Introduction: The coronavirus disease 2019 (COVID-19) pandemic has exacerbated longstanding inequities throughout the United States, disproportionately concentrating adverse social, economic, and health-related outcomes among low-income communities and communities of color. Inequitable distribution, prioritization, and uptake of COVID-19 vaccines due to systemic and organizational barriers add to these disproportionate impacts across the United States. Similar patterns have been observed within Orange County, California (OC).Methods: In response to COVID-19 vaccine inequities unfolding locally, the Orange County Health Equity COVID-19 community–academic partnership generated a tool to guide a more equitable vaccine approach. Contents of the OC vaccine equity best practices checklist emerged through synthesis of community-level knowledge about vaccine inequities, literature regarding equitable vaccination considerations, and practice-based health equity guides. We combined into a memo: the checklist, a written explanation of its goals and origins, and three specific action steps meant to further strengthen the focus on vaccine equity. The memo was endorsed by partnership members and distributed to county officials.Discussion: Since the initial composition of the checklist, the local vaccine distribution approach has shifted, suggesting that equitable pandemic responses require continual re-evaluation of local needs and adjustments to recommendations as new information emerges. To understand and address structural changes needed to reduce racial and socioeconomic inequities exacerbated by the pandemic, authentic partnerships between community, academic, and public health practice partners are necessary.Conclusion: As we face continued COVID-19 vaccine rollout, booster vaccination, and future pandemic challenges, community knowledge and public health literature should be integrated to inform similar equity-driven strategic actions. These guidelines integrate community knowledge and public health literature to inform strategic actions in equitable COVID-19 vaccine implementation at present, future COVID-19 booster vaccinations, and vaccine distribution efforts for future pandemics.21 racial/ethnic minority populations in OC are disproportionately affected by inequities in the social determinants of health that shape poorer health outcomes than non-Latino white counterparts.23 Given that OC is the sixth most populated county in the United States, OC's 34% Hispanic/Lation, 21.7% Asian, 0.4% Native Hawaiian and other Pacific Islander, 2.1% black or African American, and 1.0% American Indian and Alaska Native population represent >1.8 million individuals.21 In addition, despite OC's percentage of persons living in poverty being slightly lower than the national average , this encompasses >300,000 individuals.21Despite being a majority–minority county since 2004,24–26 Within California, a major factor in this higher rate of infection rests in the over-representation of low-income communities and communities of color in front-line public-facing low-wage essential jobs such as farm work, cleaning and janitorial, and food industries.27 This has left 5.7 million of these workers at heightened risk of COVID-19.28During the COVID-19 pandemic, low-income communities and communities of color within OC have experienced higher rates of SARS-CoV-2 infection and COVID-19 morbidity and mortality, similar to patterns observed nationwide.29 is essential workers' limited access to health services and/or sick days. The state's initial move toward age-based vaccine priority did not center vulnerable essential workers and left various community labor organizations and labor unions concerned about vaccine equity for those whose exposure is exacerbated by existing structural racial inequity and capitalist exploitation at the core of U.S. racial capital.30–32Compounding this risk for the 19% of the state's essential workforce living in near-poverty conditionsAlthough federal organizations including the Centers for Disease Control and Prevention provided some guidance for vaccine distribution, the federal government did not establish a national infrastructure to support local distribution plans. In many cases, states have given county health departments discretion in applying vaccine eligibility categories and left distribution solutions to counties. Consequently, OC, as elsewhere, was tasked with developing and implementing its distribution plan for the COVID-19 vaccine.34 In the absence of federal and societal support for evidence-based public health practice, health departments struggled during the pandemic's first year to implement equitable COVID-19 mitigation, testing, and vaccination strategies.36Moreover, politically induced attacks on evidence-based public health efforts and the officials who promulgate them have contributed to institutional turnover and loss of longstanding public health leaders who are critical partners in resolving the COVID-19 pandemic.37 Rather, it is about bouncing forward by centering community members' active participation in creating the OC public health and safety interventions needed to eliminate health inequities and to build health equity.37–39In May of 2020, the Orange County Health Equity COVID-19 community–academic partnership (see Acknowledgments section for list of partner organizations) was formed to bring together community-based health equity leaders and health equity academic partners to elevate the need for and guide local COVID-19 equity response and recovery initiatives. The partnership was formed under the belief that building local resilience in response to the pandemic is not about bouncing back to prepandemic conditions.40–42When local actions reflect this orientation, we believe that OC will be better positioned to (1) refrain from increasing and exacerbating harm to local communities of color, indigenous communities native to the land and who have been displaced to Orange County, low-income communities that are suffering social and economic hardships, those who are homeless or facing housing insecurity, and people who are incarcerated or detained—all of whom have faced disproportionate negative impacts of COVID-19 and (2) spur and foster social cohesion, inclusion, and trust in our public health and safety pandemic responses in ways that increase the power, participation, and long-term vitality of such communities.43; and urge the implementation and maintenance of mask mandates. As COVID-19 vaccine development rapidly progressed and the first emergency use authorization of a vaccine to combat SARS-CoV-2 was issued by the U.S. Food and Drug Administration in December 2020,44 we shifted our collective focus toward vaccine equity.Building upon our vision, members worked together in the early months of our partnership to inform strategic actions to improve equitable access to COVID-19 testing, data, and communication; support the training of contact tracers and resident-led COVID-19 responseThis shift occurred as part of our continued process of identifying priority areas in advocating for equitable COVID-19 recovery. In the following section, we describe our partnership processes and approach to advocating for equitable vaccine allocation, distribution, outreach, communication, and data monitoring through the development and dissemination of a vaccine equity tool centered around the pivotal role of community-based knowledge and localized context in shaping recommendations.The Orange County Health Equity COVID-19 community–academic partnership implemented several strategies to center our work around the voices of community partners while attending to potential power imbalances between academic and community partnership members. Throughout this stage of the partnership, we held biweekly meetings during which community and academic partners cocreated agendas with items suggested by community partners taking priority in terms of both order discussed and time allotted.Depending on community partner availability and capacity to attend partnership meetings, academic partners would help to focus meetings on agenda items most relevant to those present. Academic partners' roles during these meetings centered on facilitating dialogue between community partners, listening to and identifying themes within community partners' experiences, and codeveloping action plans to address these themes. Academic partners would also connect with members who were unable to attend to share updates and create space for additional feedback.Ultimately, academic partners aimed to support community partners' priorities—especially when navigating difficult conversations where further power imbalances may exist or when leveraging resources available through the university would be beneficial. One example of how this has been operationalized is by academic partners facilitating conversations with local policy leaders with whom community partners expressed unease around discussing relevant issues freely due to underlying political considerations that did not constrain academic voices in the same way. This partnership and vaccine equity advocacy strategy is classified as exempt by the University of California, Irvine.In January 2021, shortly after COVID-19 vaccine rollout began, partnership members amplified longstanding concerns regarding vaccine inequities unfolding in OC communities of color, indigenous communities, and low-income communities.12 OC's local public health department's  initial vaccine rollout efforts focused on prioritizing the vaccine to adults 65 years of age and older—regardless of race/ethnicity, socioeconomic position, or zip code46;—implemented vaccine sign-up through a mobile application that was only available in English for the first three weeks of the vaccine rollout47; and only made the vaccine available at super points of distribution (Super POD) such as Disneyland and a small private university located in an affluent region.46 This initial rollout rendered vaccination sites and vaccines accessible to more economically, technologically, and time resourced communities that were English language dominant.45As a microcosm of trends nationwide,Although aware of vaccine shortages and OCHCA's multifaceted response efforts, partnership members emphasized the crucial need to strengthen the vaccine equity approach, with particular attention to communities experiencing the greatest negative impacts of COVID-19. Partnership members engaged OCHCA by calling for vaccine equity in spaces such as OCHCA-designated task force meetings, one-on-one discussions between county officials and partnership members, and partnership planning meetings.After these efforts, community partners acknowledged OCHCA's progress toward improving equitable vaccine access , while emphasizing that more needs to be done as the populations they serve were still facing inequitable vaccine access. Community partners invited our partnership to develop the OC COVID-19 vaccine equity best practices checklist  with hop48–51After listening carefully to the concerns raised by community partners, academic partners responded to community members' invitation and began to draft the checklist. The checklist draft underwent multiple reviews by community and academic partners through an iterative process. Thus, the final checklist was generated through the synthesis of community-level knowledge in the form of partnership member input that prioritized the perspectives of community leaders, a strategic review of the literature regarding equitable vaccine distribution considerations, and health equity guides produced by similar coalitions in other regions of California.Based on the literature and priority areas emphasized by community partners, we organized the OC COVID-19 vaccine equity best practices checklist to highlight vaccine equity best practices in four key areas: vaccination data, allocation and distribution, access, and community outreach and communication. Within the checklist, each focus area is presented with an overarching statement defining its fundamental purpose.For example, the focus area of “vaccine data” is delineated as the need for distributors to “Provide real-time data regarding vaccine access and uptake to monitor and address vaccine inequities.” Furthermore, the focus area of “access” is defined as the need to “Reduce barriers and provide the necessary resources for communities to achieve equitable access to the COVID-19 vaccine.”51–53To make each focus area's overarching goal actionable, we provide four to five explicit and measurable items pivotal to equitable development and implementation of vaccine distribution programs at each level of the social ecological model.55 was addressed in checklist items pertaining to community outreach and communication and vaccine data. Specifically, “Communicate messages that promote anti-racism, anti-xenophobia, and anti-hate as it relates to COVID-19. Act on this messaging by providing/identifying methods to report transgressions and include this information” and “Make vaccine distribution data readily available and comprehensible to members of the community.”For example, historically rooted exclusion from and mistrust of medical and public health systems by members of minority and immigrant populationsPolicy- and community-level barriers are addressed in the item, “Create multilingual, culturally sensitive, and literacy-appropriate education and outreach strategies available across multiple media platforms and discussion formats designed to reach the most impacted communities.” Although the underlying issues at the root of inequities exacerbated by COVID-19 cannot be solved by performing the checklist items, we aimed to provide recommendations for actions that could be implemented swiftly to mitigate the ongoing inequitable outcomes of vaccine distribution in OC.To further strengthen the focus on vaccine equity, we prioritized three action steps grounded in local contextual considerations, community partner recommendations, and best practices emerging in the literature .These action steps sought to address the fundamental issues shaping barriers to equitable vaccine implementation, including the need to (1) implement an evidence-based vaccine distribution strategy that prioritizes the most affected social groups by applying an intersectional approach ; (2) engage in authentic partnership with local community leaders and improve transparency; and (3) engage in contextually, culturally, and linguistically appropriate outreach efforts to combat mistrust in science and public health institutions that have escalated in the pandemic and have been further galvanized by inequitable vaccine rollout and shutting out of information streams.To put these tools into action, we combined the checklist, a written explanation of its goals and origins, and the action steps into a memo. After feedback and iterative review by community partners followed by academic partners, partnership members were invited to endorse the memo as signatories. After careful discussion with community partners, they requested that the memo be sent on behalf of the partnership and signed by academic partners to support local community-based organizations that navigate a delicate role of advocating for change with a powerful institution that shapes the distribution of county resources. In February 2020, we sent the memo to OCHCA and the county board of supervisors with the request for continued communication regarding plans to enact the outlined distribution practices.As development of the checklist, action steps, and memo was underway, partnership members discussed balancing the significance of completeness of the vaccine equity requests versus urgency of delivering the checklist and memo to shape vaccine equity locally. The concern was that too much weight would be placed on perfecting the tool, taking away from the timeliness of its delivery. To maximize potential impact, partnership members collectively determined that the memo should be produced in a manner that prioritized prompt distribution. With this decision came the understanding that the issues addressed, and recommendations made will need to be updated over time as equity concerns evolve and local practice-based evidence expands.13 Without increased attention to best practices for equity across the areas of vaccine data, allocation and distribution, access, and community outreach and communication, communities of color, indigenous communities, and low-income communities will continue to suffer disproportionate negative outcomes.Equitable COVID-19 vaccine distribution and uptake are pivotal to successfully reducing COVID-19 morbidity, mortality, and associated inequities.Purposefully designed to support these needs, the OC COVID-19 vaccine equity best practices checklist demonstrates how vaccine equity best practices can be communicated and promoted across various stakeholder groups throughout the development, mobilization, and continued adaptation of vaccine programs. Moreover, our partnership's nascency, goals, and actions to date serve as a practical demonstration regarding authentic community–academic–practice relationships focused on health equity.56–58 Under this coordination, it stood likely that although many of the crucial equity considerations would remain the same, the approach required to appropriately advocate for and address equity concerns would change.Following the initial composition of the OC COVID-19 vaccine equity best practices checklist, California shifted to a new distribution scheme, with a major insurer managing state-wide distribution efforts—a role that lasted only a few months.Notably, authentic relationships and our practice of continual one-on-one and meeting-based check-ins among partnership members helped to further surface an underlying critical analysis of the fact that, to date, our local multicounty serving entities  have escaped our focus on vaccine equity advocacy. As contexts continually fluctuate, advocates must be ready and willing to adjust in response to shifts at local, state, and national levels.A major strength of the checklist comes from the collaborative efforts and input from diverse partnership members, which allowed for a community-centered and locally contextualized evaluation of pressing issues. In addition, our process highlights the ability to create institutionalized opportunities for community partners to be authentically involved in decision-making processes—particularly those related to advancing equity through acknowledging community-specific needs. To this end, we understand that each municipality, county, or state effort to achieve equitable vaccination faces diverse challenges, thus we do not anticipate the content of this tool to be uniformly appropriate across diverse settings. Yet, the actions outlined here may prove a useful technique in advocating for change.59–61 Other partnerships see these same COVID-19 equity opportunities and have formed or shifted their focus with an aim to address similar challenges.63In addition, we recognize that partnerships that equitably bring together community, academic, and public health practice partners have the potential to increase understanding of how racial, social, and economic inequities unfold locally and develop and implement opportunities for structural intervention.Although we are acutely aware that the development and distribution of our checklist do not solve the systemic barriers responsible for COVID-19 inequities at their root, our partnership and this advocacy tool were a decisive response to the prevailing emergency environment that unevenly impacted historically marginalized communities. In addition to advocating for equity when addressing the immediate threats posed by inequitable vaccine distribution in OC, our partnership aims to broaden its scope of advocacy efforts to address structural drivers of health inequities in OC. The equitable relationship between community, academic, and practice partnership members is pivotal to achieving this goal.Furthermore, as was discussed in several of our partnership meetings, community–academic–practice partnerships are critical to amplifying the voices of community-based organizations who are otherwise vulnerable given often delicate relationships based on inequitable power relationships between affected communities and governmental institutions. Utilizing the less vulnerable positionality of universities to advocate for community-identified priorities is essential to advocating for equity until authentic partnerships between community-based organizations and governing institutions can be achieved.59–61In addition, partnerships or coalitions that bring together representatives of multiple affected communities can transform common dynamics created by racial capitalism, which often segregate issues and position organizations and initiatives to compete against each other for perceived scarce resources and thus impede collective action. By providing a space that supports and connects diverse community-based organizations and initiatives through inclusive conversations regarding shared and disparate needs, community–academic–practice partnerships can strengthen relationships across organizations, initiatives, and groups; foster a collective identity; and advance mutual equity initiatives such as the OC COVID-19 vaccine equity best practices checklist.15 Officials in charge of COVID-19 vaccine distribution plans should aim to engage in authentic and equitable partnerships with community-based organizations and coalitions to gain insight into locally contextualized community needs.Health inequities effecting low-income communities, communities of color, and indigenous communities across the United States as a result of the COVID-19 pandemic will persist if vaccines are not equitably allocated, distributed, and accepted within the most impacted communities.Our partnership provides an example of an approach to effectively integrate these groups into critical dialogue and strategic planning regarding each element of an equitable vaccine strategy. Partnerships such as ours are not only necessary within the immediate landscape of COVID-19 vaccinations but will also need to continue so that we can understand and address the structural changes necessary for the reduction of both existing and forthcoming health inequities.1–9 These effects relate to risk of exposure, limited access to resources to protect against transmission, and related morbidity and mortality—all of which are grounded in longstanding racial, social, and economic inequities.1–9 Although COVID-19 vaccinations have become more widely available, systemic and organizational barriers have furthered disproportionate impacts through the inequitable distribution, prioritization, and uptake of vaccines.64The COVID-19 pandemic has had disproportionate negative social, economic, and health-related impacts on various marginalized communities throughout the United States.Authentic partnerships between community-based organizations and initiatives, academic institutions, and public health practitioners are essential to understanding and addressing structural change needed to reduce and eventually eliminate inequities. Our partnership developed the OC COVID-19 vaccine equity best practices checklist to guide a more equitable vaccine approach with an emphasis on the pivotal role community-based knowledge and localized context should have in shaping recommendations.This checklist was developed to provide guidance within the immediate emergency setting focusing on local public health actions that were not being implemented within OC. Similar collective approaches and advocacy will be necessary as we face continued COVID-19 vaccine distribution, booster vaccinations, and future pandemic events.


Gerontologists have long documented how age is associated with political participation. However, few studies have considered how macrocontextual factors shape participation across the life span. Moreover, very few studies have dealt with political engagement and aging in emerging democracies, including those in Africa. This study addresses those gaps, integrating the most recent three waves of Afrobarometer survey data (2011–2018) with country-level data from the freedom house (i.e. freedom index). Findings reveal that, at the individual level, an age gap widens for engagement in protests and shrinks for electoral and non-electoral political participation. When the political context is considered, however, we find that political freedom softens the drop-off of protest behavior at later ages. For electoral and non-electoral political participation, we find that freer countries lessen the expected growth in engagement across the life span. The study implies that political oppression shapes the links between age and political behaviour, but the processes seem different depending on whether they are engaging in risky (where the age gap widens) or non-risky (where the age gap shrinks) political forms of engagement.


C. trachomatis (CT) infections leads to an increased burden on human reproductive health, especially in middle- and low-income settings. Early detection and management of these infections could play a decisive role in controlling this public health burden. The objective of this review is to provide an insight into the evolution of diagnostic methods for CT infections through the development of new molecular technologies, emphasizing on -omics’ technologies and their significance as diagnostic tools both for effective patient management and control of disease transmission.Chlamydial genital infections constitute significant sexually transmitted infections worldwide. The often asymptomatic status of Narrative review of the diagnostic methodologies of CT infections and the impact of the introduction of -omics’ technologies on their diagnosis by review of the literature.Various methodologies are discussed with respect to working principles, required specifications, advantages, and disadvantages. Implementing the most accurate methods in diagnosis is highlighted as the cornerstone in managing CT infections.Diagnostics based on -omics’ technologies are considered to be the most pertinent modalities in CT testing when compared to other available methods. There is a need to modify these effective and accurate diagnostic tools in order to render them more available and feasible in all settings, especially aiming on turning them to rapid point-of-care tests for effective patient management and disease control. Chlamydia trachomatis (CT) infections impose a huge burden on human sexual and reproductive health. They are considered to be the most significant causes of bacterial sexually transmitted infections (STIs) worldwide [orldwide –3. The Worldwide . Accordiorldwide . As CT iChlamydiaceae, because of its association with ocular trachoma and a wide array of genital tract manifestations in both males and females [ompA gene encoding for MOMP; thus the cultivation of cell lines is no longer required. The various genovars were named using similar letter-based nomenclatures as for serovars. Some of the typing methods involved are ompA sequencing, multilocus sequence typing (MLST), multilocus variable number tandem repeat analysis (MLVA), and whole-genome sequencing (WGS). In addition to the 19 serovars mentioned above, genovars Ja and L2b have been recognized [Chlamydia is an obligate intracellular pathogen characterized by a distinctive life cycle involving dual forms: an infectious extracellular elementary body (EB) and an intracellular reticulate body (RB) for replication. These bacteria grow only within the specialized vacuoles called inclusions in eukaryotic cells. CT comprise the most popular species in the family  females . Origina females , 8. To dcognized . The varSeveral risk factors are reported that may contribute to developing CT infections. The sexually active younger age group of < 25 years is considered to be at a higher risk of acquiring the infection. Other major factors include multiple sex partners, inconsistent usage of barrier methods with new sexual partners, past history of STIs, and exposure to commercial sex partners , 11. GenDuring pregnancy, CT infections have been mostly associated with adverse outcomes such as recurrent miscarriages, stillbirth, premature rupture of membranes, preterm labor with low birth weight, and postpartum endometritis. Maternal infection can be transmitted to the neonate during vaginal delivery, through contact with infected cervical secretions, potentially leading to neonatal inclusion conjunctivitis, otitis media, and infant pneumonia. Long-term respiratory sequelae may be significant in these children when followed-up , 17.Being a silent pathogen, most of the infections remain concealed and may later be revealed to have influenced the reproductive health, causing female sterility, which in turn brings with it an associated financial burden. Adequate screening programs need to be implemented in order to diagnose such infections. CDC recommends annual screening for chlamydia among sexually active females aged < 26 years and in older females associated with risk factors, although there is no emphasis on routine screening in men , 15. HowFrom all the above, it becomes clear that the real challenge in managing CT infections is to identify these silent infections. This requires efficient diagnostic methods with adequate sensitivity and specificity, which, when implemented as part of screening programs, can contribute to detecting new cases and controlling the transmission of infections. This review focusses on the currently available diagnostic approaches in detecting chlamydial infections, with special emphasis on the significance of -omics’ technologies in molecular testing.CT diagnosis involves various direct and indirect laboratory-based methods whose efficiency depends primarily on the adequate collection and transportation of clinical specimens. As chlamydia is an obligate intracellular bacteria, specimens collected must involve host cells harboring the pathogen, especially when direct methods are involved in testing . Also, tGenital swabs are known to be the most commonly collected specimens for the detection of chlamydial infections. Dacron, rayon, cotton, and calcium-alginate tipped swabs are generally preferred , 21. In Genitourinary specimens collected from various anatomical sites can influence diagnosis owing to the difference in their bacterial load. Michel et al. have elaborated on the importance of bacterial load in varied specimen types and their association with clinical symptoms . In men,Cervical cytological examination using Papanicolaou (Pap) smears has been among the early techniques adopted in screening for cervical cancer. This simple, rapid, readily accessible method is also known to provide clues in diagnosing STIs, with the associated inflammatory changes . SeveralIsolating CT on cell cultures is considered to be the most optimal method of detecting urogenital infections caused by the organism. Because of the high specificity of this technique, it had been referred to as the “gold standard” for CT detection in the past. These cell culture methods require invasive specimens like endocervical and urethral swabs which must be collected precisely to include host cells with viable organisms. They must be transferred in suitable transport media like sucrose-glutamate phosphate buffer and transported at a temperature < 4 °C within 24 h of collection. If the processing within 24 h is not secured, transport at a temperature of − 70 °C on dry ice is required . Cell liDue to these drawbacks of culture methods in CT diagnosis, various other methods, which are non-culture based, have been developed and implemented. Methods demonstrating CT antigens from clinical specimens are among these improved techniques. As these methods do not require viable bacteria, highly specific procedures involved in specimen collection and transport, as needed for culture samples, are not necessary. Also, the advent of these methods has made CT testing possible in laboratories lacking the capability to maintain cell cultures.Direct fluorescence antibody (DFA) test performed directly on clinical specimens is one of the most useful diagnostic methods available. This uses fluorescein isothiocyanate-labeled monoclonal antibodies directed against chlamydial lipopolysaccharide (LPS) or MOMP of CT to detect chlamydial inclusions. Using monoclonal antibody reagents specific for MOMP of CT has led to 98–99% specificity and 80–90% sensitivity in comparison with cell culture methods. Very high specificity shown by the DFA test owes to it being dependent on visualizing distinctive morphology and staining properties of chlamydial inclusions as in cultured cells. This test is relatively rapid and can be performed in about 30 min. It does not depend on a temperature-controlled transportation system. But this technique demands expertise and time in examination and interpretation of results, thus confining its use in low volume setups. However, due to its high specificity, it has been used as a confirmatory test for positive results obtained by other non-culture tests , 19, 21.Enzyme immunoassays (EIAs) based on the detection of LPS genus-specific antigen was introduced later in diagnostics. These tests are based on LPS which is more abundant and soluble than MOMP. The major drawback of these techniques is the cross-reaction of LPS-specific antibodies to LPS of other gram-negative bacteria, leading to false-positive results. In order to improve specificity, certain blocking assays were developed which consist of re-testing the previously positive results in the presence of monoclonal antibodies specific to chlamydial LPS. As a result, these confirmatory tests increased the specificity of EIAs from 97 to > 99% rendering them as appropriate screening tests, even in a population with low prevalence , 19. TheThe role of serological techniques in CT diagnosis is very limited, as they have been proven to be inadequate. The chlamydial antibody response is either delayed or absent in some individuals. Most of these techniques detect anti-chlamydial antibodies directed against LPS antigen which is a genus-specific antigen. Often these tests fail to differentiate antibodies produced against different species of chlamydia. As a result, these tests are not recommended as part of screening programs. However, they may be of diagnostic importance in chronic and invasive infections like in cases of PID and sexually acquired reactive arthritis (SARA) where direct detection of CT from genital specimens is not possible but found to be associated with increased anti-chlamydial antibody titers , 8.Some of the serological tests evaluated for the diagnosis of chlamydial infections are the microimmunofluorescence (MIF) test and enzyme-linked immunosorbent assay (ELISA). The MIF test was believed to be the most sensitive serological test, which even presented species and serovar-specific responses. This was regarded as the “serological gold standard” for chlamydial testing . HoweverTraditional methods for the diagnosis of CT infections have several disadvantages including low sensitivity, the requirement of invasive specimens, longer duration for performance and reporting, and associated high cost. Also, these tests may produce false-negative results, which can in turn lead to the spread of infections and increase in complications. These limitations point out the need to develop tests with higher sensitivity and specificity, in order to be used either as standard standalone reference tools or in combination with other traditional methods. The introduction of these methods led to a drastic leap in CT diagnosis, enabling better identification of cases and management.Neisseria gonorrhoeae from a single specimen [Commercially available nucleic acid hybridization (NAH) methods have been used in laboratories with high specimen load. The most commonly used method is the PACE 2 test . This method uses a chemiluminescent DNA probe that hybridizes to a species-specific sequence of chlamydial 16S rRNA. Another DNA probe test developed in the later years is the PACE 2C which simultaneously detects both CT and specimen . One othspecimen . These rspecimen , 46, 47.specimen . SpecialThe development of nucleic acid amplification tests (NAAT) has been regarded as a major breakthrough in CT diagnostics and has utterly transformed the field. These tests can detect chlamydial DNA or RNA based on amplification technology and have almost succeeded in replacing cell culture methods, being referred to as the “new expanded gold standard.” These tests demonstrate high sensitivity, as they are efficient enough to detect the presence of a single nucleic sequence in the clinical specimen and are at the same time highly specific. Additionally, these tests have introduced the concept of non-invasive sampling for diagnosing genital CT. Studies have shown their efficacy in detecting CT in self-collected vaginal swabs and urine specimens , 49, 50.The major role of NAAT technologies in chlamydial diagnostics was appreciated with the development of polymerase chain reaction (PCR) and ligase chain reaction (LCR) techniques. Both of these methods involve the amplification of nucleotide sequences within a cryptic plasmid . In 1993Following PCR and LCR, newer molecular technologies have been developed for CT diagnosis, which include the transcription-mediated amplification (TMA) test, the APTIMA Combo 2 , and the strand displacement amplification-based ProbeTec . The TMA test is directed against ribosomal RNA, which works as an isothermal system using enzymatic target amplification and chemiluminescent detection in a single tube format . FDA hasAnother FDA cleared NAAT is the Cepheid Xpert CT/NG , a rapid, easy-to-use, cartridge-based real-time PCR. This assay has been proven to be highly effective in testing urogenital specimens from both symptomatic and asymptomatic population . EquallyN. gonorrhoeae in the same assay using a single specimen and therefore enabling better patient management. However, the requirement of well-trained personnel for testing specimens and the necessity of sophisticated laboratory infrastructure for most of these assays have limited their usage to high-end settings. Additionally, the high financial cost of these methods has prevented their popularity in resource-poor setups.NAATs have been the most competent methods devised for CT diagnosis with high sensitivity and specificity values. The fact that non-invasive samples are accepted for testing has favored the use of these methodologies in screening asymptomatic population. These are found to be considerably rapid, while providing accurate results. They are even considered efficient enough to replace culture methods in testing specimens implicated with legal issues. Most of these test formats are designed to detect both CT and Though a highly conserved pathogen, genetic variation involving a 377 base pair deletion in the cryptic plasmid has been noted for CT. This variant denoted as nvCT was first reported from Sweden in 2006 by Ripa and Nilsson and has been associated with reduced transmission duration. Earlier versions of some commercial NAAT systems had failed to detect this variant as the targets for these NAATs were located in the acquired deletion site of the plasmid. While designing in-house PCRs or using commercial NAAT systems for CT detection, ability to detect this variant should be considered to avoid false-negative results –68.“Point-of-care” testing (POCT) in a public health setting provides major advantages compared with the often cumbersome sample logistics of medical laboratories. The relatively low costs of the testing equipment allow more installations closer to the patient. This also leads to a faster availability of the results for the patient and medical care team and, in eligible cases, an immediate commence of treatment. The so achieved initiation of treatment during the first visit to the health facility could result in higher rates of follow-up.The availability of POCT in clinical settings is considered essential in controlling chlamydial infections. According to WHO, such tests should be affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end-users (ASSURED criteria) . ChlamydImmunochromatography-based rapid diagnostic tests (RDTs) detecting chlamydial LPS antigen have been developed, which require minimal logistics and deliver rapid results enabling treatment of positive cases immediately. But poor sensitivity made them inappropriate for testing both symptomatic and asymptomatic patients –73. To oThe sensitivity, specificity, advantages, and disadvantages of various diagnostic tests available for CT detection are summarized in Table The advent of -omics’ technologies has led to a major revolution in diagnostics of a wide array of clinical conditions. These assays can prove to be instrumental in managing various infectious diseases, as they could reveal critical details of the pathophysiological mechanisms involved in health and disease. These methods in combination with bioinformatics have contributed immensely to understanding the pathophysiology and complex interplay between hosts and pathogens. The wider term “-omics” encompasses a range of technologies for analyzing and detecting genes (genomics), messenger RNA (mRNA) (transcriptomics), proteins (proteomics), and metabolites (metabolomics) from clinical specimens, which could prove to be important biomarkers in various clinical conditions. These technologies, especially when utilized in an integrated manner, could evolve as powerful diagnostic tools in clinical medicine , 80.The utility of -omics’ in CT diagnosis has evolved over the years. Despite its high infection rates and impact on human health, the unique biology of these obligate intracellular bacteria has not been well explored until the introduction of -omics’ investigations. These modern modalities have been fundamental in shedding light on the natural history, evolutionary aspects, and complex pathophysiology of this mysterious pathogen, thus paving new ways to the improvement of patient care , 82.Genomics, the first and most widely known -omics’ technology, studies the genetic profile of organisms either with respect to the whole genome or by targeting exonic coding regions or analyzing single nucleotide polymorphisms (SNPs) . These hompA gene using restriction enzymes, where each genotype produces fragments of varying lengths that are electrophoretically identified. This technique is more focused on looking into the epidemiological patterns of various genovars of CT but fails to perform in infections with mixed genotypes and to identify single nucleotide changes and possesses low discriminatory power [RFLP involves the typing of the amplified ry power . Variousry power –87.Hybridization methods have been the superior technologies in detecting mixed CT infections and in epidemiological studies. Advanced types in use are reverse line blot hybridization, reverse dot blot hybridization, and microsphere suspension array hybridization. These techniques are based on hybridization of the amplified DNA with probes labeled on nylon membranes or carboxylated beads and results are noted either by blot formation or by analyzing the signals generated . StudiesThe discriminatory power of CT typing has been increased with the introduction of high-resolution typing methods like MLVA and MLST , 10. SevWGS has emerged as a vital tool for public health surveillance, molecular epidemiology of infectious diseases, and determination of antimicrobial drug resistance. This method is known to provide higher resolution and accuracy than the classical molecular typing methods. In CT diagnosis, this technology has been a promising solution in recognizing recurrent and persistent infections, thus helping to improve clinical management and disease control . ClarityEven with genomic studies providing highly accurate data, the information generated is mostly static and not functional . AdvanceProteomics, the qualitative or quantitative study of the proteome, which comprises the entire set of proteins of an organism, could help develop new diagnostic and prognostic biomarkers. Though transcriptional profiles provide insight into host–pathogen relations, post-transcriptional alterations could generate modified host proteins which can be effectively evaluated by proteomics . ChlamydMetabolomics, the most recent layer of the -omics’ cascade, focuses on the quantitative estimation of metabolites produced either during the host–pathogen interaction or during interventions. These can prove to be significant biomarkers indicative of infection, as well as in monitoring the effectiveness of therapy . This fiThe advantages and disadvantages of the various -omics’ technologies discussed above are summarized in Table Considering the disadvantages associated with individual -omics’ methodologies, their implementation in clinical practice is not yet well established. In very few instances, these methodologies have been found to outpace the already available testing strategies . With reThe accurate and cost-effective diagnosis of STIs, while keeping in mind that a syndromic approach is needed, poses a major challenge, particularly in developing and underdeveloped countries. Implementing highly sensitive and specific POC tests that are rapid, accessible, and affordable is a necessity in all settings. The diagnosis of CT genital infections has come a long way, from the less-sensitive culture techniques to the non-culture methods, to the advent of highly accurate molecular technologies. -omics’ technologies, especially when used in an integrated manner, can be very promising solutions. Better awareness of their diagnostic and prognostic value, supporting infrastructure and available research studies for further validation, is necessary for the success and widespread implementation of these technologies in CT diagnosis and management.


Autophagy is a conserved cellular degradation system that maintains intracellular homeostasis. Cytoplasmic components are engulfed into double-membrane vesicles called autophagosomes, which fuse with lysosomes, and resulting in the degradation of sequestered materials. Recently, a close association between autophagy and the pathogenesis of metabolic diseases and ageing has become apparent: autophagy is dysregulated during metabolic diseases and ageing; dysregulation of autophagy is intimately associated with the pathophysiology. Rubicon (Run domain Beclin-1 interacting and cysteine-rich containing protein) has been identified as a Beclin-1 associated protein. Notably, Rubicon is one of few negative regulators of autophagy whereas many autophagy-related genes are positive regulators of autophagy. Rubicon also has autophagy-independent functions including phagocytosis and endocytosis. In this mini-review, we focus on the various roles of Rubicon in different organs in the settings of metabolic diseases and ageing, and discuss its potential role as a promising therapeutic target. The prevalence of metabolic diseases and age-related diseases is significantly increasing in the world . TherefoRubicon has been discovered as a negative regulator of autophagy in 2009 . BesidesClass III phosphatidylinositol-3-kinase (PI3KC3), which consists of three core components, Vps34, p150, and Beclin-1 , plays aRubicon directly binds with UVRAG and Vps34 (one of PI3KC3 complex components), and suppresses their function, which impedes the maturation of autophagosome . In addiIntriguingly, knockdown of Rubicon also promotes autophagosome formation , suggestRecently, several components of autophagic machinery are closely associated with vesicular trafficking, including phagocytosis and endocytosis . Atg8s aWhat is the significance of the role of Rubicon that promotes LAP and LANDO while suppresses autophagy? There is a limit to the lysosomal degradation capacity, and lysosomal overload could lead to lysosomal membrane permeabilization, which causes the release of lysosomal enzymes into the cytosol and activates the cell death pathway. Therefore, it is tempting to speculate that Rubicon might balance the activity of autophagy, phagocytosis and endocytosis, which are independent lysosomal degradation pathways, and in order to avoid lysosomal overload.Independent of autophagy, LAP and LANDO, Rubicon is reported to be associated with the exacerbation of microbial infection by suppressing the innate immune response. After viral infection, the expression of Rubicon is increased and Rubicon suppresses interferon response by directly binding to NF-κB essential modulator (NEMO)  or interWhereas autophagy has been reported to be protective against various metabolic disease, recent studies have elucidated that autophagy is dysregulated in these settings (reviewed by ). In 201Besides NAFLD, it is reported that there is a close relationship between Rubicon and myocardial infarction . Nah et Regarding diabetes mellitus, Aoyama et al. verified the role of Rubicon in pancreatic β cells using pancreatic β cell-specific Rubicon knockout mice, however, Rubicon deletion did not affect glucose homeostasis under a normal diet, and high fat diet . As for Caenorhabditis elegans (C. elegans), Drosophila and mammals, and suppression of Rubicon in C. elegans promotes longevity via autophagic activation , glp-1 mutant  and eat-2 mutant  in C. elegans, indicate the decreased expression of Rubicon at the transcriptional level, and leading to the longevity via activation of autophagy (Furthermore, this study elucidated that various longevity paradigms, including utophagy . CalorieC. elegans and Drosophila. How Rubicon in neuronal cells regulate the ageing process in the whole body? Neuronal cells are known to communicate with peripheral organs via neural circuit as well as humoral factor. Recently, accumulating evidence has elucidated that autophagy-related molecules have an important role in intercellular communication by promoting secretory autophagy (We further investigated which organs of Rubicon are responsible for lifespan extension and clarified that specific knockdown of Rubicon in neuronal cells extended lifespan in utophagy  and/or eutophagy . TherefoIs Rubicon merely a bad guy in age-related disease? We answered the question in 2020 . This stC. elegans accumulate during ageing and knockdown of ELT-5 and ELT-6 extends lifespan (A recent report has identified that Rubicon affects male fertility: Rubicon deficiency in Sertoli cells causes defective spermatogenesis via accelerated autophagic degradation of GATA4, which is an essential transcription factor for Sertoli cell function . This relifespan . ConsideThe autophagy-independent function of Rubicon has also been identified to contribute to the pathogenesis of age-related disease. Heckmann et al. reported that deficiency of Rubicon-dependent LANDO exacerbates the pathogenesis of Alzheimer disease . RubiconWhereas Rubicon inhibition could be a promising therapeutic strategy, several concerns exist. Rubicon inhibition in the whole body might have side effects including lipodystrophy and infertility due to excessive autophagy, and exacerbation of autoimmune diseases, infectious diseases and neurodegenerative disease due to LAP and LANDO inhibition. To overcome these side effects, several issues should be resolved.First, the regulatory mechanism of Rubicon during ageing should be clarified. Interestingly, there is a close relationship between Rubicon expression and the occurrence of age-related disease: Rubicon expression increases in the liver and kidney during ageing, which leads to the exacerbation of NAFLD, and kidney fibrosis. On the other hand, Rubicon expression decreases in adipose tissue during ageing, which leads to the exacerbation of lipodystrophy, and ectopic fat accumulation . TherefoSecond, it is critical to identify the region in the brain responsible for lifespan extension by Rubicon suppression. As noted above, Rubicon inhibition in the whole brain might have side effects including Alzheimer disease. Therefore, the site-specific inhibition approach is important to avoid side effects. Recent reports have elucidated that the hypothalamus or neural stem cells regulate ageing . It is tFinally, considering the relationship between Rubicon and diseases, human evidence is completely lacking at present. Future studies should accumulate human evidence.Rubicon is a cell biologically interesting molecule that plays specific functions: Rubicon is one of few negative regulators of autophagy whereas Rubicon is indispensable for LAP and LANDO. There remain many questions unanswered. What is the regulator of Rubicon during ageing? How Rubicon exerts its various functions properly in response to various situations? Future studies to answer these questions will provide clues to a novel treatment for metabolic diseases and age-related diseases.


Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by bone marrow dysfunction and increased risk of transformation to leukemia. MDS represent complex and diverse diseases that evolve from malignant hematopoietic stem cells and involve not only the proliferation of malignant cells but also the dysfunction of normal bone marrow. Specifically, the marrow microenvironment—both hematopoietic and stromal components—is disrupted in MDS. While microenvironmental disruption has been described in human MDS and murine models of the disease, only a few current treatments target the microenvironment, including the immune system. In this review, we will examine current evidence supporting three key interdependent pillars of microenvironmental alteration in MDS—immune dysfunction, cytokine skewing, and stromal changes. Understanding the molecular changes seen in these diseases has been, and will continue to be, foundational to developing effective novel treatments that prevent disease progression and transformation to leukemia.  Myelodysplastic syndromes (MDS) occur when mutant hematopoietic stem cells (HSCs) proliferate, giving rise to dysplastic myeloid progeny and ineffective hematopoiesis . There iSF3B1 may cause a slowly progressive MDS, others, including Rat sarcoma virus (RAS)-pathway mutations, are associated with increased blast counts and, therefore, higher risk of progression to Acute Myeloid Leukemia (AML), which occurs when myeloblasts account for greater than 20% of nucleated cells in the bone marrow [Some of these initiating mutations are advantageous for the HSCs and allow for continued expansion, while they are detrimental to functional hematopoiesis . What ene marrow ,15.IDH) mutations are epigenetic modifiers and do not have a proliferative advantage per se [SRSF2) mutations influence the splicing machinery and require additional proliferative mutations [Notably, several common mutations do not alone provide the clone with proliferative advantage, with HSCs that are defective in their growth and differentiation in vitro and that engraft poorly in immunodeficient recipient mice . For exae per se ,18. Simiutations . These fThe manifestations of MDS are due both to the proliferation and aberrant differentiation of mutated, malignant HSCs and their progeny, as well as the failure of normal bone marrow as clonal MDS cells take over. The complex interplay between these two categories of cell populations ultimately determines the course of the disease . At its Exploring the individual contributions of these components, as well as the significant interactions between them, will be the focus of this review. Immune dysfunction is manifested both by clonal MDS cells and previously normal mature immune cells. Cytokine production in MDS is also altered—not only by MDS cells and normal hematopoietic cells but also by stromal bone marrow cells. Finally, beyond aberrant cytokine production, stromal cells in the bone marrow microenvironment contribute significantly to disease development and progression via multiple mechanisms. The overlapping nature of these three components highlights a core principle of MDS—there is no one mutation, alteration, or microenvironment that alone initiates or facilitates disease. Rather, interactions between multiple endogenous and clonal cell populations are responsible. Given its complexity, much is still unknown regarding MDS pathogenesis; however, this review aims to synthesize key research related to immune cell behavior within the MDS microenvironment, cytokine production patterns in MDS, and stromal cell contributions to disease development and progression.Hematopoietic stem cells are impacted by their surroundings . From chImmune cells existing in the bone marrow microenvironment are altered in the presence of MDS, specifically macrophages, dendritic cells, Natural Killer (NK) cells, B cells, and T cells . ResearcMacrophages interact with and regulate hematopoietic stem cells ,23,24. IDendritic cells (DCs) represent a key component of the low-risk proinflammatory immune response. DCs are antigen-presenting cells and thus stimulators of T cells. Antigens from apoptotic HSCs in low-risk MDS are presented by DCs, therefore triggering induction of T-cell activation against normal HSCs. The ensuing apoptosis of normal HSCs contributes to the dominance of clonal MDS-derived HSCs and disease progression .Natural Killer (NK) cells are responsible for the lysis of unwanted cells, and therefore have intrinsic antitumor activity. In high-risk MDS, there are decreased numbers of NK cells, allowing further clonal proliferation. In low-risk MDS, however, it appears that NK cells are cytotoxic to clonal MDS precursors, thereby curbing disease progression and presenting an opportunity for therapeutic intervention for myeloid malignancies including MDS ,32.B lymphocyte dysregulation is seen in autoimmune disorders; therefore, researchers have hypothesized similar findings in MDS. While some evidence has shown downregulation of B-cell signaling pathways in MDS, further research is needed to clearly delineate this relationship .Multiple subtypes of T lymphocytes are altered in the setting of MDS. Firstly, regulatory T cells (Tregs) play separate and unique roles in low-risk versus high-risk MDS. In low-risk MDS, low numbers of Tregs allow T-cell activation and apoptosis. In high-risk MDS, however, it has been found that high numbers of Tregs inhibit the ability of the endogenous immune system to respond to clonal cells, thereby allowing for MDS cell proliferation . SecondlTP53 mutations have an expansion in MDSCs, with an associated decrease in bone marrow infiltrating cytotoxic T cells [Myeloid-derived suppressor cells (MDSCs) are becoming recognized as key cell populations derived from the normal innate immune system that, in the presence of the tumor, which often recruits these cells by secreting chemokines, provide immunosuppressive signals that suppress the adaptive immune response to cancer. This population has long been recognized in solid tumors and is beginning to be studied as part of the immune microenvironment in MDS. As in solid tumors, the data published to date show that MDSCs interfere in tumor immunity by suppressing cytotoxic T cells . In addi T cells . The autAlong with alterations in endogenous immune cells, clonal MDS immune cell progeny contribute to an inflammatory environment that favors ineffective hematopoiesis. MDS-derived monocytes, macrophages, dendritic cells, and neutrophils all behave aberrantly.Monocytes isolated from MDS patients have been shown to have a unique phenotype. Matrix metalloproteinases (MMP) are enzymes responsible for extracellular matrix remodeling and the production of cytokines. MMP-9 specifically has been found to play a role in the mobilization of HSCs and is a known secretory product of monocytic cells . ResearcTET2) is the most frequently mutated gene seen in MDS (Tet2-deficient murine macrophages had a constitutive expression of lipopolysaccharide (LPS)-induced genes, suggesting that loss of normal Tet2 function contributes to an upregulated inflammatory state and alteration of the microenvironment [MDS-derived macrophages also play a role in disease progression. Tet methylcytosine dioxygenase 2 , interferon-gamma (IFN-y), interleukin-6 (IL-6), interleukin 1β (IL-1B), and interleukin 8 (IL-8) are found in high levels in MDS . CytokinTNF-a is a proapoptotic cytokine and, therefore, likely contributes to the rapid cell death seen in lower-risk diseases . As wellIn MDS, alterations in transforming growth factor-beta (TGF-B) signaling, including a loss of inhibitory factors, have been shown to cause constitutive pathway activation and suppression of hematopoiesis . IL-6 anWhile exact mechanisms of action are still unknown, data suggest many cytokines exert their pro-MDS effects via the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway. NF-kB is a transcription factor that is a critical regulator of immune defense. Basal NF-kB activation is present in healthy HSCs, however, its constitutive activation induces bone marrow failure ,59. In MThese elevated cytokines also interact with immune cells, altering their behavior and contributing to many functional changes described in the prior section. For example, Tregs, which activate apoptosis in low-risk MDS and inhibit the immune response in high-risk MDS, also secrete TGF-B . TGF-B, TET2 and DNMT3A clones [TRAF6 overexpression as a mechanism of cytopenia and myelodysplasia [IL-6 is a key inflammatory cytokine that likely contributes to CHIP and MDS not only by driving clonal dynamics but also by directly mediating hematopoietic dysfunction. For example, IL6 modulates the expansion of mutated A clones ,65. Moreysplasia .MDS also appears to trigger activation of the NLRP3 inflammasome, an endogenous inflammatory system. As noted previously, apoptosis is a hallmark of lower-risk MDS. However, recent research has highlighted similarities in apoptotic gene expression between MDS patients and healthy controls, therefore suggesting that other cellular death pathways contribute to the cell lysis seen in the disease . SpecifiIncreasing evidence suggests that cytokine levels can be predictive of clinical outcomes in certain circumstances. A study of plasma cytokine levels in MDS patients found that increased levels of IL-6, chemokine (C-X-C motif) ligand 10 (CXCL10), and Interleukin 7 (IL-7) predict shorter overall survival independent of cytopenias. IL-6 and CXCL10 were also predictive of worse leukemia-free survival, suggesting that elevated levels of these cytokines may contribute to leukemic progression . ElevateDespite this evidence, utilizing cytokine profiles as prognostic tools is challenging due to the multiple mutations and etiologies that may drive the overproduction of a given cytokine in MDS . Indeed,Given the important role cytokine skewing plays in altering the inflammatory microenvironment, cytokines represent an opportunity for therapeutic intervention. However, low- and high-risk MDS have different cytokine profiles, and therefore, require distinct treatment approaches. For example, therapies targeting proapoptotic cytokines may be beneficial in low-risk MDS but not in high-risk diseases. As TNF-a is a key proapoptotic factor, monoclonal antibodies targeting TNF-a such as Infliximab and Etanercept may theoretically be beneficial; however, studies testing their use in low-risk MDS have found variable to minimal impact ,78.Beyond monoclonal antibodies, lenalidomide has been found to reduce TNF-a, IL-6, and IL-1B levels and is approved for the treatment of the del(5q) subset of MDS—one of the most common cytogenetic alterations seen in MDS . It alsoWhile it is clear that certain cytokines are upregulated or downregulated in MDS, and therapeutic modulation of cytokine levels can be moderately effective in managing disease, there is still much that is unknown about why and how certain cytokines impact disease progression. Namely, the impact of common MDS mutations  on cytokThe ability of the bone marrow microenvironment to regulate hematopoietic stem and progenitor cells is well-established. While not the focus of this review, more information on these interactions can be found in recent review articles, including those by Ho et al., 2020, Pinho et al., 2019, and Galán-Díez et al., 2018 ,83,84.MDS is no longer considered a disorder of only hematopoietic cells but rather a dysfunction of the entire bone marrow, including mesenchymal components. Early research looking at bone cell function in MDS patients found close relationships between hematopoietic and stromal cells . MineralIn fact, functioning osteohematopoietic interactions and stromal support are necessary for maintaining healthy hematopoiesis. Mesenchymal stromal cells (MSCs) are critical to hematopoietic differentiation. This link has been further demonstrated by the fact that MDS-derived MSCs are unable to provide stromal support, contributing significantly to hematopoietic deficits seen in MDS. Human bone marrow fibroblast colony-forming units (CFU-F) assays performed demonstrated reduced CFU-F counts in MSCs from MDS patients compared with those purified from healthy controls. MSCs from these MDS samples also had impaired growth in culture and were not able to be maintained at as high passages as samples from healthy controls. MDS-derived MSCs also were more likely to senesce. As evidence of the molecular differences underlying these observed phenotypic changes, differential methylation was observed between the MDS-derived and healthy bone marrow MSCs. Specifically, hypermethylation was seen in promoter regions of DNA within the MDS MSC samples, resulting in the silencing of genes associated with key cellular processes. These deficits translate into decreased support of stem cell populations; in a long-term coculture with CD34+ hematopoietic stem and progenitor cells (HSPCs), MDS-derived MSCs compared with MSC controls had substantially reduced ability to provide HSPC support. This deficiency was able to be restored when the CD34+ HSPCs were then cocultured with healthy MSCs. The molecular mechanisms of this defective support include differential cytokine expression by MDS-derived MSCs and shifting of HSPCs into the resting phase of the cell cycle by MDS MSCs .Dicer1 deletion demonstrated that a dysfunctional stromal environment could initiate myelodysplasia [Dicer1 deletion downregulates the production of miRNA, which is essential to hematopoietic differentiation and maturation. By introducing this gene deletion into mesenchymal osteolineage cells of mice via Cre recombinase, the effects of Dicer1 gene deletion on osteoprogenitor cell growth and differentiation could be studied. The deletion was seen in MSCs but not in HSPCs, and classic features of human MDS were observed. The osteogenic population was adversely impacted, similar to findings in previously mentioned studies, with decreased CFU-F counts and reduced levels of differentiation markers such as osteocalcin. However, this study uniquely demonstrated the causal link between altered MSCs and hematopoiesis, with mutant mice experiencing profound anemia and thrombocytopenia with altered hematopoietic cell morphology. Moreover, the bone marrow cellularity of these mice was normal to hypercellular, and HSPC populations were not adversely impacted in terms of count or function. These mice also had reduced B cells and increased myeloid cells. All of these findings are characteristic of human MDS. Several of these mice subsequently developed AML—another key feature of progressive MDS. Researchers then transplanted HSPCs from these mutant mice into wild-type mice and were able to demonstrate normal cell counts and a lack of myelodysplasia, showing the inability of HSPCs alone to trigger an MDS phenotype. However, coculturing MSCs from mutant mice with normal HSPCs in vitro demonstrated morphologic and functional changes in the hematopoietic cells, supporting the idea that altered osteogenic cells directly alter HSPC function.Not only is MSC support needed for functional hematopoiesis, but further research by Raajmakers et al. utilizing a murine model of ysplasia . Dicer1 The ability of MSCs to drive hematopoietic failure in humans, however, has not yet been established and is a challenge to study. Xenotransplantation of human MDS into mouse models is a method that hypothetically would allow for longitudinal, controlled examination of the interactions between the stroma and hematopoietic cells throughout the disease course. Yet, the engraftment of human MDS cells to murine models has proved difficult by several studies, perhaps suggesting the importance of the microenvironment in the propagation of the disease. Engraftment has shown to be improved, however, with the addition of stromal cells ,89. FurtThe concept of donor cell leukemia (DCL) provides further support for the importance of the stromal environment in initiating hematopoietic failure. DCL occurs when a patient transplanted with supposedly normal donor cells develops hematologic malignancy, but the donor does not. Researchers have speculated that dysregulation of the bone marrow microenvironmental niche alters the differentiation and proliferation pathways of HSPCs, possibly to such an extent that they become leukemogenic . CytokinRecent research provides further rationale for targeting the bone marrow microenvironment as a therapeutic strategy. Using a murine model of MDS, Balderman et al. found that normal cells exposed to the same microenvironment present in MDS acquire myeloid skewing . RejuvenThe potential of the bone marrow microenvironment as a therapeutic target in MDS was recently demonstrated by the approval of Luspatercept, an activin receptor type IIB, for the treatment of low-risk MDS. Wobus et al. found that bone marrow-derived stromal cells from patients treated with Luspatercept, who had improved erythropoiesis, were superior in their ability to support normal hematopoiesis, with normalization of CXCL12 production in the MDS microenvironment . This stThe exact mechanisms underlying how stromal cells interact with and influence hematopoietic cells to contribute to their failure in MDS is an active area of research. Activation of Wingless-related integration site (Wnt) signaling in MSCs has been shown to drive bone marrow failure. Secondly, as previously discussed, shifts in cytokine production by both non-MDS and clonal MDS immune cells contribute to the MDS phenotype; however, research has shown that cytokine skewing by mesenchymal cells, including cytokine production initiated by the NF-kB pathway, is also relevant. Ultimately, it is likely the interplay between these various mechanisms that is integral to disease development. FRZB) gene—was hypermethylated, and therefore its expression was suppressed in the stromal cells of MDS patients, allowing increased activation of the Wnt pathway. Furthermore, primary MDS-derived MSCs had increased activation of this pathway. The Nup98-HoxD13 (NHD13) fusion gene has been identified in subsets of patients with MDS; when mice transgenically express this gene under the control of the Vav promoter, they develop prolonged cytopenias, eventually progressing to leukemia [NHD13 mice are frequently used to model MDS, its phenotype is more representative of low-proliferative Acute Myeloid Leukemia (AML) than MDS. In this experiment, NHD13 mice were crossed with constitutively-active human B-catenin mice models (S33Y), and the bone marrow of these mice was transplanted into irradiated recipients who were followed for disease progression. Most NHD13/S33Y mice died from myeloid leukemia, whereas other nonmutant mice died of a less aggressive MDS-like phenotype, suggesting the importance of Wnt/B-catenin activation in leukemic transformation. To apply these findings to human MDS samples, researchers determined the frequency of transcription of genes downstream of the Wnt pathway in healthy controls and CD34+ cells from the bone marrow of MDS patients. Many of these genes were found to be overexpressed in the MDS patients compared to controls. Furthermore, higher-risk MDS cases (as determined by blast counts) had higher levels of transcription of gene products downstream of the Wnt pathway, suggesting an almost dose–response effect of pathway activation. These findings were also extremely clinically relevant—patients who had a high level of Wnt pathway activation had a median overall survival of 2.95 years compared with a median survival of 5.24 years in those with a low Wnt activation profile. Taken together, these data highlight the importance of Wnt/B-catenin activation in disease progression and leukemic transformation [The Wnt/B-catenin signaling pathway is critical in determining cellular fate, migration, and proliferation. This was initially observed in embryonic development and the importance of the pathway in establishing the body axis . More releukemia ,98. Whilormation .Apc) is a negative regulator of the Wnt signaling pathway, and prior research by this group demonstrated that mice deficient in Apc developed MDS. Researchers were then able to show that loss of a single copy of the Cttnb1 gene  was able to prevent MDS in this same Apc-deficient mouse model, suggesting that this pathway is not only able to induce MDS when upregulated, but that suppression of the pathway can inhibit MDS development. For further validation of these findings, pyrvinium—an FDA-approved medication that inhibits the Wnt pathway—was given to these Apc-deficient mice, and MDS development was likewise prevented [Further evidence of the importance of this pathway comes from Stoddart et al., who found that inhibition of the Wnt/B-catenin pathway can prevent MDS development. Adenomatous polyposis coli , and Inhibin subunit beta A (INHBA), which are known suppressors of hematopoiesis .Initiation of inflammatory programs via other activation mechanisms appears to contribute to the MDS phenotype as well. CD271+ cells are mesenchymal bone marrow cells that exist in close proximity to CD34+ HSPCs. Transcriptional analysis was performed on sorted MDS stromal cells and found enhancement of inflammatory signatures in CD271+ cells. Overexpression of inflammatory cytokines, and those known to be negative regulators of hematopoiesis, including IL-6 and IL-8, were found in MDS stromal cells compared with normal controls. This data both supports the idea that stromal cell cytokine production has the potential to cause the hematopoietic failure seen in MDS and that spatial location of cells relative to one another may matter, as the CD271+ cells are in close proximity to HSPCs .Finally, MSCs may also modulate immune populations in MDS. For example, Serhan et al. found that MSCs from patients with MDS could alter monocytes, inducing phenotypic and metabolic changes consistent with MDSCs, which could then suppress NK cells. . These dAs Pronk and Raaijmakers, 2019 highlight, these mechanisms underlying mesenchymal–hematopoietic interactions overlap and influence one another, creating a microenvironment prime for bone marrow failure. For example, Wnt signaling is upregulated by inflammatory cytokines and activated by NF-kB. Moreover, NF-kB impairs the proliferation of MSCs and promotes cellular senescence. Cell senescence induces the production of inflammatory cytokines, which, in turn, upregulates Wnt signaling pathways. Therefore, these mechanisms not only directly mediate mesenchymal cells’ ability to support hematopoiesis but also influence other inflammatory processes, creating spirals that result in an exponentially more toxic microenvironment .As this review has highlighted, the impact of various components of the bone marrow microenvironment on MDS development and progression is an exciting and active area of research with clinical and therapeutic implications. Several critical areas of research are important to explore, especially relating to the role of key MDS mutations . For exaAlterations in the three core components discussed in this review—immune cells, inflammatory signals and cytokine production, and the stromal microenvironment—contribute to the range of disease phenotypes and clinical manifestations seen with myelodysplastic syndromes. The mutational and clinical heterogeneity in MDS presents a challenge to developing a single sweeping and successful therapeutic intervention. Therefore, understanding precisely the inflammatory and microenvironmental changes seen at various disease stages with various driving mutations is necessary to construct targeted treatments to combat the proinflammatory environment of the disease and ultimately halt disease progression.


Malnutrition is highly prevalent in inflammatory bowel disease (IBD) patients and disproportionately affects those admitted to hospital. Malnutrition is a risk factor for many complications in IBD, including prolonged hospitalization, infection, greater need for surgery, development of venous thromboembolism, post-operative complications, and mortality. Early screening for malnutrition and prompt nutrition intervention if indicated has been shown to prevent or mitigate many of these outlined risk factors. There are many causes of malnutrition in IBD including reduced oral food intake, medications, active inflammation, and prior surgical resections. Hospitalization can further compound pre-existing malnutrition through inappropriate diet restrictions, nil per os (NPO) for endoscopy and imaging, or partial bowel obstruction, resulting in “post-hospital syndrome” after discharge and readmission. The aim of this article is to inform clinicians of the prevalence and consequences of malnutrition in IBD, as well as available screening and assessment tools for diagnosis, and to offer an organized approach to the nutritional care of hospitalized adult IBD patients. Inflammatory bowel disease (IBD) is a chronic debilitating inflammatory disorder of the gastrointestinal tract characterized by a relapsing and remitting course. The etiology of IBD is incompletely understood, but includes an imbalance between pro-inflammatory and anti-inflammatory signaling arising from genetic susceptibility and environmental triggers . The twoIBD is often diagnosed in young adulthood or middle age, resulting in a large burden of disability-adjusted life years that is now improving as more effective treatments become available . AdvanceMalnutrition is a frequently under-recognized complication of IBD despite its high prevalence ranging between 20 and 85% . AlthougIn the case of IBD, malnutrition can result from a number of different mechanisms see  includinSarcopenia is a nutrition-associated disorder characterized by reduced muscle mass and strength. There are multiple contributors to sarcopenia in IBD, including chronic inflammation, malnutrition, and physical inactivity . SarcopeThere is a wealth of data pertaining to malnutrition in hospitalized patients as a whole, and while some IBD-specific data exist, this area is still a growing field of interest. The prevalence of malnutrition is high among patients admitted to hospitals in general, with studies demonstrating a prevalence of up to 45% ,17. A laThere are significant adverse clinical implications of underdiagnosing malnutrition in IBD patients. We summarize the current state of knowledge and make best practice suggestions based on expert opinions that practitioners can readily apply to optimize nutritional and clinical outcomes in hospitalized IBD patients. The primary objective of this article is to serve as an expert narrative review to inform clinicians of the prevalence and consequences of malnutrition in IBD, available screening and assessment tools for diagnosis, and its use during hospitalization, and to offer an organized approach to the nutritional care of hospitalized adult IBD patients. We employed a rigorous search strategy for our narrative review for nutrition interventions . We searMalnutrition in hospitalized patients is associated with prolonged length of stay, higher readmission rates after discharge, greater rates of complications, infections, and increased mortality ,20,21. ASpecific to IBD, malnutrition is disproportionately higher in those admitted to hospitals ,24. MalnPatients with severe IBD frequently have restricted oral intake due to their symptoms. Long-term avoidance of specific foods was reported to be as high as 77% in a recent study and can lead to micronutrient deficiencies even in the absence of weight loss . When IBEvidence suggests that identifying malnutrition and introducing a nutritional intervention can improve outcomes in hospitalized patients as a whole ,31. WhilDefining the malnutrition construct as it applies to IBD remains challenging, largely due to a lack of validated tools for its diagnosis. The prevalence is high, but estimates are variable due to use of different tools. An ideal gold standard would include elements of history, physical examination, anthropometric measurements, laboratory markers, body composition, and assessments of physical and mental functioning. While such a comprehensive assessment would capture the spectrum of malnutrition in IBD, it is not practical and could not be universally applied. In clinical practice and even in research settings, malnutrition in IBD is usually diagnosed using nutritional assessment tools (NAT); however, many of these are not yet comprehensively validated in IBD.Various NAT are available and validated in general medical conditions, including bedside global assessment, bedside muscle measurements, bedside body composition analysis, and cross-sectional measures of body composition. Bedside global assessments include elements of clinical history, diet history , physical examination, and laboratory values. The Subjective Global Assessment (SGA) is one of the most widely used bedside global NAT, and has been well validated in general populations . In IBD,Bedside muscle measurements include hand grip strength (HGS), mid-upper arm circumference (MUAC), and mid-upper arm muscle circumference (MUAMC). HGS has been shown to be an effective, quick, and convenient parameter to predict functional status and muscular health in CD patients compared to healthy controls . HGS corNAT focused on identifying altered body composition include bioelectrical impedance analysis (BIA), which can be conducted at the bedside, or radiologic tools such as dual-energy x-ray absorptiometry (DEXA), computer tomography (CT), and magnetic resonance imaging (MRI). Body composition analysis estimates lean body mass and can be used to determine the presence of sarcopenia. The practical use of DEXA and BIA are limited in a hospital environment due to extra resources including equipment and time, or potential adverse effects such as radiation. Currently, body composition analysis tools are primarily being used for research. While CT and MRI are frequently performed on patients with IBD for assessment of their disease, software for calculations of body composition from such imaging is not universally available. Thus, with resource or time constraints, HGS and MUAC are more portable and easily obtained in a hospital and outpatient environment compared to DEXA or BIA, which require specialized equipment. However, clinicians may not have the tools and time to perform these bedside measurements, which are likely most useful when taken serially.2 or the combined finding of unintentional weight loss with reduced BMI  or reduced fat free mass index (FFMI)  [2. The American Society of Parenteral and Enteral Nutrition (ASPEN) criteria for malnutrition require two or more characteristics including insufficient energy intake, weight loss, loss of muscle mass, loss of subcutaneous fat, and localized or generalized fluid accumulation that may sometimes mask weight loss or diminished functional status as measured by HGS [ESPEN criteria to diagnose malnutrition require either a low BMI of <18.5 kg/mr males) . Fat fred by HGS .2 for females and <17 kg/m2 for males [However, ESPEN and ASPEN criteria may not capture all components of malnutrition in IBD, especially micronutrient deficiencies that may arise due to malabsorption, selective avoidance of food groups, or high losses. Until a gold standard for malnutrition in IBD is created, we recommend that a diagnosis of malnutrition in IBD not require a low BMI nor unintentional weight loss. We suggest if an IBD patient has a minimum of two criteria below, a diagnosis of malnutrition should be considered. These include food restrictions/avoidance, active luminal disease, symptoms of nausea/vomiting/diarrhea/poor appetite beyond one week, unintentional weight loss >5% in 3 months, HGS < 20% percentile based on age/gender, or low FFMI < 15 kg/mor males . These bWhile a formal nutrition assessment in all inpatients would appear to be the optimal approach, this is not feasible due to limited resources. Nutritional screening tools (NST) are rapid and simple evaluation tools that can be completed by any healthcare team member, and even the patient. Screening tools are designed to detect risk for protein and energy malnutrition, and/or predict whether malnutrition is likely to occur under present or future conditions . A positFamiliar and validated NST include the Malnutrition Universal Screening Tool (MUST) and Nutrition Risk Screening (NRS-2002). MUST was initially developed for outpatient settings where serious confounders of the effect of malnutrition are relatively rare . HoweverBoth the NRS-2002 and MUST factor in BMI, amount of weight loss in a specified time, any reduced nutritional intake, and assessment of whether the patient is acutely ill. Unlike MUST, the NRS-2002 incorporates an age adjustment score of ≥70 years old, outlines specific medical conditions to stratify disease severity, and quantifies ranges of reduced food intake in a one-week span. Kondrup et al. outline the specific scoring parameters of these two screening tools . A recenA 2019 systematic review explored various NST used in IBD patients including MUST, NRS-2002, Malnutrition Inflammation Risk Tool (MIRT), and Saskatchewan IBD-Nutrition Risk (SaskIBD-NR tool) . The sysWe recommend that all IBD patients with a new diagnosis, flare, or complication associated with their IBD, including hospitalization, infection and surgery, be screened for malnutrition. Patients with active disease have the highest risk of malnutrition, especially in newly diagnosed IBD . IdentifHospitalized IBD patients screened for risk of malnutrition should have comprehensive nutritional assessments involving a dietitian and a nutritional management plan that is well-developed. Focus groups highlighted that physicians do not screen for malnutrition frequently enough and miss opportunities to intervene early in malnourished IBD patients . Given tNutritional assessment and therapy should be initiated within 24 h of hospital admission in patients assessed as positive for malnutrition risk. Nutrition intervention can consist of one or more of: oral nutritional supplements (ONS), enteral nutrition (EN), peripheral parenteral nutrition (PPN), or central parenteral nutrition (CPN). A proposed algorithm for nutrition support is presented in When safe and feasible, using the oral or enteral route is the preferred method for nutrition support, and oral feeding is preferred to tube feeding. Advantages of using the gut to deliver nutrition support include lower cost, avoidance of intravenous (IV) access related complications, maintenance of epithelial barrier integrity, immune modulation, and effects on the microbiome . ONS areEN is indicated in patients who cannot meet nutritional needs orally but have a sufficiently intact gastrointestinal tract for micro- and macronutrient absorption. Reasons for inadequate oral intake can include malabsorption, active inflammation, diarrhea, anorexia, nausea, vomiting, and abdominal pain. There are several formulations available for enteral  nutrition: polymeric , semi-elemental , and elemental . Standard polymeric formulas are generally recommended as first-line when using EN. However, certain patients who have significant malabsorption or maldigestion can benefit from predigested semi-elemental or elemental formulas. These predigested formulas are higher in osmolarity and are not palatable, and best infused slowly using a feeding tube. In patients who can eat and drink but are not meeting their needs orally, supplemental EN can be used to infuse nutrients slowly at a controlled rate to reduce intolerance related to bolus feeding. Supplemental EN can also be used only overnight in order not to interfere with spontaneous daytime oral intake. Even in patients with CD who present with symptoms of partial bowel obstruction due to intestinal stricture, EN can be used if tolerated, especially if enteral access can be obtained distally to the stricture . Even ifIn patients with CD who present with complications requiring surgery, such as bowel resection for active or fibrostenotic disease or enterocutaneous fistula, internal fistula, or intra-abdominal abscess, there is emerging evidence regarding the benefit of pre-operative EN. In a recent case control study of patients with CD undergoing surgery, Yamamoto et al. demonstrated that compared to no nutritional intervention, a period of two or more weeks of pre-operative EN with an elemental diet was associated with a significant reduction in risk of post-operative wound and anastomotic complications . In anotWhile the usual indication for EN in adult hospitalized patients is to treat malnutrition, there is also evidence for its use as a primary therapy for inflammation in CD. Exclusive enteral nutrition (EEN) is an acceptable first line treatment for pediatric CD . HoweverIt is accepted that malnourished patients who cannot meet nutritional needs by the oral or enteral route should have parenteral nutrition (PN) initiated . PN invoPN implementation should be considered if a patient is meeting less than 50–60% of nutritional needs via the gut . PN has Although guidelines state that there is insufficient high-quality data from well-controlled observational and randomized controlled trials to support the routine use of PPN, clinical experience points to scenarios in which PPN can be utilized to optimize nutrition for hospitalized IBD patients . PPN canIn such a setting of short-term partial restriction of oral intake in a malnourished patient, PPN is a logical intervention. Rapid supplementation via PPN can be expedited as a central line is not required. As delays in central line insertion can take between 24 and 72 h at some institutions, PPN can also serve as a bridge to longer-term CPN.CPN is indicated in patients with significant malabsorption, short bowel syndrome, intestinal obstruction, high output fistula, ileus, intractable vomiting, or uncontrolled severe gastrointestinal bleeding. It is also indicated in those who would benefit from PPN, as described above, but in whom PPN is contraindicated or not feasible, such as those with difficult peripheral IV access, severe malnutrition, fluid restrictions, or metabolic derangements, and those in whom the duration of PN therapy is anticipated to be more than 10–14 days. CPN can also be used in patients who meet criteria for short-term PPN use but happen to have a central line in place already. However, a lumen dedicated for PN use is recommended to reduce infection risk . Even inMalnutrition increases the risk of post-operative complications in patients with IBD, including wound and infectious complications, as well as mortality . Nutritional interventions, both enteral and parenteral, were demonstrated to ameliorate this risk ,69,70,71Post-operative follow-up of IBD patients with intestinal resections is key to reducing the risk of new or progressive malnutrition. As outlined in Poor nutrition, often worsened during hospitalization, is thought to contribute to a phenomenon termed “post-hospital syndrome”, which is a generalized transient vulnerability after hospital discharge which leads to higher morbidity and an increased rate of readmission for the same or other causes . ContinuIn Alberta, Canada, a unique multidisciplinary High Risk Malnutrition Clinic led by gastroenterologists who are also physician nutrition specialists and dietitians was developed specifically to optimize nutrition care of malnourished patients with digestive diseases, including IBD . This unOur narrative review highlighted high-priority areas for consideration in the nutritional management of hospitalized IBD patients see  below. W(A)No gold standard definition of malnutrition in IBD and lack of validated NST and NATThis applies to both hospitalized and community IBD populations. There is a wide range in the reported prevalence of malnutrition in IBD due to a lack of well-validated tools and wide variability in disease location and severity. A set of disease-specific yet encompassing criteria for malnutrition in IBD remains to be determined. Until further high-quality data are available, it is essential that all IBD patients admitted to hospitals be screened for malnutrition utilizing either the NRS-2002 or MUST NST with prompt nutritional interventions if at moderate to high risk of malnutrition.(B)Limited high-quality data for the use/timing of PN, including total versus supplemental, and central versus peripheralWhile there are widely agreed-upon parameters for CPN use and timing, these are based on studies in non-IBD populations, using non-IBD specific nutritional risk assessment tools. Further studies of PN use, including timing and dosing, are warranted. There is a lack of data for when and how PPN can be optimized in patients with IBD. There are critical points early in hospital admission where patients may benefit from PPN as a bridge or supplement to longer-term therapy, but there are no data to guide this use. How IBD patients may benefit from PN requires further research.(C)Paucity of data on the impact of nutritional interventions on hospitalized IBD patientsWhile there is growing literature on the impact of nutrition interventions in improving IBD peri-operative risks, there remains a lack of high-quality studies for benefits of nutrition interventions on other outcomes of interest . Various nutritional therapies including ONS, EN, PPN, supplementary PN, and CPN were explored in this review, and recommendations for the sequenced use of nutritional therapies were provided based on data from other conditions.In summary, we outlined the high prevalence of malnutrition in IBD arising from its unique pathophysiology and the importance of malnutrition screening in all hospitalized IBD patients. We provide an expert opinion on diagnosing malnutrition in IBD that does not require weight loss or low BMI. Finally, we suggest approaches to nutrition therapies for the hospitalized IBD patient. Future prospective studies should prioritize these important areas of research to advance our understanding of how to best treat malnutrition in IBD.


Sarcina ventriculi is a rare gram-positive coccus increasingly reported in patients with a history of delayed gastric emptying or gastric outlet obstruction and is sometimes seen in association with emphysematous gastritis and perforation. We report a case of a 67-year-old male who presented with epigastric pain. CT imaging and cholangiopancreatography were concerning for pancreatic neoplasia. Upper endoscopic ultrasound-guided fine needle aspiration cytology of a perigastric lymph node confirmed metastatic adenocarcinoma of pancreatic origin, and cocci arranged in a tetrad fashions characteristic of Sarcina ventriculi were noted. To our knowledge, this is the first reported case of Sarcina ventriculi in an FNA of metastatic pancreatic carcinoma in a perigastric lymph node. These organisms likely represent carry-through contaminants from the transgastric approach of the endoscopic FNA. Sarcina ventriculi is an anaerobic, gram-positive coccus with characteristic morphological features such as basophilic staining [Sarcina organisms can survive in very low pH environments where they form their distinctive morphological features. At higher pH levels, organisms are noted to be larger and varied in shape and size [Sarcina ventriculi has been reported to have an association with delayed gastric emptying and is sometimes implicated as a causative factor in gastric perforation [Sarcina ventriculi organisms were incidentally noted on cytological examination of a perigastric lymph node biopsy performed for staging and diagnosis of adenocarcinoma of the pancreas.First described in 1842 by John Goodsir [staining , and tetand size . Sarcinaforation  and emphforation . In addiA 67-year-old man with type 2 diabetes presented approximately one year prior to diagnosis with complaints of intermittent sharp epigastric pain and bloating. In the interim, he had undergone CT imaging, upper and lower endoscopy, laboratory testing, and magnetic resonance cholangiopancreatography at outside institutions. A nonenhancing lesion of the neck of the pancreas had been noted and was favored to represent necrotizing pancreatitis. During this timeframe, he was unsuccessfully treated with a proton pump inhibitor, pancreatic enzyme replacement, gabapentin, and acetaminophen. He experienced an approximate 50-pound unexplained weight loss during this interval and ultimately sought care at Mayo Clinic.Sarcina ventriculi  and the pyruvate decarboxylase gene, a unique metabolic pathway in Sarcina, can also be used [Our patient presented with epigastric pain and bloating. These presenting symptoms have been described in patients with symptoms . Upper eorganism , 14, 15. be used  to confiSarcina ventriculi is debated as it has been identified in clinically asymptomatic patients as well as in patients with life-threatening conditions like emphysematous gastritis and gastric perforations. Treatment with antibiotics and proton pump inhibitors have been reportedly used in symptomatic individuals [Sarcina ventriculi with pancreatic adenocarcinoma, but identification of these organisms warrants mention, as knowledge of their presence may provide a better global understanding of an individual patient's condition. Sarcina ventriculi organisms have been only rarely reported in cytology specimens, including urine cytology, esophageal brushings, and patients undergoing endoscopic ultrasound-guided biopsy evaluation for diagnosis and staging of gastric adenocarcinoma [Sarcina ventriculi in an FNA of metastatic pancreatic carcinoma in a perigastric lymph node. As mixed bacterial forms were present, and minimal accompanying inflammation was seen, carry-through organisms from the gastric lumen were favored.The pathologic role of ividuals , while iividuals . Currentarcinoma –19. To o