Stopes International, works in the district to provide regular community outreach to distribute contraceptives in selected villages within the district, including the provision of LARCs and non-reversible methods.Methods used for data collection included focus group discussions (FGDs) and key informant interviews (KIIs). We conducted four FGDs with men and women (N = 26) recruited directly from one of the three villages with the support of a VHT. Groups were separated by age and gender to ensure participants could speak openly . Inclusion criteria included being from the selected communities, being of reproductive age , considering oneself married, speaks Luganda, not currently pregnant, and having an unmet need for family planning. An unmet need for family planning was defined as wanting to delay pregnancy for at least a year but not currently using an effective method of contraception. These criteria were chosen so that we could assess barriers to contraceptive use generally and specific to COVID-19 among community members at risk for unintended pregnancy and wanting to delay pregnancy, but not using a contraceptive method following the 3-month lockdown. Potentially eligible individuals recommended and introduced to study staff by the VHT completed a brief screening assessment to confirm eligibility with two research assistants. Research assistants obtained written informed consent from eligible and interested participants.Two facilitators experienced in qualitative research and trained in this study’s focus group protocol moderated the groups, with one leading facilitation and the other taking detailed field notes. The interview guide explored barriers and facilitators to contraceptive use generally and included questions specific to the effect of COVID-19 on pregnancy intentions, risk of unintended pregnancy, access to family planning services, and the ability to use contraception. It also elicited participants’ recommendations for ways to improve family planning delivery during future COVID-19 or other outbreaks/lockdowns. The facilitator conducted the focus groups in Luganda, the local language. The group sessions lasted approximately 90 min each and were audio recorded. Participants received light refreshments during the sessions, and 7,000 Ugandan Shillings (~ $2 USD) for their participation. The audio recordings of FGDs were translated from Luganda to English, translated verbatim, and reviewed by a second party.In addition, we conducted 15 KIIs with community leaders and family planning stakeholders. Key informants were identified in collaboration with the District Health Team, and recommendations from other local health stakeholders, such as VHTs and the HCIII In-Charges. We sought individuals from the community that could provide expert insight into the local health system, community norms, and other barriers or facilitators to contraceptive use. Thus, KII participants included midwives and other health workers involved in the provision of family planning, VHTs, and community leaders, including religious and elected leaders. We selected KII participants from all three villages and the respective HCIIIs. A trained interviewer conducted one-on-one, in-person interviews in English. Like the FGD guide, the interview guide aimed to elicit determinants of contraceptive use, the effects of COVID-19 on contraceptive use, and recommendations to improve service delivery, but questions were tailored for relevance for each KII. Individuals received 15,000 Ugandan Shillings (~ 4 USD) for their participation. Audio recordings were transcribed and double-checked by a second party. The data that support the findings of this study are available from the corresponding author upon reasonable request.Data were analyzed thematically . Study tTable The 15 KII participants are listed in Table Study participants described significant disruption to the delivery of family planning services and barriers to accessing health services due to COVID-19. These effects were mainly related to the ramifications of COVID-19 lockdown or prevention measures, as opposed to direct effects of COVID-19. The COVID-19 lockdown measures had effects on individual  and community enabling factors . Health workers and community members confirmed COVID-19’s direct effect on health service utilization among the community due to fear of infection. Contextual predisposing factors, including poverty and those related to gender inequity, worsened the effect of COVID-19 lockdown on barriers to contraceptive use. Taken together, the data supports COVID-19 prevention measure’s effect on barriers to contraceptive use and other factors that might increase the risk of unintended pregnancy . These findings are detailed below, and depicted in Fig. poverty and gender inequity. For example, the broader context of community poverty exacerbated the effect of COVID-19 on the enabling factor of income (discussed in the next section). As one participant in the women’s focus group (age 36) explained, existing problems related to poverty were made worse by COVID-19:Respondent: Where [we] operate from to earn [is] locked up. Even if you want to go to the village, it will not work; COVID disorganized everything. Free land is not there anymore, poultry was attacked by different diseases especially the local ones, which we used to operate as free range. The goats were also attacked by several diseases. There is no place where someone can say they can work from or deal in and get some money. All things are disorganized.Moderator: Have all these things come because of COVID 19 or…?Respondent: No, they have been there but then COVID has just worsened it all.COVID-19 lockdown restrictions exacerbated existing contextual predisposing factors related to gender inequity. Men’s opposition to family planning, reinforced by numerous gender norms linking men’s status to large family size, was one of the most salient barriers to women’s contraceptive use irrespective of COVID-19. As explained by a council representative, “The opposition [to family planning] – I see it on men’s side. The men are not seriously in support of it. Actually, [the] majority of men strongly disagree with their spouses on the matter of family planning, both Muslims and Christian” . Power imbalances between men and women, an outcome of inequitable gender norms, result in male control of family planning decision-making. Consequently, all participants agreed that it is common practice for women in the community to use family planning in secret, but health workers explained how COVID-19 stay-at-home orders disrupted women’s ability to do so. The following quotation from a female healthcare worker demonstrates how stay-at-home orders have reduced women’s autonomy to use contraceptives:“A woman will come and tell you that, ‘Musawo [Doctor], please hurry up because I lied to my husband that I had just gone to the neighbor’s place.’ So when you hear that, you leave whatever you are doing and decide to go and attend to this mother. So, this lockdown has really affected them because the man is always around and he is so inquisitive. When they go to the health facility they are asked why they have delayed, he [man] even times her. He can say you went in the morning, how come you have spent all that time?”In addition, the COVID-19 lockdown intersected with barriers to family planning underpinned by COVID-19 has mainly brought violence in homes” . Further, IPV was an expected response to finding one’s wife using family planning in secret, thus potentially diminishing women’s use during COVID-19, as one woman explained: “Physical abuse is not supported in the community, but if it concerns family planning, most men support the abuse” .Also related to gender inequity, while participants generally looked down on IPV, they agreed that it was still prevalent in the community, with some male participants considering it necessary in some situations. The stay-at-home orders were said to have resulted in an increase in IPV in general: “Respondent 1 (age 42): Most men are at home; they lack what to do and they are all over their wives all the time and [the] majority of women are pregnant now.Respondent 2 (age 36): I have nothing different [to say] but we are bored of men being around us all the time.Moderator: Okay, but what has led to these women getting pregnant during the COVID lockdown?Respondent 1: We used to count our days [calendar method] and things would be okay but today they [men] are around us all the time. You would spend your 10 days or more without being disturbed but today they [men] are home all the time.Gender inequity also intersected with the COVID-19 pandemic to increase women’s risk of unintended pregnancy through the increased occurrence of sex due to men being at home more; see excerpt below from the women’s FGD.This and other narratives imply that women may not be interested in having more sex, but due to inequitable gender norms, have less say over its occurrence than men in a relationship.income, an individual enabling factor, in turn affecting service access. As one female focus group participant (age 23) stated, “For me, this lock down has affected me in my wellbeing and income; all [of] my business has gone down. We are so poor in this lock down.” Participants explained how the pandemic’s effect on income created new financial barriers to accessing health services, particularly in not having money to cover the cost of transportation to the clinic, another enabling factor, or the cost of methods at private clinics, as the following exchange between participants from the younger women’s focus group illustrates:Participant 1 (age 24): Now the method can be due but when you don’t have money for transport to go to the health centre and get the new dose or even for the injectaplan. So, the next thing is to conceive because you are married but the family planning method expired.Participant 2 (age 23): Also, such women may get money for transport and may fail to get money for the injectaplan [at private clinics] or fail to get transport but remember they escape from home to go for it to a health centre. So, you cannot walk; you will be delayed – bearing in mind that you escaped from home, you need to run and come back soon.COVID-19 had a negative effect on gender; women that use family planning in secret tend to have more difficulty getting to the clinic.This narrative again intersects with the contextual predisposing factor of lower capacity due to fewer health workers available and shorter hours. Moreover, a common misconception in the community was that facilities were closed completely, resulting in poor attendance, as one female health worker explained: “New [contraceptive] users were even more affected. They thought that family planning services were also locked-down. It's mainly after a period of about two months that we started to see some of the new users.” Moreover, health worker participants described fear of COVID-19 transmission, as well as a lack of protective gear, as a barrier to the delivery and receipt of services. As one female health worker explained, “It [COVID-19] has really affected me because most of my work has been door-to-door, so it worries me to move to people’s homes because I can infect them or they can infect me." Health workers described the risks of being without protective masks, gloves, thermometers, and hand sanitizer throughout the 3-month lockdown. As another male VHT explained, this also negatively affected client attendance: “The family planning clients fear to meet health workers and vice versa because they don’t have masks.”Community enabling factors were prominent barriers to contraceptive use pre-dating COVID-19, including contraceptive method and equipment stock outs, low provider capacity to provider LARCs such as IUDs at HCIIIs, clinic wait time, and the perception of poor treatment from providers. Facilitators, irrespective of COVID-19, included community outreach strategies, such as VHT outreach and collaboration with Marie Stopes International for pop-up clinics. COVID-19 worsened existing and created new barriers to family planning within community enabling factors. For example, during lockdown, family planning services had strict transportation ban that restricted all public transportation , which negatively affected the community’s access to health facilities; those that do not live in walking distance rely on public transportation to get to the clinics. Some transportation exceptions were made for “essential services,” such as for pregnant women reaching the clinic. Although the District provided transportation for healthcare workers to reach the facilities, the transportation ban still challenged the movement of healthcare workers, making it more difficult to get to the facilities for work, and prohibited the distribution of contraceptives and family planning education through community outreach from Marie Stopes International and VHTs. The community relies on quarterly outreach from the non-governmental organization, Marie Stopes International, to provide contraceptives not offered at the local clinic because of limited provider capacity to offer LARCs and frequent stock outs. This outreach was restricted during lockdown.“Between March and June, we stopped the outreaches because there was a total ban on gatherings. In that quarter, we never held any outreaches and the outreach is our biggest clinic because people know that is when Marie Stopes, that has the experience, is coming. So, in that quarter the uptake was low, people were not coming out and they also had their own challenges with transportation to the facility” .COVID-19 lockdown’s effect on mobility was the most salient community enabling barrier to family planning delivery. Lockdown included a “The lock down affected our mobilization and counseling because of the rules of social distancing. We could not access community members.” VHTs who normally mobilize the community, providing individual and group outreach, education, and short-term contraceptives, were prohibited from working in the community.“As health workers, we remained open but working for fewer hours than full days. Of course, we kept receiving complaints from the community about the inaccessibility of the patients who wanted us to help them, but we could not help; we had no means to reach out to help them. The VHTs would bring complaints from the community expecting quick feedback on how we could reach out to communities but we had no means to do it. Besides, we had to adhere to the guidelines from the government” .In addition, as explained by one male VHT, the movement of VHTs was restricted during lockdown: a lack of emergency preparedness, with no protocols in place to ensure client needs could be met during the pandemic, and government restrictions did not include an exception for outreach of family planning services.As the above quotation explains, there was Health workers, VHTs, and community leaders recognized a high need for family planning in the community based on a shared perception that contraceptive use in the community was low alongside high fertility and limited resources to support large families. While women endorsed family planning much more than men, both women and men perceived a need for family planning from a socioeconomic standpoint; there was consensus that families should only have the number of children they can afford to care for. In addition, women discussed the health and social benefits of spacing their children. While participants recognized that COVID-19 lockdown measures had a negative effect on access to contraceptives, and thus, a need for increased access to services during the pandemic, they did not express any new need for or reasons for wanting contraceptives related to COVID-19 itself. That is, women and men in the FGDs did not say COVID-19 changed their pregnancy intentions or the spacing and timing of their future pregnancies.As described above, participants perceived that COVID-19’s effects on predisposing and enabling factors likely resulted in lower contraceptive use through restrictions that affected health service delivery and access. As such, risk of unintended pregnancy was thought to have increased due to a lack of access to services, but also through the increased occurrence of sex. Further, participants perceived a rise in IPV at home due to COVID-19, which could further diminish women’s autonomy of contraceptive use and ability to refuse sex with one’s husband, also contributing to risk of unintended pregnancy.“There should be prior arrangements to equip health facilities by [the] government with other stakeholders, with means of transport like motorbikes, vehicles, and the government should train staff for outreaches in case of similar eventualities as this one [COVID-19 lockdown].” As this quotation from a female health worker exemplifies, recommendations included making special provisions for transportation that would allow family planning users to continue to access services through public transport in the event of a COVID-19 transportation ban, as well as strengthening community-based family planning (CBFP). Participants suggested adapting existing CBFP approaches so that they could be safely implemented during a COVID-19 outbreak, including continued distribution through Marie Stopes International and VHTs. Health workers, VHTs, and community members were in support of building VHT capacity to deliver methods, such as injectable contraceptives irrespective of COVID-19, and noted that it would be particularly beneficial during a COVID-19 outbreak. An external project had previously trained some VHTs in the district in injectable contraceptives, but implementation became inconsistent after the project ended.Participants’ recommendations to improve contraceptive use in the context of COVID-19 centered on strengthening emergency preparedness and response. “The community dialogues, in my view, can easily create hostilities and conflicts in homes. Remember that in our community men discourage family planning use because of religion and culture. The men will have to also attend those dialogues, or someone would tell them what transpired in those meetings. I see that this kind of strategy would not be effective, unless you first provide counseling and education [for] men separately and women separately and make sure that their spouses are in agreement.”Recommendations for new CBFP approaches to ensure contraceptive access amid COVID-19 included the creation of a voucher system that would link governmental facilities to private shops/pharmacies, allowing people to access contraceptives from local shops for free. Participants explained that bringing free methods to nearby shops would reduce barriers to access during a COVID-19 outbreak, including transportation barriers and partner disapproval, as one could access the private shops more discreetly. Participants also recommended mobile services, such as pop-up tent clinics or delivering methods as part of community dialogues, which would simultaneously generate demand for family planning. Participants discussed how such venues could be set up to incorporate COVID-19 prevention protocols in an outdoor or semi-outdoor space, allowing for ventilation and social distancing. While VHT and private shop distribution were viewed favorably by women and health workers because they could allow women to access contraceptives without their partners’ knowledge, FDGs and KII narratives made clear that issues of gender inequity and male control over contraceptives need to be considered when planning more public outreach events. Specifically, engaging and sensitizing men was viewed as essential to the success of such efforts, as the following quotation from a male VHT demonstrates:We need psychosocial counseling [for] health workers because everyone was in panic saying, ‘I might infect my family.’ The In-Charge had to explain to the people all the time that they had to work.” Pointing to health system weaknesses that pre-date the pandemic, providers also recognized the need for capacity building and refresher trainings on family planning services generally, as the following exchange between a male health worker and the moderator exemplifies:Respondent: I think training is key, because if we are aware that indeed the disease is spread, and if you have the protective equipment, I think that gap can be reduced.Moderator: So, you are saying sensitizing health workers or educating them about the outbreak and equipping them?Respondent: Yes, and also providing confidence to them [health workers].Moderator: What else?Respondent: Even training on the skills, for example, in general; it may not be for COVID-19 but like this refresher training, like in general, inserting IUDs and also training us on managing side effects.Finally, health workers emphasized the need for protective gear  and COVID-19-related health worker training. Training was requested to ensure methods could be delivered while minimizing risk of transmission, but health workers recognized the need for training to also reduce fear and panic, as a male health worker stated, “This qualitative study used the Andersen Behavioral Model of Health Services (ABM) to understand the effects of COVID-19 on family planning barriers and facilitators in a rural area of Uganda. Participants described significant disruptions to the delivery of family planning services, and access to health services generally, due to COVID-19 during and after Uganda’s first government-mandated COVID-19 lockdown in 2020. COVID-19 had direct effects on enabling factors at the individual-level, such as reduced income negatively affecting individual’s access to transportation and private services, but the larger consequences were at the community-level, with the suspension of CBFP being particularly disruptive. In addition, COVID-19 exacerbated contextual predisposing factors that served as barriers to family planning before the pandemic, including poverty and those related to gender inequity, such as IPV and power inequities between men and women that diminish women’s ability to refuse sex with their husband and their autonomy to use contraceptives. Recommendations to improve family planning service delivery in the context of COVID-19 centered on emergency preparedness and strengthening existing community distribution and creating new ways to safely deliver contraceptive methods directly to communities during future COVID-19 lockdowns. Although this qualitative study is not a direct measure of COVID-19’s effect on contraceptive use or unintended pregnancy, this study highlights potential pathways in which COVID-19 may directly  and indirectly  result in unintended pregnancies via reduced contraceptive use, increased sex from men being at home, and increased IPV.While a number of commentaries and modeling studies exist that project the effects of COVID-19 on women’s reproductive health outcome , few stuTo our knowledge, the present study is one of the first qualitative studies to examine the potential effects of COVID-19 on family planning in a LMIC. Thus, this study adds context to the limited quantitative research that exists by illuminating different ways in which COVID-19 and its consequential lockdown and prevention measures have affected family planning barriers and exacerbated existing gender disparities and health system weaknesses. Narratives from our community sample described COVID-19’s disruption to community resources that enable access to family planning services . This included reduced facility capacity and community outreach disruptions, as researchers predicted early in the pandemic , 14, 41.These findings point to a lack of emergency preparedness that would allow for the continued and safe provision of family planning services amid an infectious disease outbreak or other humanitarian crises. Participants’ recommendations highlighted the need for COVID-19 training and capacity building, access to protective supplies, and client access/transportation to the facility by deeming family planning an essential service. In addition, community members and health workers alike in our sample recommended the strengthening of CBFP to bring services safely to communities. Mobile services, VHTs , and distribution of methods through local pharmacies and shops were strategies recommended by our sample to deliver family planning while limiting facility-based contact and reducing barriers to care resulting from travel restrictions. These methods are considered High Impact Practices (HIPs), or evidence-based strategies for family planning , and havOur findings suggest that the success of CBFP during a public health or other humanitarian crisis, however, would be contingent on simultaneous efforts to increase male support for family planning. In this rural community, men largely opposed family planning and had a strong influence over women’s autonomy to use contraceptives. CBFP would make contraceptive use more public, reducing women’s ability to use contraceptives in secret. These findings align with a large literature demonstrating the need to increase male support of family planning and women’s use of reproductive healthcare generally in order to increase service uptake in LMICs –51. PartThese findings point to the need to address the underlying gender inequities that already impede women’s use of contraceptives in rural settings. Interventions to engage men in family planning or sexual and reproductive health services have had mixed success –57. HoweReaders should interpret the findings of the present study with the study’s limitations in mind. While this study can shed light on the effects of COVID-19 on family planning barriers and facilitators in rural LMIC settings, the findings may not be generalizable to other dissimilar settings. This qualitative study explores the perceived effects of COVID-19 on family planning outcomes but is not a direct measure of these outcomes. For our focus groups, we sampled men and women of reproductive age with an unmet need for family planning . Thus, these participants were able to share their reasons for non-use, including those related to COVID-19, but do not provide insight into barriers to continued use of contraceptives among current users.As the COVID-19 pandemic continues to devastate LMICs globally, and inequities in global vaccine distribution continue to favor high-income countries , efforts


Bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) is a bacterial second messenger that affects diverse processes in different bacteria, including the cell cycle, motility, and biofilm formation. Its cellular levels are controlled by the opposing activities of two types of enzymes, with synthesis by diguanylate cyclases containing a GGDEF domain and degradation by phosphodiesterases containing either an HD-GYP or an EAL domain. These enzymes are ubiquitous in bacteria with up to 50 encoded in some genomes, the specific functions of which are mostly unknown.ori) and terminus (ter) of replication and at ter while the EAL-encoding genes peaked near ori. The patterns were more complex in the Rhizobiales, but the GGDEF-encoding genes were biased for localization near ter.We used comparative analyses to identify genomic patterns among genes encoding proteins with GGDEF, EAL, and HD-GYP domains in five orders of the class Alphaproteobacteria. GGDEF-containing sequences and GGDEF-EAL hybrids were the most abundant and had the highest diversity of co-occurring auxiliary domains while EAL and HD-GYP containing sequences were less abundant and less diverse with respect to auxiliary domains. There were striking patterns in the chromosomal localizations of the genes found in two of the orders. The Rhodobacterales’ EAL-encoding genes and Rhizobiales’ GGDEF-EAL-encoding genes showed opposing patterns of distribution compared to the GGDEF-encoding genes. In the Rhodobacterales, the GGDEF-encoding genes showed a tri-modal distribution with peaks mid-way between the origin (The observed patterns in the chromosomal localizations of these genes suggest a coupling of synthesis and hydrolysis of c-di-GMP with the cell cycle. Moreover, the higher proportions and diversities of auxiliary domains associated with GGDEF domains and GGDEF-EAL hybrids compared to EAL or HD-GYP domains could indicate that more stimuli affect synthesis compared to hydrolysis of c-di-GMP.The online version contains supplementary material available at 10.1186/s12864-022-09072-9. Gluconacetobacter xylinus [Bis-3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) is a second messenger that was first described for its role in regulating cellulose biosynthesis in  xylinus , 2, but ′-5′-cycl xylinus . Cellula xylinus . There a xylinus , 8 which xylinus , 10. PDE xylinus , 12. In  xylinus , with a  xylinus  or dimer xylinus . However xylinus .C-di-GMP levels can be controlled via transcriptional and translational regulation of gene expression, or through post-translational modification of the synthesis and degradation enzymes as a quicker response. Auxiliary domains can be present on the enzymes and include sensory, signalling and protein binding domains, and these can allow for rapid adaptation . CellulaSinorhizobium meliloti with plant roots [Caulobacter crescentus [Rhodobacter capsulatus and Dinoroseobacter shibae [In the Alphaproteobacteria, c-di-GMP has been examined for its role in many different processes, such as the symbiosis of nt roots  and relant roots , which int roots .The CtrAnt roots . It has nt roots , 24, butescentus , 25 and r shibae , 27. C-dr shibae , 29. Ther shibae .ori) are replicated earlier and are therefore temporarily present in higher copies than genes that are closer to the terminus of replication (ter) [Bacillus subtilis, where it was shown that the temporal copy number imbalances due to the opposite localization of genes encoding members of a regulatory network influenced its output [ori, and methylation status can affect regulatory protein binding and transcription [ctrA initiates is only activated in the hemi-methylated state in C. crescentus [The chromosomal positioning of genes can affect their functions in different ways and have effects on multiple cellular processes. For example, gene location can influence the spatial distribution of proteins within cells due to transcription-coupled translation . Positioon (ter) . An exams output , 34. Addcription . For exaescentus . It seemescentus .C-di-GMP-modulating enzymes are broadly distributed in phylogenetically and metabolically diverse bacteria. They are also very diverse with respect to their roles and regulation, with a wide range of stimuli affecting c-di-GMP levels, and only a small proportion of the total diversity of these enzymes has been characterized in detail . Therefohttp://pfam.xfam.org/family/PF00990#tabview=tab7; EAL: http://pfam.xfam.org/family/PF00563#tabview=tab7; HD: http://pfam.xfam.org/family/PF01966#tabview=tab7) [Protein sequences with identified EAL (PF00563), GGDEF (PF00990) or HD (PF01966) domains from genomes of bacteria within five orders of the Alphaproteobacteria  were downloaded from the EMBL website on 6 August 2020 (GGDEF: ew=tab7) . ProteinAll analyses were done in R version 4.0.3 with the appropriate packages as needed Table S.https://www.uniprot.org/uniprot/V4RSF5.txt) or EBI  [http://pfam.xfam.org/protein/A0A0N0K049#tabview=tab0). Domain annotations could not be withdrawn for all sequences due to inconsistent html path formatting, which reduced the dataset (Table https://www.uniprot.org/uniprot/A0A0N0K3V8.txt). Due to inconsistencies some sequences have different version numbers  and only version 1 was subsequently considered in such cases. All sequence identifiers and html paths can be found in Table SSequence identifiers were extracted from the EMBL fasta files and used to access the respective organism information from UniProt  . The ideet Table . The ideori) was identified for each chromosome using Ori-Finder and default settings [https://github.com/sgivan/gb2ptt#gb2ptt) from gbff files, downloaded from NCBI on 23 April 2019. Only chromosomes with one unambiguously identified ori were subsequently included in the investigation, which reduced the dataset (Table ter) was assumed to be opposite ori on the circular chromosomes [The origin of replication  were downloaded for the members of each order and their NCBI identifiers were determined. Alignments were done using MAFFT with L-INS-i option  in GeneiWe quantified the genes encoding the domains associated with c-di-GMP synthesis and degradation in members of the five alphaproteobacterial orders. This included those that contained one of the GGDEF, EAL, or HD-GYP domains or both GGDEF and EAL domains. The GGDEF and GGDEF_EAL sequences accounted for the highest proportions in all five orders at 35–48%, followed by proteins containing an EAL domain that ranged between 8.9% and 23.4% of all sequences Fig. . The HD-Rhodomicrobium and Neorhizobium), 1.7–51 , 3.6–14.2 (Phenylobacterium and Caulobacter), 3–25.4 (Croceicoccus and Novosphingobium), and 1–49  were observed in the respective individual orders.Next, the numbers of c-di-GMP-metabolizing sequences in different genera were compared by calculating the mean number of sequences per genus Fig. . The c-dFor the subsequent investigation of the numerical relationships among the various domains, all orders were analyzed Figure S, but dueDevosia, Fulvimarina and Rhizobium. Smaller additional clusters with increased c-di-GMP numbers that were less closely related were also observed. In the Rhodobacterales, the closely related genera Stapia and Labrenzia stood out with their high c-di-GMP-metabolizing gene numbers. A connection between phylogeny and c-di-GMP-metabolizing gene number could also be observed in the Rhodospirillales. Here there were three clusters of organisms that had increased gene numbers and one notable group was made up of three genera including Magnetospirillum, Magnetovibrio and Telmatosprillum. A clear connection between phylogenetic relationships and numbers of c-di-GMP-metabolizing genes was not observed in the Sphingomonadales, and it is difficult to make any statement for the Caulobacterales because of the lower genome and gene numbers.The large variability in numbers of c-di-GMP-metabolizing proteins among organisms stimulated us to investigate their evolutionary relationships. Therefore, the number of c-di-GMP enzymes present in different species was evaluated in a phylogenetic context Figure S. Some clNitrospirillum amazonense CBAmc , Rhizobium sp. NXC24  and Asticcacaulis excentricus CB 48  more c-di-GMP genes were found on the second-largest replicon and in Paracoccus denitrificans PD1222  equal numbers of c-di-GMP-metabolizing genes were found on the largest and second-largest replicons.There was a statistically significant positive correlation between chromosome size and the number of c-di-GMP-metabolizing genes in all five orders Figure S. We onlySecondary chromosomes (defined as replicons > 800 kb that are not the largest replicons in the genome) contain genes that evolve faster  and are ori) and terminus (ter) of replication. No obvious trend was observed in the Rhodospirillales, while GGDEF and GGDEF_EAL sequences seemed less prevalent near ter in the Sphingomonadales  while the EAL and GGDEF as well as the GGDEF and GGDEF_EAL pairs were distributed differently . The Rhizobiales GGDEF and GGDEF_EAL genes were also distributed differently .Comparison of the similarities of distributions among the groups of genes indicated that the Rhodobacterales EAL and GGDEF_EAL genes were similarly distributed .In the two orders with the most available data, the Rhodobacterales and Rhizobiales, there is a clear conservation of the Rhodobacterales EAL- and GGDEF_EALori are replicated earlier than genes that are close to ter, which leads to a temporary copy number imbalance between genes at these two locations [B. subtilis, the opposite location of two genes encoding components of a phosphorelay with respect to ori and ter leads to temporal copy number imbalances, and this allows spore formation to only take place at the end of the cell cycle when the balance between the regulators is restored [Vibrio cholerae, moving genes from ori to ter and thus reducing their copy number during the cell cycle has an impact on growth and infectivity [Escherichia coli [There are multiple potential effects caused by the chromosomal locations of specific genes. The observed chromosomal localization patterns revealed in this study might affect cellular c-di-GMP concentrations during the cell cycle. Genes that are close to ocations . In B. srestored , 34. In ectivity , 62. Suchia coli , althougctrA gene in C. crescentus is only active in the hemi-methylated state [ctrA, which is localized near ori, is transcribed more during DNA replication because it is hemi-methylated right at the beginning of the cycle. However, any broad role of methylation in regulating transcription of genes encoding c-di-GMP-metabolizing enzymes is currently unknown and future work is required to investigate this possibility.Another effect of localization could be manifested through DNA methylation, where the chromosomal DNA changes from fully methylated to hemi-methylated during replication. This change in methylation can affect gene transcription. For example, the p1 promoter of the ed state . Thus, cter while GGDEF sequences are biased towards ter. Additionally, the EAL and GGDEF_EAL domain-containing sequences show lower diversity and occurrence of auxiliary domains compared to the GGDEF sequences. There are several known examples in which chromosome localization of genes is important, and this can manifest in different ways such as through changes in copy number and methylation status during the cell cycle. The patterns we found support the suggestion that the chromosomal localization of c-di-GMP-metabolizing genes is important in these bacteria. Our findings also support the notion that the synthesis of c-di-GMP is more regulated and responsive to a variety of specific signals whereas its degradation might be less regulated and dependent on different stimuli.C-di-GMP-metabolizing enzymes are very diverse, and the specific roles and functions of only a few of these proteins are known. In this study new patterns and common properties for these proteins were identified in members of the Alphaproteobacteria. We systematically examined gene occurrence, localization on the genome, and the presence of auxiliary domains. In the Rhodobacterales and Rhizobiales, the EAL and GGDEF_EAL sequences, respectively, are primarily located away from Additional file 1:Figure S1. Numerical relationships between the number of GGDEF and EAL (GGDEF:EAL) sequences by genera. The ratios were calculated per genome and the mean per genus was plotted. Figure S2. Numerical relationships between the number of GGDEF and GGDEF_EAL (GGDEF:GGDEF_EAL) sequences by genera. The ratios were calculated per genome and the mean per genus was plotted. Figure S3. Numerical relationships between the number of GGDEF and HD-GYP (GGDEF:HDGYP) sequences by genera. The ratios were calculated per genome and the mean per genus was plotted. Figure S4. Numbers of c-di-GMP sequences in a phylogenetic context. Phylogenetic relationships are based on RpoB sequences. All alignments were done using MAFFT with LINS-i option. Bootstrap values based on 1000 replicates and hill-climbing nearest-neighbor interchange search were used. A. Rhizobiales. B. Caulobacterales. C. Rhodobacterales. D. Rhodospirillales. E. Sphingomonadales. Figure S5. Relationships between chromosome size and the number of encoded c-di-GMP enzymatic domains. Spearman's rank correlation was used to evaluate the significance. Only the biggest replicon, considered the main chromosome, was included in this analysis. Figure S6. Chromosomal locations of c-di-GMP-associated genes. Cumulative distributions of cdi-GMP-associated genes on the chromosomes, with lengths normalized to 100% where ori is at 0% and 100% and ter is at 50%. The red line indicates the estimate of the kernel density. In this analysis only closed genomes with one unambiguously identified ori were used. Figure S7. Secondary domains that are present along with the different c-di-GMP-associated enzyme groups. A. Shared and individual secondary domains. The c-di-GMP-modulating domains are not included in this analysis. The color code of the Venn diagram represents domain counts from highest(red) to zero (white). B. Number of sequences that have zero, one, or more than one secondary domain. Figure S8. Relationships between protein length and presence of detected auxiliary domains. The sequences with EAL and GGDEF domains were segregated based on the occurrence of auxiliary domains. The minimal amino acid lengths for proteins containing auxiliary domains (left panel) were identified as 375 for EAL proteins and 275 for GGDEF proteins (blue dashed lines). This threshold was then used to calculate the percentage of sequences without identified auxiliary domains that were shorter and longer than these minimal lengths (right panel).Additional file 2:Table S1. R packages used for analyses.Additional file 3:Table S2. Compilation of Pfam, EBI and NCBI sequence identifiers.Additional file 4:Table S3. Sequence counts by genera. The mean number of sequences is calculated per genus. Blue indicates a "genus" that is not included in the analysis because it does not represent an actual genus.Additional file 5:Table S4. Domains found in all c-di-GMP-associated protein sequences. The cyclic di-GMP-associated domains are indicated in light blue. Values >0 for other domains are indicated in red.Additional file 6:Table S5. Count of c-di-GMP-associated genes on chromosomes and plasmids. The primary chromosome is defined as the largest replicon of a genome. A "c" at the start of a column name indicates information regarding the chromosome while "p" indicates plasmids. Blue coloration indicates those genomes that have c di GMP-associated genes on secondary chromosomes or extrachromosomal replicons.Additional file 7:Table S6. Occurrence of secondary domains with cyclic di-GMP-modulating domain sequences.Additional file 8:Table S7. Frequency of association of all secondary domains that co-occur with c-di-GMP-associated domains.Additional file 9:Table S8. All domains found associated with one of the c-di-GMP-associated domains examined in this study with associated Pfam HTML paths and Pfam IDs.


Scientific Reportshttps://doi.org/10.1038/s41598-021-89473-0, published online 11 May 2021Correction to: The original version of this Article contained an error.The accession codes for the raw sequencing data generated in the course of the study were omitted from the Data Availability statement.“Data generated in this study are available in the main table, figures, and additional files.”now reads:“Data generated in this study are available in the main table, figures, and additional files, and the raw sequencing data are available at the National Center for Biotechnology Information SRA database under the accession PRJNA860974.”The original Article has been corrected.


Little is known on the burden of co-infections and superinfections in a specific setting such as the respiratory COVID-19 sub-intensive care unit. This study aims to (i) assess the prevalence of concurrent and superinfections in a respiratory sub-intensive care unit, (ii) evaluate the risk factors for superinfections development and (iii) assess the impact of superinfections on in-hospital mortality.Single-center retrospective analysis of prospectively collected data including COVID-19 patients hospitalized in a newly established respiratory sub-intensive care unit managed by pneumologists which has been set up from September 2020 at a large (1200 beds) University Hospital in Rome. Inclusion criteria were: (i) COVID-19 respiratory failure and/or ARDS; (ii) hospitalization in respiratory sub-intensive care unit and (iii) age > 18 years. Survival was analyzed by Kaplan–Meier curves and the statistical significance of the differences between the two groups was assessed using the log-rank test. Multivariable logistic regression and Cox regression model were performed to tease out the independent predictors for superinfections’ development and for mortality, respectively.Acinetobacter baumannii (CR-Ab) isolated in 47%. Overall mortality rate was 30%. Prior (30-d) infection and exposure to antibiotic therapy were independent risk factors for superinfection development whereas the development of superinfections was an independent risk factors for in-hospital mortality. CR-Ab resulted independently associated with 14-d mortality.A total of 201 patients were included. The majority  presented severe COVID-19. Co-infections were 4 (1.9%), whereas 46 patients (22%) developed superinfections, mostly primary bloodstream infections and pneumonia. In 40.6% of cases, multi-drug resistant pathogens were detected, with carbapenem-resistant In a COVID-19 respiratory sub-intensive care unit, superinfections were common and represented an independent predictor of mortality. CR-Ab infections occurred in almost half of patients and were associated with high mortality. Infection control rules and antimicrobial stewardship are crucial in this specific setting to limit the spread of multi-drug resistant organisms.The online version contains supplementary material available at 10.1186/s12890-023-02315-9. As of 28th July 2022, more than 570,000,000 people had been infected with SARS-CoV-2 worldwide, with more than 6,000,000 deaths . Around Viral respiratory illnesses predispose patients to bacterial and fungal infections; while this link has been widely described in relation with influenza pneumonia, it is still unclear the exact roles co-pathogens play in patients with COVID-19 . Indeed,During the first wave of COVID-19 pandemic, although few papers reported a low incidence of bacterial co-infections , most paWhile the rate of co-infections has an important role in deciding whether to use empiric antibiotic therapy, superinfection rate tends to be higher in patients with prolonged hospital stay, worst clinical presentation, and need for Intensive Care Unit (ICU) . Wide spIn most centers in Italy, COVID-19 patients were admitted to ICU or ordinary wards based on their clinical conditions. Italian hospitals faced an unpreceded massive inflow in a short period of time . In centTo our knowledge, little is known about the prevalence of concurrent and superinfections in this peculiar setting.Based on these premises, we carried out a study specifically targeting patients hospitalized in a respiratory sub-intensive care unit with the aims to (i) assess the prevalence and etiology of co- and superinfections, (ii) evaluate the risk factors for superinfection development and (iii) assess the impact of superinfections on in-hospital mortality.We performed a single-center, retrospective analysis of prospectively collected data on patients with COVID-19 pneumonia hospitalized in a respiratory sub-intensive respiratory care unit at Azienda Ospedaliero-Universitaria Policlinico Umberto I, Sapienza University of Rome, from November 2020 for the following 6 months, until April 2021.Inclusion criteria were: (i) diagnosis of COVID-19 respiratory failure and/or acute respiratory distress syndrome (ARDS), (ii) hospitalization in a sub-intensive respiratory care unit for > 48 h and (iii) age > 18 years. Exclusion criteria included: age < 18 years, hospitalization in a sub-intensive respiratory care unit for < 48 h and missing data.Diagnosis of SARS-CoV-2 infection was made with molecular analyses. Diagnosis of respiratory failure was made on clinical presentation and arterial blood gases (ABGs), a lung CT scan was performed to detect ground glass opacity and/or others lesions compatible with COVID-19 pneumonia. Acute respiratory distress syndrome was diagnosed on clinical and ABGs data according to Berlin definitions . The decStarting from September 2020, a sub-intensive respiratory care unit with 42 beds managed by pneumologists was set up in our 1200-bed Academic Hospital. Patients were admitted in case of severe respiratory failure and/or ARDS due to COVID-19 pneumonia requiring oxygen therapy and/or Helmet continuous positive airway pressure (CPAP) treatment or non-invasive mechanical ventilation (NIMV) . OxygenPatients required the use of continuous vital signs monitoring, and, in most cases, central venous catheter (CVC) or arterial catheters’ placement, total parenteral nutrition and, in some cases, sedation. Sedation was performed in case of non-adaptation to ventilation. Drugs administered in case of sedation were midazolam or dexmedetomidine. The level of sedation was related to the possibility of maintaining non-invasive ventilation.We managed also critically patients with septic shock, or heart failure or requiring renal replacement, in this case with integrated management with nephrology specialists. We collaborated with an infectious disease specialist to treat superinfections.Criteria for ICU-transfer was: if clinical conditions worsened despite ventilation and clinical care, an anesthesiologist consultation was required, and patients were transferred to ICU in case orotracheal intubation was needed.Main differences with ICU setting were: (i) we did not manage intubated patients; (ii) most patients were not sedated and (iii) we have a different number of nurses, with one nurse assisting almost height patients during the work shift (nurse/patient ratio 1:8); (iv) ICU was managed by anesthesiologists.Nasopharyngeal swab samples were collected, and SARS-CoV-2 RNA was detected by using real time RT-PCR assay .All patients received oxygen therapy and/or HFNC or CPAP or NIMV treatment based on respiratory failure severity.Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), or carbapenem resistant (CR) gram-negative bacilli] colonization was systematically performed by means of rectal swab, at admission to the sub-intensive respiratory unit and every 7 days afterwards.Screening for multi-drug resistant (MDR) bacteria .As shown in Table At multivariable analysis, independent risk factors for in-hospital mortality were the development of superinfections , age > 65 years , CCI > 5 , lymphocytes count < 750/mmc , use of sedation  and NIMV  , were older  and had a higher rate of diabetes, chronic ischemic heart disease, chronic heart failure, atrial fibrillation and malignancy, with a higher Charlson Comorbidity Index (CCI) (p = 0.0001)  and antibiotic exposure in the previous 30-d before superinfections development  were more common in the Infection group. Given the severity of COVID-19 pneumonia, corticosteroid treatment was similar in both groups . Colonization with MDR pathogens was higher in the group of patients with super-infections . No differences were observed for pronation and sedation use in the two groups, whereas NIMV was more frequent in patients developing superinfections (23.9% vs 6.4%).Mortality rate was higher in patients with superinfections . Furthermore, they presented a longer in-hospital stay .At multivariable analysis, prior (30-d) infection  and exposure to antibiotic therapy in last 30-d  were independent risk factors for new onset superinfections development (Table Clostridioides difficile infection (1.5% each).Overall, 46 patients (22%) developed superinfections, with the different causative pathogens shown in Supplementary Table 1. Several patients experienced > 1 infective episode and therefore we recorded 64 infections. Primary BSI was the most frequent superinfection with 23 episodes (35.9%), followed by pneumonia (29.6%), urinary infections (28.5%), CR-BSI (3.1%), skin/soft tissue infections and Acinetobacter baumannii (CR-Ab) isolated in 11 (47%) of them. Others MDR involved were MRSA, VRE and CR Klebsiella pneumoniae (CR-Kp) . All the strains of A. baumannii were resistant to meropenem; all but one exhibited colistin susceptibility. Cefiderocol susceptibility was available only for 6 strains; all the tested strains were susceptible at disk diffusion method (Supplementary Table2).First infective episode was diagnosed after a median of 14.5 days (IQR 7–22) from sub-intensive unit admission. In 23 cases (40.6%), MDR pathogens were detected, with CR A. baumannii as etiologic agents of first infective episode , p = 0.018]  co-infections were uncommon while the development of superinfections occurred in almost one quarter of patients and were independently associated with mortality; (ii) MDR organisms were isolated in approximately 40% of patients, with CR-Ab accounting for almost half of cases and (iii) CR-Ab as the causative agent of the first infective episode was associated with early (14-d) mortality.To the best of our knowledge, the prevalence and characteristics of superinfections in COVID-19 patients hospitalized in a respiratory sub-intensive care unit are not well understood and represent a significant knowledge gap in literature data.Staphylococcus aureus [Indeed, from the spread of SARS-CoV-2 pandemic, several studies reported the rates of bacterial, viral and/or fungal infections in COVID-19 patients, especially in ICU. A study conducted during the first pandemic wave reported a high rate of incidence and prevalence of co-infections during COVID-19 . This ars aureus , thus juMycoplasma pneumoniae as the etiologic agent in all the cases, although concerns were raised about diagnosing M. pneumoniae infection in COVID-19 patients only by means of serology diagnostic testing. These data are in accordance with our previous data collection referred to the first pandemic wave in which Mycoplasma pneumoniae was responsible for the 1.1% of co-infections [Nevertheless, according to the following literature –26 co-infections  and sustfections  the possOverall, 46 patients (22%) developed superinfections. Our data seems in line with literature , 30 in tChong et al. reported an overall time to diagnose pulmonary superinfections of 10 days (2–21 days) from initial hospitalization and 9 days (4–18 days) after ICU admission . LikewisCOVID-19 severity could in part explain the association between critical conditions and superinfections. Ripa M et al. concluded that factors associated with superinfections were low baseline lymphocyte count, baseline P/F ratio, and ICU admission , while FWe reported an overall mortality rate of 30%, with a higher rate in patients with superinfections, especially when sustained by CR-Ab. The development of superinfections represented an independent risk factor for in-hospital mortality, together with age > 65 years, CCI > 5, severe COVID-19 and a count of lymphocytes < 750/mmc on admission. In a previous study conducted in 24 ICUs, trends of clinical, ventilatory and laboratory parameters throughout the entire ICU stay showed different slopes between survivors and non-survivors . FurtherA. baumannii and CR-K. pneumoniae were detected in 40.6% of cases, with CR-Ab, a common colonizer in the ICU setting, isolated in almost half of cases. This prevalence could be explained by considering that patients hospitalized in our sub-intensive unit presented several risk factors usually associated with A. baumannii infections, such as prolonged hospitalization, need of intensive care, presence of devices, older age, prior colonization, and prior use of antibiotics [As reported for patients hospitalized in ICU –35, in tibiotics .Pseudomonas aeruginosa and Acinetobacter baumannii have been increasingly reported during the hospital stays of patients with severe COVID-19 [MDR in Gram-negative bacilli represent a major threat to human health and significantly contribute to global deaths attributable to and associated with bacterial antimicrobial resistance . With thCOVID-19 .P. aeruginosa, A. baumannii and S. maltophilia [Indeed, a recent study by Falcone and colleagues during the first pandemic wave in Italy (March–April 2020) showed that MDR organisms caused 65.1% of superinfections, slightly higher than our report. Interestingly, the rate of MDR infections increased during the hospital stay, from 50% in the first 7 days up to 70.4% if hospitalized for > 30 days. Although the majority of MDR organisms were represented by CRE, the increase during hospitalization mostly regarded non fermenters such as tophilia .These findings are in line with other reports showing that more than 60% of patients with COVID-19 who had a bacterial infection carried a highly resistant organism . FurtherMore recently, national Italian data evaluating superinfections caused by CRE in hospitalized patients with COVID-19 from March to December 2020 found that the majority of infections occurred in the ICU, although almost one third of episodes occurred in medical wards; however, no data on sub-intensive respiratory units was present. Thirty-day mortality was 33.3% .A. baumannii infection in COVID-19 patients [In a previous study, we evaluated the impact of COVID-19 on MDR Gram-negatives BSIs in a single ICU: while before pandemic patients were more likely to present CR-Kp BSIs, after COVID-19 CR-Ab BSIs were more commonly observed . Furtherpatients .Candida auris [Acinetobacter baumannii seems to be the main pathogen involved, with several outbreaks reported worldwide [Many hospitals have reported MDR outbreaks in ICU during the COVID-19 pandemic, mostly caused by Gram-negative bacteria or da auris . Carbapeorldwide , 43.Another Italian study compared colonization and infection rates before and after COVID-19; authors found that in the COVID-19 period, the incidence rate ratios of colonization and infection with CR-Ab increased 7.5- and 5.5-fold, respectively. Genome sequencing showed that all CR-Ab strains belonged to the CC92/IC2 clonal lineage Pseudomonas aeruginosa or Acinetobacter baumannii) was observed, particularly in settings where enhanced infection control policies and/or antimicrobial stewardship programs were not reported. Furthermore, there was a small increase in the proportion of infections due to resistant Acinetobacter spp [More recently, a systematic review and meta-analysis analyzed the impact of the COVID-19 pandemic on MDR organisms across different healthcare settings by providing data on antimicrobial resistance before or during the COVID-19 pandemic. Authors found that the pandemic was not associated with a change in the incidence or proportion of MRSA or VRE; conversely, although not significant, an increase of resistant Gram-negatives  in patients with severe influenza, which accounted for up to 22–28% of total cases of pulmonary co- and superinfections and were associated with a high mortality rate [In our study, MRSA was detected in 6/64 (9.4%) infective episodes, mostly represented by BSIs. Our results are in line with those recently observed by Falcone et al., who found that MRSA accounted for 5.5% of superinfections episodes . Conversity rate .The low percentage of pulmonary Aspergillosis (1.9%) is in line with the data recently observed in our hospital context, where we found COVID-19 associated pulmonary aspergillosis (CAPA) in only a minority of ICU patients . The incOur study presents some limitations: (i) this is a single-center retrospective observational study with a limited number of patients, that does not include all hospitalized patients; (ii) patients usually received empiric antibiotic therapy at admission, so that the prevalence of co-infections could be possibly underestimated, even if administering antibiotics was a common practice worldwide in that phase of pandemic and we reported similar prevalence of literature; (iii) lack of control group, such as COVID-19 patients hospitalized in ordinary wards or ICUs or patients without COVID-19; (iv) we were unable to investigate with specific microbiological analyses whether the isolation of CR-Ab was a cluster. Moreover, we developed this study in an emergency setting in the middle phase of the second wave, so we should acknowledge that the hospital surge during the pandemic may have affected patients’ outcome.Nevertheless, our study offers important explanations on the prevalence and clinical impact of superinfections in COVID-19 patients hospitalized in a highly intensive care setting other than ICU. Accordingly, we believe that additional studies specifically focused on respiratory sub-intensive care units are warranted, in order to elucidate similitudes and differences, if any, between this special setting and the ICU.In conclusion, we showed that in a COVID-19 respiratory sub-intensive care unit, superinfections were common and represented an independent predictor of mortality. Conversely, co-infections had a low prevalence, supporting an antibiotic sparing strategy in the first phase of SARS-CoV-2 infection. Indeed, exposure to antibiotic therapy was independently associated with superinfections’ development. MDR organisms were found in almost half of patients with superinfections, and CR-Ab as the causative agent was associated with high mortality. For all these reasons, infection control rules and antibiotic stewardship programs represent critical points also in sub-intensive care units to limit the spread of MDR organisms.Additional file 1. Supplementary Table 1. Pathogens isolated for type and site of infection. Supplementary Table 2. Antimicrobial susceptibility testing of the 11 CR-Ab isolates causing superinfections.


TP53 gene. We report the first case of endometrial cancer after yolk sac tumor with LFS.Li-Fraumeni syndrome (LFS) is a rare autosomal dominant disease with high penetrance caused by a germline variant of TP53 c.844C > T, p.( R282 W) with NM_000546.5 variant, a class 5 (C5) variant. This is the first reported case of a yolk sac tumor accompanied by subsequent endometrial cancer that is associated with LFS.The presented female patient underwent right adnexectomy at age 23 because of a yolk sac tumor of the ovary. At the age of 27, the patient was diagnosed with endometrial adenocarcinoma, received cytoreductive surgery and chemotherapy. Given that her personal cancer history along with a strong family history of cancer, her father passing away from lung cancer at age 48 and her grandmother dying of ovarian cancer at age 50, the patient was referred for genetic counseling and testing. Genetic screening revealed a heterozygous pathogenic TP53 pathogenic variation which expanded types of tumor that can be presented in patients with LFS. This case highlights the importance of genetic testing for patients with malignant tumors, as well as patients with a family history of malignant tumors. And our case highlights the necessity of screening for gynecologic tumor in LFS patients.We reported a first case of an endometrial cancer after yolk sac tumor patient with a tumor family history of harboring the germline The online version contains supplementary material available at 10.1186/s12905-023-02426-9. TP53 gene, located on the 17p13.1 chromosome that codes for p53, the most commonly inactivated protein in human cancer. Germline TP53 variants most often occur in the DNA-binding domain, resulting in the production of a dysfunctional p53 protein [TP53 gene encodes a transcription factor that controls the expression of multiple genes, which activate cell cycle arrest for genomic repair or apoptosis, depending on the level of DNA damage [Li-Fraumeni syndrome (LFS) tends to be a widespread, early-onset cancer associated with a germline variant in the A damage . LFS is A damage , 5. WomeA damage . HoweverA 28-year-old female presented with right abdominal discomfort for two weeks in 2017 (age 23) without previous gynecologic abnormalities. Computed tomography (CT) revealed a 14 cm solid cystic mass in the right adnexa without other imaging abnormalities , PR (40% +), P53 (60% +), Ki-67 (30% +), cytokeratin 8/18 (90% +), MLH1( +), PMS2( +), MSH2( +), MSH6( +) and NapsinA negative  and carboplatin for 2 cycles. At the beginning of the third cycle of paclitaxel, the patient showed allergic symptoms including facial flushing, urticaria and labored breathing, so we changed the protocol to bevacizumab, liposomal doxorubicin, and carboplatin for 6 cycles. She eventually received 8 cycles chemotherapy and received maintenance therapy with bevacizumab q 21 days for 16cycles as a meta-analysis showed there were significant differences in progression-free survival (PFS) between patients receiving chemotherapy combined with and without bevacizumab [otherapy . In lighTP53 c.844C > T, p.( R282 W) with NM_000546.5 was confirmed as a germline variation, the rest were verified as the somatic variations, Table For molecular diagnosis, the Qiagen DNeasy Blood & Tissue Kit  was used to extract genomic DNA (gDNA) from FFPE tissues, following the manufacturer’s protocol. Qubit  and agarose gel electrophoresis were used to detect DNA concentration and quality. gDNA 250 ng) was used to construct the sequence library using the method described in previous literature . The hyb50 ng wasTP53 c.844C>T, p.( R282 W) with NM_000546.5 was found in a germline variant, a class 5 (C5) variant, inheritance of which is autosomal dominant. Her father and grandmother suffered from malignant tumor at a young age, it is very likely that her patrilineal family carried the TP53 gene mutation and passed it on to the patient, she got YST accompanied by subsequent EC that is associated with LFS, these two tumors rarely occur in a patient with LFS, this case represented a new finding that extends the clinical scope of LFS; since her father and grandmother had passed away, family verification could not be performed, we confirmed the mutation as a germline heterozygous variation  gene were not detected, as the same as microsatellite instability. Through this germline analysis, we suggested her mother and other patrilineal relatives received the germline testing, which would help the whole family discover or eliminate the risk of tumor in time. Sanger sequencing was performed on her mother in order to rule out carrying the TP53 variant during the follow-up, and the result was negative. It was further proved that her paternal family carried the TP53 variant. She is undergoing gynecological pelvic examinations, tumor markers and regular imaging for the progression of her disease. In addition, regular gastroenteroscopy, head and abdominal CT scans are also included during follow up to rule out other tumors.The timeline of major events in the patient’s treatment is shown in Table TP53 variant (p.R282 W). LFS patients are prone to ovarian and endometrial cancers [TP53 germline variants. TP53 variants are often inherited, and family history is still the key criterion for considering LFS [Patients with LFS have a significant lifetime cancer risk –13. Here cancers  while thring LFS .TP53 is the most mutated gene in tumors with some hot spots. In LFS, most TP53 variants are located in the highly-conserved regions of exons 5–8 of the DNA-binding domain, especially in exons 7 and 8 [TP53 variant in this patient is that a cytosine-to-thymine transition leads to a missense arginine-to-tryptophan transition at amino acid 282 (p. R282W) within exon 8. The R282W germline variant has previously been observed in cohorts that tend to develop mixed adenoneuroendocrine carcinoma of the gallbladder and breast cancer [ 7 and 8 . The spet cancer , 18. Thet cancer , this vat cancer . An in vt cancer . The R28TP53 variantal status and p53 protein expression by immunostaining [Pathological consequences of p53 variant include loss of normal p53 function, dominant-negative variants that can alter wild-type p53 function, and even a rare form of translocation defects with cytoplasmic accumulation and nuclear exclusion, particularly in certain regulatory domain variants . In partstaining . p53 immstaining . A cohorPTEN, frequently detected in EC and often appears in 80% of Cowden syndrome, has the most somatic copy number variations [PTEN is part of the PI3K/AKT/mTOR pathway regulation [PTEN function in EC, via inactivating variant, deletion, or loss of protein expression, is associated with elevated levels of phosphorylated AKT [PTEN in EC was 66.42%. Loss or alteration of PTEN occurs in 45% of EC and is more commonly found in endometrioid EC than in other histological subtypes. A study showed that the TP53 variant frequency in serous endometrial carcinomas (> 90%) differentiated them from the endometrioid subtypes (11.4%) [TP53-mutated endometrial carcinomas is uterine serous carcinoma [TP53 variant also have non-silent variants in PTEN, compared to only 2.6% serous tumors with non-silent TP53 variants, although TP53 variants are not restricted to serous tumors, the co-existing PTEN variants in the endometrioid cases suggest a distinct tumorigenic mechanism [In addition, the current patient carried six types of somatic cancer variants, especially riations , 26. PTEgulation . Loss ofated AKT . The Can (11.4%) , anotherarcinoma . Howeverechanism , which iTP53 germline variants [TP53 germline variants. This paper reports for the first time, a LFS patient with a rare ovarian germ cell tumor. The current understanding of YST at the molecular level is very limited, despite recent cancer genomic characterization efforts. The genomic landscape, evolutionary pattern, and chemoresistance-related mechanisms of this disease are largely unknown, due to lack of molecular evidence in this kind of tumor, further researches are needed with clinical trials and studies dedicated to better understanding the rare gynecological tumor molecular profile and pathogenesis.Ovarian tumors reported in cases of LFS usually correspond to common epithelial tumors . It had variants . HoweverTP53 variant carriers. The results showed that approximately 49% of people carrying with TP53 variants developed a subsequent cancer within 10 years of the first cancer. The average age-specific risk of developing a second cancer was comparable to the risk of developing a first cancer [TP53 variant had an increased risk of developing a second cancer; 30 years after the first cancer was diagnosed, the cumulative probability of developing the second cancer was 57% [In the National Cancer Institute’s LFS study, Phuong L. Mai reported the risk assessment of the first and subsequent cancers and the annual cumulative risk of the first and second cancer in t cancer . Hisada  was 57% . In this was 57% .Genetic counselling and predictive testing should be offered to patients fulfilling the classic LFS criteria as well as to their relatives, with intensified cancer screening if LFS is confirmed. This patient was very cooperative with our recommendation for genetic testing, and she understood that it is very important to her prognosis. As increasingly comprehensive genetic testing is provided to individuals who do not display a confirmed syndrome phenotype or family history, a wider range of aberrant expressions associated with germline variants of cancer-susceptibility genes may be realized. When pathology results show aberrant p53 patterns, as in this case, germline testing should be performed, particularly in young patients.This study represents the first reported case of a young, female patient with LFS who developed EC after a YST, highlighting the importance of screening and surveillance in hereditary cancer-susceptibility syndromes. This case highlights the importance of genetic testing for patients with malignant tumors, as well as patients with a family history of malignant tumors. Genetic screening not only provides enhanced cancer monitoring to improve the prognosis of patients, but also supports individualized and risk-based treatment decisions.Additional file 1: Supplementary Table 1. The genes list of the 688 genes detected in this case.


In this paper, we propose a novel approach to utilize silicon nanowires as high-sensitivity pH sensors. Our approach works based on fixing the current bias of silicon nanowires Ion Sensitive Field Effect Transistors (ISFETs) and monitor the resulting drain voltage as the sensing signal. By fine tuning the injected current levels, we can optimize the sensing conditions according to different sensor requirements. This method proves to be highly suitable for real-time and continuous measurements of biomarkers in human biofluids. To validate our approach, we conducted experiments, with real human sera samples to simulate the composition of human interstitial fluid (ISF), using both the conventional top-gate approach and the optimized constant current method. We successfully demonstrated pH sensing within the physiopathological range of 6.5 to 8, achieving an exceptional level of accuracy in this complex matrix. Specifically, we obtained a maximum error as low as 0.92%  using the constant-current method at the optimal current levels (1.71% for top-gate). Moreover, by utilizing different pools of human sera with varying total protein content, we demonstrated that the protein content among patients does not impact the sensors’ performance in pH sensing. Furthermore, we tested real-human ISF samples collected from volunteers. The obtained accuracy in this scenario was also outstanding, with an error as low as 0.015 pH unit using the constant-current method and 0.178 pH unit in traditional top-gate configuration. Metabolic acidosis in patients admitted to the intensive care unit (ICU) is commonly associated with various causes and unfavorable outcomes. Studies indicate that the rate of pH change in blood may serve as a better predictor of ICU mortality compared to other metabolic indicators ,2,3,4. LIn this context, field-effect transistors (FETs) present excellent potential for the development of wearable and continuous biosensors. They offer advantages such as miniaturization, low power consumption, and the ability to develop label-free bioassays, enabling continuous monitoring. Specifically, fully depleted silicon nanowires (SiNWs) have emerged as promising candidates for integrating a multiplexed biosensing platform ,6,7,8.The objective of our work is to lay the groundwork for the development of wearable and continuous biosensors capable of detecting pH and potentially other biomarkers in human ISF using silicon nanowire transistors a. To achPrevious studies have demonstrated the use of silicon nanowire dual-gate field-effect transistors for ultra-high sensitivity pH sensing ,10. HoweThe fabrication of silicon nanowire arrays on Silicon-On-Insulator (SOI) substrates have been performed at CEA LETI . In our All electrical measurements were performed on a Keithley 4200A. The silicon nanowires ISFETs were cleaned with 99% ethanol and abundantly rinsed with deionized water before each experiment.We propose to investigate a constant drain current operating method for the SOI field-effect transistor, wherein the front gate, back gate, and source terminals are biased with a constant voltage, and the output drain voltage is monitored as the output voltage while the drain current is kept constant a. Our fiREF b. Througnversion a, the trOn the contrary, when operating in strong inversion, the SiNWs transistor can function as a pH sensor with a lower sensitivity but an expanded linear range, particularly when biased in the linear operation region , it leads to a charge perturbation on the oxide surface, which, in turn, is counterbalanced by a modification in the channel conductance or carrier density. In our particular use case, where the drain current flowing in the channel is maintained constant, any alteration in conductance triggers an automatic adjustment in the drain voltage. To establish a relationship between the shift in drain voltage and the pH change in the solution, we can express it using the following equation:The creation of an all-region model of the pH sensor while working in constant current operation is not straightforward. When a pH shift occurs in a solution  three times in PBS 1X at 150 mM. The dilution factor of three was chosen to replicate the concentration of total protein content typically found in human interstitial fluid, which accounts for approximately one-third of the total plasma protein content ,15,16. IS1), Human serum H4522 (S2), and 6914 from Sigma-Aldrich (S3). These samples were diluted three times in PBS 1X at 150 mM. We tested these samples on the same silicon nanowire sensors, and their pH values were determined by back-calculation using the calibration curve obtained from the first set of ISF-like samples. The pH values obtained were then compared to those obtained using a benchtop pH meter (Fisherbrand accumet AE150).The selected pH range corresponds to what is believed to be the relevant range for interstitial fluid, as suggested by clinicians and literature, which considers the interstitial fluid pH to vary between approximately 6.5 and 8 under physiopathological conditions ,18. AddiFinally, real human ISF samples were collected from three different healthy volunteers a. Each vTo compare the pH sensitivities of the silicon nanowires in both “buffer” and “ISF-like” matrices, we conducted voltage sweeping experiments in top-gate configuration. The reference voltage was swept from −0.5 V to 1.5 V using an external Ag/AgCl reference electrode immersed in the liquid under test. The applied back-gate voltage , were injected for 120 seconds. The pH of these “unknown” samples was back-calculated using the pH calibration curve and compared to the readings from the benchtop pH meter.Following the sensitivity assessment in voltage sweeping mode, we proceeded to real-time pH sensing in top-gate configuration. The reference voltage was biased at 0.2 V, the drain voltage at 0.5 V, and both the back gate and the source were biased at 0 V. The drain current was recorded over time. The ISF-like calibrators were injected for 120 seconds, forming a “pyramid” as the pH transitioned from high to low. One of the calibrator samples was retested after the pyramids to assess the accuracy of the calibration curve. Subsequently, three different ISF-like samples with unknown pH  was collected from three healthy volunteers, similarly to other works ,20. The To assess the pH accuracy of our SiNWs platform in real human ISF, we prepared four ISF-like calibration solutions following the method described in the previous section. For each testing method, we generated a calibration curve using these solutions and subsequently tested the “unknown” ISF sample. All experiments were conducted using the same SiNWs sensor.Initially, we tested the SiNWs in voltage sweeping mode in top-gate configuration. In ISF-like solutions, we observed a lower sensitivity a compare fitting b.Following the voltage sweeping test, we proceeded to real-time pH sensing in top gate configuration. We followed the same protocol as described in the previous section, injecting the ISF-like calibrators for 120 seconds to form a pyramid shape, followed by injection of the “unknown” ISF sample c. The pHThe results of this section are summarized in Surprisingly, the pH of the pooled human interstitial fluid sample was notably higher than anticipated, measuring at 8.60. Previous works have measured pH in skeletal muscles interstitial fluid using microdyalisis and found values between 6.9–7.4 , others In summary, our study presents a novel approach to use silicon nanowires ISFETs as high-sensitivity pH sensors for wearable applications. By fixing the current bias of silicon nanowires and monitor the resulting drain voltage as sensing signal, we were able to outperform the accuracy of conventional top-gate methods. We provided a phenomenological explanation on how this constant current method works and what parameters influence the sensing performance. It was demonstrated that by fine tuning the injected current levels and operating the transistors in different regimes, we can optimize the sensing conditions according to different sensor requirements. Typically, we deduced that the weak inversion regime demonstrates superior sensitivity but poor stability and sensing range, while the strong inversion regime shows poorer sensitivity but superior stability and sensing range. We established that the moderate inversion regime provided the optimized balance for pH sensing by offering high sensitivity and good stability across a practical pH range. Additionally, our proposed approach simplififes the requirements of the wearable readout circuit, by enabling voltage measurement instead of current at nanoamper range.In our study, we found that human serum diluted three times in PBS 1X exhibited a similar total protein content to human ISF. We validated this threefold difference in total protein content by conducting BCA assays on three different human ISF samples. Using the three times diluted human serum as ISF-like calibrators, we successfully measured pH in ISF-like solutions with high accuracy (0.07 pH unit) through a constant current method, which outperformed conventional top-gate methods (0.13 pH unit). Subsequently, we applied the constant current method to real human ISF sensing, obtaining a pH value of 8.60 for the pooled human ISF sample. This value was notably higher than what is typically reported in the literature for ISF in general, making us the first group to measure dermal interstitial fluid pH using silicon nanowire ISFETs. Further research is needed to explore the pH baseline and dynamics of dermal human interstitial fluid, as well as to assess the long-term stability and potential applications of our constant current method for continuous and real-time biosensing.


The development ofnew methods and protocols for thesynthesisof biologically active substances remains one of the most importantpillars in organic chemistry, and one of these privileged structuralmotifs are allylic alcohols. The method of choice to date for thesynthesis of these is the Nozaki–Hiyama–Takai–Kishireaction. We describe here a valuable alternative to the synthesisof allylic alcohols via 1,2-metallate rearrangement. In this work,various vinyl boronic esters with different functional groups havebeen applied in the Hoppe–Matteson–Aggarwal reaction.In addition, two monoterpenoids were constructed via this convergentsynthetic strategy. This setup mimics the NHTK reaction2 albeit with reversed polarities, where the alcohol is maskedas a Hoppe anion acting as the nucleophile, while the vinyl compoundfunctions as the electrophile  synthesis,making this disconnection approach even more useful  analogues were designed to reflect the situation inpolyketide frameworks. For example, TIB esters 18, ethers/enol ethers (29-31), and aromatic residues (32-35) were used in any matched or mismatched cases. A tendency of increasing yieldwith the degree of substitution was identified (52–79%). Thedecisive factor for this observation is probably the enhanced migrationability in the 1,2-metallate rearrangement due to the inductive effectof the aliphatic substituents. Only fully substituted vinyl boronicester 26 stands out by showing a comparable yield tounsubstituted 23 (50% vs 52%), possibly due to the sterichindrance in the ate-complex overruling the increased migration ability.At the beginning of our studies,vinyl boronic esters 23-27 with dif23-272a. The e8. After protection of the generatedalcohol, the vinylsilane unit in 44 could be used innumerous reactions such as electrophilic substitutions, Heck-likereactions, or transmetalations and could thus represent an importantbuilding block in total synthesis.14 Inaddition to the vinylsilane unit, enol ether functions were also introduced.Here, the observed yield of the cyclic enol ether 46 (39%)was significantly lower than that of the open-chain enol ether 45 (64%). The reaction of TIB ester 18 and moderate yield (45%).The low selectivity in the case of the TIB ester could be attributedto π–π interactions between the TIB group and thearomatic substituent of the vinyl boronic ester. As a result of thisattractive interaction, the seven-membered transition state ate-II could occur during ate-complex formation, which would favorthe ate-complex formation under inversion. In 2017, the group of Aggarwalinvestigated the SE2′ reaction of allyl-boronatesby treating the Hoppe anion derived from TIB ester 21 in the presence of (+)-sparteine with elongated vinyl boronic ester 52.8 In contrast to our observationswith the directly substituted vinyl boronic esters, they did not observean inversion process. Therefore, it can be assumed that both the directconjugation to the double bond and the enormous rigidity are crucialfor the inversion. This inversion process probably runs in strongcompetition with the usually observed retention and could explainthe low selectivity for the TIB ester. This attractive interactionis not possible in the case of the Cb group; therefore, no inversionprocess can occur here, and accordingly, the excellent selectivity(≥19:1) induced by (+)-sparteine is maintained. Based on theseobservations, the other vinyl boronic esters with aromatic substituentswere converted with carbamate 22 and in an excellent selectivity(≥19:1). In addition to the simple phenyl substituent, electron-richand -poor aromatic substituents were introduced in moderate to goodyields .A remarkableobservation was made when vinyl boronic esters witharomatic substituents were used in the 1,2-metallate rearrangement3 in comb21 with vin2115 and (−)-rosiridol(56)16 were constructed via our Hoppe–Matteson–Aggarwalprotocol (Rhodiola rosea (golden root) and Rhodiola sachalinensis.17 Sachalinol A (55) was first described by the group of Kadota in 2001.15a The strong cytotoxic property and good anticanceractivity18 make Sachalinol A (55) an attractive synthetic target. Rosiridol (56) isthe aglycone of the MAO inhibitor rosiridin and thus also a syntheticallyvaluable target.19 The synthesis of thesetwo natural products will be carried out via a convergent approachof the previously described Hoppe–Matteson–Aggarwalprotocol, requiring in both cases the literature-known vinyl boronicester 58.20In order to show that this chemistry can be applied tonaturalproduct classes other than polyketides, the two monoterpenoids (−)-sachalinolA -sparteine and subsequent treatment with vinyl boronic ester 58 (78% o2s)20 afforded allylicalcohol 62 in a good yield of 68% over two steps andin excellent selectivity (≥19:1). The synthesis of sachalinolA (55) was completed by global deprotection using TBAFin an overall yield of 35% (6 steps lls).The first step in the synthesis of TIB ester etone 60 5. Additi59 requiredfor the synthesisof (−)-rosiridol (56) was carried out under phasetransfer conditions22 with commerciallyavailable homoallylic bromide 63 (sBuLi and (+)-sparteinewith vinyl boronic ester 58 (78% o2s)20 afforded allylic alcohol 64 in a very goodyield of 69% after oxidation. Deprotection of the primary TBS etherwith TBAF afforded (−)-rosiridol (56) in an overallyield of 51% (5 steps lls).The synthesis of TIBester omide 63 6. The su23 However, the use of the corresponding carbamates again led to theusual excellent selectivities. Moreover, the applicability of ourprotocol in convergent synthesis planning was impressively demonstratedby the total synthesis of two monoterpenoids. Thus, to the best ofour knowledge, our synthesis of sachalinol A (55) representsthe shortest synthesis of this cytotoxic compound to date.24 Accordingly, this protocol of the Hoppe–Matteson–Aggarwalchemistry represents a synthetically very valuable alternative tothe NHTK reaction.Inconclusion, our protocol of the Hoppe–Matteson–Aggarwalchemistry allowed us to convert a broad range of vinyl boronic estersinto allylic alcohols in consistently good yields and excellent selectivities.Only the vinyl boronic esters featuring aromatic substituents in combinationwith the TIB group were an exception. Here, in addition to the usualretention, inversion was also observed during ate-complex formation.This observation clearly contradicts the established thesis that boronicesters react exclusively under retention.