Cervical cancer is the leading cause of female cancer mortality in Botswana with the majority of cervical cancer patients presenting with late-stage disease. The identification of factors associated with late-stage disease could reduce the cervical cancer burden. This study aims to identify potential patient level clinical and sociodemographic factors associated with a late-stage diagnosis of cervical cancer in Botswana in order to help inform future interventions at the community and individual levels to decrease cervical cancer morbidity and mortality.There were 984 women diagnosed with cervical cancer from January 2015 to March 2020 at two tertiary hospitals in Gaborone, Botswana. Four hundred forty women (44.7%) presented with late-stage cervical cancer, and 674 women (69.7%) were living with HIV. The mean age at diagnosis was 50.5 years. The association between late-stage (III/IV) cervical cancer at diagnosis and patient clinical and sociodemographic factors was evaluated using multivariable logistic regression with multiple imputation. Women who reported undergoing cervical cancer screening had lower odds of late-stage disease at diagnosis  compared to those who did not report screening. Women who had never been married had increased odds of late-stage disease at diagnosis  compared to women who had been married. Women with abnormal vaginal bleeding had higher odds of late-stage disease at diagnosis  compared to those without abnormal vaginal bleeding. HIV was not associated with a diagnosis of late-stage cervical cancer. Rural women who consulted a traditional healer had increased odds of late-stage disease at diagnosis compared to rural women who had never consulted a traditional healer .Increasing education and awareness among women, regardless of their HIV status, and among providers, including traditional healers, about the benefits of cervical cancer screening and about the importance of seeking prompt medical care for abnormal vaginal bleeding, while also developing support systems for unmarried women, may help reduce cervical cancer morbidity and mortality in Botswana. Cervical cancer affects women across the globe, with a disproportionately higher burden of morbidity and mortality in low- and middle-income countries (LMICs) . In 2018Cervical cancer screening aims to prevent invasive cancer –10. OtheIn Botswana, an upper-middle-income country in SSA, cervical cancer is the leading cause of female cancer deaths , 23. HowThis study aims to identify potential clinical and sociodemographic factors associated with a late-stage diagnosis of cervical cancer in Botswana in order to help inform future interventions at the community and individual levels aimed at decreasing cervical cancer morbidity and mortality.We abstracted data from questionnaires administered during the initial consult visit and medical records for women with invasive cervical cancer who had consented to participate in research studies , 31 at PData collected at the time of cancer diagnosis included patient/sociodemographic and clinical factors , HIV status, and utilization of antiretroviral therapy (ART)). Additional clinical data was abstracted from medical records regarding clinical factors, such as stage, pathology, and CD4 count. Place of residence was characterized as urban or rural based on the sub-district of the participant’s reported residence .Cervical cancer stage at diagnosis was based on the International Federation of Gynecology and Obstetrics (FIGO) staging system , 34. FIGDescriptive statistics for each variable of interest were examined for the entire study sample, and by cervical cancer stage at diagnosis (early vs. late). Differences between early- and late-stage disease were examined using Pearson’s chi-squares test for categorical variables, Fisher’s exact test for small sample sizes, and Student t-tests for continuous variables. Multivariable logistic regression was used to examine potential risk factors associated with late- versus early-stage disease. Variables included in the multivariable model were determined based on purposeful selection, review of the literature, and clinical relevance. We assessed missing data, patterns, and reasons for missing data. To account for missing data, we performed multiple imputation with chained equations (MICE) for the multivariable logistic regression model, assuming data were missing at random –37. We aThis study, “Treatment and Outcomes of Patients Presenting with Cancer in Botswana,” was approved by the University of Pennsylvania as part of the Botswana-University of Pennsylvania Partnership (IRB: 820159 IRB#7 Penn) and by the Ministry of Health and Wellness of the Republic of Botswana (HPDME 13/18/1).Between January 2015 and March 2020, 1,007 women were diagnosed with cervical cancer. We excluded 16 women with prior cervical carcinoma in situ and seven women with recurrent disease, resulting in the inclusion of 984 women in this study. Sociodemographic and clinical characteristics are shown in Table Table There were no significant associations between living in an urban residence versus living in a rural residence and having ever visited a traditional healer and having late-stage cervical cancer at diagnosis in the multivariable model. However, in our cohort, more rural women than urban women (11% vs. 5%) had consulted with a traditional healer. Table 3 at diagnosis, and 96.2% (n = 640) reported being on ART.Table Table This large study of women with cervical cancer in Botswana aimed to identify potential clinical and sociodemographic factors associated with a late-stage diagnosis of cervical cancer in Botswana. Our study showed that prior cervical cancer screening was associated with decreased odds of having late-stage cervical cancer at diagnosis, whereas experiencing abnormal vaginal bleeding and having never been married were associated with an increased odds of having late-stage cervical cancer at diagnosis. Having HIV was not associated with having late-stage cervical cancer at diagnosis. Furthermore, results suggested that women living in rural areas who visited a traditional healer were more likely to be diagnosed with late-stage cervical cancer.Screening has been shown to lead to an earlier diagnosis of cervical cancer in high-income countries with established screening programs, and screening has also been shown to be effective in low resource settings , 9, 38. Early-stage asymptomatic cervical cancer can be detected through screening, but in the absence of screening, patients with cervical cancer can present with clinical symptoms including abnormal vaginal bleeding and post-coital bleeding . ReportsIn our study, there was no significant difference between WLWH and women without HIV with regard to their likelihood of presenting with advanced stage cervical cancer. Some studies have recognized HIV as a risk factor for late-stage cervical cancer at diagnosis , 19, yetOur study saw a decrease in late-stage cervical cancer cases among women who had been married. Similarly, a study from Nepal  noted thAreas of residence may also impact health care. For example, living in a rural area versus living in an urban area has been investigated as a potential contributing factor for women with late-stage cervical cancer at diagnosis , 18, 19.This large study investigating late-stage cervical cancer at diagnosis in Botswana includes detailed demographic and clinical information on patients in Botswana collected over a five-year period, but it does have several potential limitations and challenges. It is important to note that, due to the cross-sectional study design, no decisive conclusions can be made about the temporality or causality among the study variables and late stage diagnosis. Patients were enrolled at PMH, a public tertiary hospital with oncology services, and at GPH, a private tertiary hospital with the only chemo-radiation oncology center in Botswana; thus, all patients who need radiotherapy should be sent to GPH. We were unable to account for patients who were not diagnosed with or treated for cervical cancer outside of these two facilities. In addition, the study collected data at the time of diagnosis and is therefore subject to recall bias, social desirability bias, and potential unmeasured confounding and missing data. Unfortunately, due to the retrospective nature of the study, we lacked important information and were unable to account for confounders including education level, knowledge and awareness of cervical cancer, and cervical cancer screening. To account for any bias due to missing data, we conducted MICE for the primary analysis –37. ResuOur results highlight patient level factors associated with late-stage cervical at diagnosis and indicate potential areas for intervention to mitigate the cervical cancer burden in Botswana. Our findings show that cervical cancer screening for women in Botswana is associated with the early detection of cervical cancer, particularly in women with HIV. Future efforts to include women without HIV and women who have not been married in cervical cancer screening efforts could result in the earlier detection of cervical cancer in these groups. Future early cervical cancer detection efforts should emphasize cancer symptom awareness and early detection through cervical cancer screening, and should also include traditional healers in the cancer care continuum.


Immunocompromised patients are at higher risk of surgical site infection and wound complications. However, optimal management in the perioperative period is not well established. Present systematic review aims to analyse existing strategies and interventions to prevent and manage surgical site infections and other wound complications in immunocompromised patients.A systematic review of the literature was conducted.Literature review shows that partial skin closure is effective to reduce SSI in this population. There is not sufficient evidence to definitively suggest in favour of prophylactic negative pressure wound therapy. The use of mammalian target of rapamycin (mTOR) and calcineurin inhibitors (CNI) in transplanted patient needing ad emergent or undeferrable abdominal surgical procedure must be carefully and multidisciplinary evaluated. The role of antibiotic prophylaxis in transplanted patients needs to be assessed.Strict adherence to SSI infection preventing bundles must be implemented worldwide especially in immunocompromised patients. Lastly, it is necessary to elaborate a more widely approved definition of immunocompromised state. Without such shared definition, it will be hard to elaborate the needed methodologically correct studies for this fragile population. Surgical site infection (SSI) is a global health issue , 2. BothOther wound management and dressing techniques have been evaluated in the literature trying to reduce SSI incidence, but no definitive results have been obtained. Moreover, management of perioperative steroids, immunomodulatory drugs and additional “wound-healing” impacting medication is far from being fully understood , 12, 13.Basing on the existing literature, five principal areas of interest were investigated: (1) specific intervention in transplanted patients and in patients under immunomodulatory therapy undergoing surgery, (2) perioperative management of drugs affecting wound healing/SSI, (3) oncological patients operated under chemotherapy, (4) wound dressing in immunocompromised patients, and (5) high-risk patients undergoing general or emergency surgery.This systematic review was conducted based on PRISMA methodology . SSI wasAn immunocompromised host is a patient presenting an impaired or weakened immune system; this does not allow a normal response to infections.Congenital conditions Infected by human immunodeficiency virus (HIV) who developed acquired immunodeficiency syndrome (AIDS)Hematologic malignancyPatients affected by intrinsic immune conditions considered immunodeficiency along with one between “solid malignancy or solid organ transplanted patients or inflammatory disease/rheumatologic disease” plus the concurrent assumption of immunomodulatory drugs or chemotherapyPatients in a physiologic or pathologic condition that is accompanied by any degree of immunodeficiencyAcquired conditions:Immunocompromised patients are defined as follows , 17:ConPatients conditions. Low serum albumin concentration, older age, obesity, smoking, diabetes mellitus, and ischemia secondary to vascular disease or irradiationSurgical risk factors. Prolonged procedures and inadequacies in either the surgical scrub or the antiseptic preparation of the skinPhysiological states. Trauma, shock, blood transfusion, hypothermia, hypoxia, and hyperglycemiaBeside the properly defined immunocompromised patients, many other ones present a mix of conditions, surgical risk factors, and physiological states which increase the risk of SSI and contribute to define the high-risk population . These cIncluded trials are those about IP and high-risk populations analysing benefit of iNPWT over SD or other dressing/management techniques, techniques for suturing the surgical incision, and the effect of steroids, immunomodulatory, or other drugs affecting wound healing and infection development.A systematic search was conducted from January 2000 to March 2020, for all articles on immunocompromised patients and surgical site infection in MEDLINE via PubMed, The Cochrane Library, and Scopus by two reviewers  independently. The following terms were used: surgical site infection; SSI; immunocompromised; immunosuppressed; wound; primary, secondary closure; iNPWT, combined with AND/OR. Given that definition of immunocompromission is wide and there is no consensus, the search included also terms as “HIV”, “AIDS”, “transplanted”, and “chronic steroid therapy” with synonyms and MeSH terms. Manual and reference-text research for additional relevant studies was allowed. All duplicates, articles in a language other than English, and animal studies were removed. Abstracts were screened and not relevant studies were removed; then, full-text assessment of the articles was performed. Randomized controlled trials (RCTs), meta-analysis, prospective, and retrospective studies that included rate of SSI as the first outcome, length of hospital stay (LOS), and other surgical complications were included. Studies with surgical specialties other than general and colorectal  were excluded, unless they specifically deal with immunocompromised patients.Studies on SSI in adult population (> 18 years old) in general surgery where high-risk patients were assessed even if there was no formal definition of immunocompromised state were included . Case reports were excluded. Case series with less than 30 patients were also excluded unless they reported relevant results  studies on the same topic were excluded, conversely small “unique” RCTs on anti-vascular endothelial growth factor (VEGF) therapy were included).In case of disagreement between the two senior reviewers , the consensus was reached by discussion, if there was no consensus a third reviewer was sought (FCa).Research results were reported according to PRISMA flow chart in Fig. There is a paucity of studies focusing on immunocompromised patients and specifically on SSI prevention and management. Relevant studies to be included are listed in Table As said however, under the category of immunocompromised patient, or even under the definition of patient at high risk of developing SSI, we found a myriad of different specific conditions that have been differently investigated by literature.Given this characteristic of the research, the systematic review of literature enlightens specific answers to specific questions, leaving some areas uncovered. At present, from literature, it is possible to obtain precise data about the following topics, while the general frame can be only deducted by interpretating specific suggestions.Role of prophylactic antibacterial therapy and preventive methodsImmunosuppressant therapy and SSI in kidney transplantSkin closure and SSI in kidney transplantSurgical drain and SSI in kidney transplantIn transplanted patient, precise data exist regarding the following:Oncological patients under chemotherapy and SSIInflammatory bowel disease under immunomodulatory therapy and SSIRegarding drugs that may affect wound healing literature explores:iNPWT and SSI in transplanted patientsiNPWT and SSI in patients under immunomodulatory drugsiNPWT and SSI in high-risk patientsThe effect of incisional negative pressure wound therapy has been investigated in the following conditions:SSI in transplanted patients carries several consequences, with a consistent increase in graft loss rates, morbimortality, LOS, and costs –21. ConsGurusamy et al.  presenteP = 0.39) and per-protocol  analyses between the two groups. Furthermore, no significant differences in multiple secondary outcomes were recorded.The role of antibiotic prophylaxis in transplantation is strongly debated, especially considering the increasing emergence of multidrug-resistant bacteria, and its utility is questioned , 31–33. A randomized trial on kidney transplanted (KT) patients investigated the rate of SSI in patients treated with a sirolimus- or tacrolimus-based therapy . Both grA recent prospective observational study investigated 104 patients with their incision closed “partially” with interrupted stitches at 4 cm apart from each other with no drains placed. Patients were permitted to shower at postoperative day one. No patient experienced SSI infection either immediately postoperatively or at follow-up. Patient satisfaction scores ranked up to 99% .It is unclear if the placement of surgical drainage in KT lessens or increases the SSI rate. The rationale would be the reduction of deep fluid collections and the access to prompt diagnosis in the event of complications . No randSome drugs may impair wound healing, an effect that could be enhanced in the immunocompromised population. Oncological patients undergoing surgery while in therapy with VEGF inhibitors (i.e. bevacizumab) may be exposed to higher surgical site events. Patients affected by inflammatory bowel disease (IBD) may assume drugs that impair wound healing and may favour surgical site complications.Data from two randomized trials , 40 wereThe long half-life of the bevacizumab (up to 20 days) must be considered. Its immediate suspension in face of emergency surgery could not reduce its circulating level on time . The susA retrospective analysis of patients undergoing surgical procedures for Crohn’s disease (CD)  comparedA retrospective cohort trial showed as perioperative administration of infliximab or steroids may not be associated with increased morbidity if compared to their non-administration in patients with CD undergoing surgical procedure . HoweverA recent systematic review analysed the feasibility of iNPWT for the treatment of wound complications in KT . A totalNo literature exists about the application of NPWT in patients undergone to other solid organ transplant. However, it is reasonable to consider its possible efficacy in reducing postoperative wound complication in all the patients undergone to solid organ transplant other than kidney.A retrospective analysis of high-risk patients (including patients on immunomodulatory drugs or steroids) investigated the benefit of iNPWT –49. PatiAn RCT  analysed 50 patients undergoing general or emergency abdominal surgery whose incisions were treated with or without iNPWT . A benefThe same study group presented a retrospective analysis with different results where they observed reduction of SSI with iNPWT vs. SD  .An RCT analysed 123 high-risk patients allocated to receive either iNPWT or SD after pancreatic surgery . The oveA randomized trial on 284 patients  compared SD vs. iNPWT in open colorectal procedures. No difference was found in the 30 days SSI rate in the two groups . 9.3% of patients were either immunocompromised or on chronic steroid therapy, and they were equally distributed through the two groups [Two large multicenter randomized trials on trauma population undergoing surgery for lower limbs fractures found no difference in SSI rate in patients treated with iNPWT vs. SD , 58. In The great discordance between results of these trials inspired several meta-analyses. However, in some of these papers, immunocompromised and high-risk patients were mixed together with other cohorts. Indicative results may be obtained from those meta-analyses; however, specific indications cannot be finalized.Strugala et al. took into account several papers on spinal, orthopaedic, breast, and vascular surgery . They obAnother meta-analysis focused on iNPWT in colorectal and abdominal elective and emergency surgery . ResearcIn 2019, Zwanenburg et al. conducted a meta-analysis with a meta-regression taking into account the wide heterogeneity within the RCTs. They found that iNPWT decreased SSI rate. They did not stratify results for various surgical categories, but they did it for surgical field conditions  . In the Lastly, Kuper et al.  conducteThe definitions of the IP are multiple and heterogeneous . SeveralReducing SSI in elective and emergency abdominal surgery has always been matter of great attention. This is exceptionally true in the IP where SSI morbidity and mortality are higher.A prompt recognition of the IPs allows to insert them in a high-risk category and, thus, to establish the correct preventive measures. However, as we can extract from the transplantation experience , it is dIn the same time, commonly used practices like antimicrobial prophylaxis could be inefficient to prevent SSI in IPs –33. FurtWHO 2016 guidelines on SSI ); moreovOne year later, the WHO guidelines, in an international multidisciplinary consensus group , elaboraIn the present systematic review, 7 RCTs, 2 meta-analysis, and 2 retrospective studies on iNPWT were included. Four RCTs found no benefit with iNPWT while 3 found an advantage; 2 meta-analysis found benefit in iNPWT, while two found no benefit at all. In retrospective studies, data on the incidence of SSI in standard dressing groups or iNPWT did not match the RCTs results. The reason for these differences, especially when found in RCTs must be investigated.Some RCTs or meta-analysis evaluated a mixed and different population from the one matter of the present study as trauma patients  or otherNo blinding in assessing SSI within groups was adopted. The Centers for Disease Control (CDC) criteria  were useNPWT unlikely causes harm, but its application lengthens intervention and costs are elevated. Cost evaluation must be attentively evaluated . Each paA similar phenomenon has been observed in meta-analysis. Strugala et al.  reportedA meta-analysis of cohort studies (patients before and after the implementation of SSI-management-bundles) analysed more than 8500 patients . None ofTwo large trials on iNPWT are actually ongoing , 72; howThe use of surgical incision partial closure by distanced interrupted stitches in transplanted patients demonstrated very good results  and it mDrain placement is debated and probably needs more investigation. In KT, the drain placement has always been done on patient-by-patient evaluation or by protocol of the different centres. Retrospective trials showed no advantage for drain placement  in reducPatients on steroids experience impaired wound healing  and deveIsmael et al.  conducteWHO guidelines for SSI suggest against the suspension of those drugs which may impair wound healing in a statement graded as conditional and with low quality of evidence . The recIt has been demonstrated as Rapamycin inhibits early- and mid-stages angiogenesis, but not late-stage angiogenesis or lymph-angiogenesis . This daNo suggestion can be given regarding suspension of the immunomodulatory drug since evidence are lacking in favour or against it. However, surgeons must be aware of the possible complications and the various surgical strategies to reduce the risk.This work presents as main strength the fact that it for the first time systematically analysed the existing literature about the topic and resumed all the data investigating SSI prevention and management in immunocompromised and high-risk patients. Moreover, it attempts to give precise indications to those who are in charge to manage such a frail patient.As a counterpart, the present paper has several limitations: it lies in the proposal of finding an answer to a topic that carries multiple questions. There is no standardized definition of IP and it is difficult to find a single intervention that will lower the SSI rate in this population; therefore, the range of different topics included in our systematic review is wide. In many studies, IP are mixed to other cohorts and high-risk patients. In the existing literature, there are several RCTs on iNPWT, while other interventions are less investigated: there is no cunning in observing that economic and resource setting may influence the production of RCTs on iNPWT.Applicability of the results is often sub-optimal given that few studies exist on IP-specific population and somehow is necessary to consider evidence derived from studies analysing similar cohorts of patients.Strict adherence to SSI infection preventing bundles must be implemented worldwide especially in immunocompromised patients. Partial skin closure can be used to reduce SSI in this population. A clear role of antibacterial prophylaxis in IPs should be ruled out. There is not sufficient evidence to definitively suggest in favour of prophylactic negative pressure wound therapy. The use of mTOR and CNI in transplanted patient needing ad emergent or undeferrable abdominal surgical procedure must be carefully and multidisciplinary evaluated. Lastly, it is necessary to elaborate a more widely approved definition of immunocompromised state. Without such shared definition, it will be hard to elaborate the needed methodologically correct studies for this fragile population.


Sarcopenia is a common geriatric disorder characterized by decreased muscle strength, low muscle mass and poor physical performance. This aging-related skeletal muscle deterioration leads to adverse outcomes and severely impairs the quality of life of patients. The accumulation of dysfunctional mitochondria with aging is an important factor in the occurrence and progression of sarcopenia. Mitochondrial quality control (MQC) fundamentally ensures the normal mitochondrial functions and is comprised of four main parts: proteostasis, biogenesis, dynamics and autophagy. Therefore, any pathophysiologic factors compromising the quality control of homeostasis in the skeletal muscle may lead to sarcopenia. However, the specific theoretical aspects of these processes have not been fully elucidated. Current therapeutic interventions using nutritional and pharmaceutical treatments show a modest therapeutic efficacy; however, only physical exercise is recommended as the first-line therapy for sarcopenia, which can ameliorate skeletal muscle deficiency by maintaining the homeostatic MQC. In this review, we summarized the known mechanisms that contribute to the pathogenesis of sarcopenia by impairing normal mitochondrial functions and described potential interventions that mitigate sarcopenia through improving MQC. Senescence is a natural process of aging associated with degeneration of physical functions. The gradual loss of muscle mass, strength and function is one of the most important hallmarks of aging. Muscle mass starts to slightly decline from the age between 30 to 40 years of age along with a reduction in muscle function . The redThe term sarcopenia was coined by Irwin Rosenberg in the 1980s to describe an age-dependent decline in muscle mass and its adverse effects on human health . In 2019Mitochondria have strong impacts on the maintenance of cellular viability, including ATP production, oxidative phosphorylation (OXPHOX) homeostasis, calcium buffering and apoptosis. Therefore, healthy quality control is crucial for the preservation of intracellular homeostasis of muscle cells with aging. The MQC includes mitochondrial proteostasis, biogenesis, dynamics and autophagy , 18. OrcThe incidence rate of sarcopenia in the mid-life and elderly population varies according to different age, operational definitions, regions and ethnicities -31. A nuMitochondrial quality control is an elaborate and complicated network in eukaryocytes for maintenance of mitochondria homeostasis in eukaryotes by means of four core processes: mitochondrial proteostasis, biogenesis, dynamics and autophagy. Mitochondrial dysfunction amplified by defective quality control processes is gradually regarded as the major pathophysiologic mechanism of sarcopenia .Mitochondrial proteostasis plays an essential role in retaining the dynamic balance between new protein synthesis and impaired protein degradation. Imbalanced proteostasis leads to the accumulation of unnecessary and defective proteins, which further adversely impacts multiple physiological functions, including skeletal muscle activity .An intricate mechanism for the removal of misfolded and dysfunctional proteins is present in muscle mitochondria. The ubiquitin-proteasome system (UPS) and autophagy-lysosome system (ALS) are two predominant pathways to selectively eliminate damaged mitochondrial proteins , 38; UPSThe synthesis of new and properly functioning proteins is an important part of protein turnover in mitochondria. An increasing number of extensively studied regulators of protein synthesis in skeletal muscle are likely to be associated with the pathogenesis of sarcopenia. Akt is the key element of the PI3K/Akt/mTOR signaling pathway and acts as a pivotal mediator in the homeostasis of muscle mass through the mTOR/p70S6K and FoxO3/MuRF-1 and MAFbx pathways to regulate protein synthesis and degradation, respectively . MoreoveProtein dyshomeostasis may induce muscle mass and strength insufficiency in sarcopenia. UPRmt-related genes were significantly decreased in the muscle of sarcopenia patients, including those encoding mitochondrial heat shock proteins (HSP) and proteases . In addiMitochondrial biogenesis is a multistage process that generates new mitochondria . Mitochomnd2 (-/-) mice manifested low muscle mass, poor physical function and sarcopenia phenotypes. Intriguingly, HtrA2 apparently regulates mitochondrial biogenesis in sarcopenia via the differential expression of Nrf-1/2, but not PGC-1α.Mitochondrial biogenesis and its regulators collectively participate in the pathophysiologic changes in sarcopenia. In an interethnic study of human sarcopenia, it was demonstrated that expression profiles of PGC-1α, ERRα and other coactivators were reduced in sarcopenic individuals . PGC-1α,+-dependent deacetylase [+ levels was reduced [+ levels and thus activate NAD+-dependent SIRT1 [PGC-1α is a critical regulator that maintains muscle homeostasis and has attracted considerable attention due to the studies of its potential effects in aging-associated diseases . In mammcetylase , can dircetylase . It has cetylase . In sarc reduced , while Ant SIRT1 , 78. Mornt SIRT1 . It is tMitochondria are highly dynamic organelles that constantly fuse with surrounding mitochondria and split into daughter mitochondria . MitochoSeveral proteins that mediate mitochondrial dynamics are found to be dysregulated in skeletal muscle. For instance, Drp1 knockdown was found to induce muscle atrophy , 88. CurThese aforementioned studies demonstrated the prominent role of abnormal mitochondrial dynamics in muscle aging and sarcopenia ; howeverEntire mitochondrion can be degraded by autophagosomes with a fused lysosome through an intricate catabolic process termed autophagy. Mitophagy is an exceptional type of macroautophagy that primarily mediates the selective removal of the damaged or superfluous organelles and protein aggregation to maintain mitochondrial homeostasis , 97. TraBiological aging adversely affects mitophagy homeostasis in various organs and systems -112 and Additionally, mitochondrial autophagy, dynamics and biogenesis are closely associated with each other. Mitophagy and mitochondrial dynamics are interrelated because the conversion between fusion and fission is a prerequisite for mitophagy . PINK1 cIn summary, the maintenance of homeostatic MQC is attributed to the synergistic regulation of mitochondrial proteostasis, biogenesis, dynamics and autophagy in senescence. Disruption of quality control homeostasis results in accunulation of dysfunctional mitochondria that negatively affects skeletal muscle health in older individuals and may even lead to sarcopenia.Physical activity, especially resistance exercise training, has been recommended as the first-line intervention to manage sarcopenia according to the evidence-based clinical practice guidelines .Physical activities that set off skeletal muscle contraction against resistance can be defined as resistance exercise. Resistance exercise increases muscle mass, elevates muscle strength and improves the performance of physical exercise in elderly sarcopenia patients , 126. SeIn addition to resistance-based exercise, endurance training is involved in the maintenance of MQC in skeletal muscle as well, including mitochondrial protein synthesis , 137, miCurrent studies indicate that physical exercises enhance muscle performance and relieve sarcopenic manifestations, in part due to optimized MQC. Currently, consistent recommendation of specific physical exercise for sarcopenia patients is not available; however, exercise plans should correspond to patient abilities and rehabilitation goals to achieve the best therapeutic effect.Certain limitations for physical exercises in patients with sarcopenia include increased risk of fractures and falls in older adults and poor adherence to long-term training programs . Therefo+ was detected in sarcopenia, cardiovascular aging and neurodegenerative diseases [+ may become a novel strategy for alleviating sarcopenia. Moreover, urolithin A, as one of the end-products of ellagitannins (ETs) and ellagic acid (EA), was also found to induce mitophagy and improve muscle functions in aged mouse, which probably becomes a promising nutritional supplementation for sarcopenia [Recently, several bioactive compounds or drugs have been identified as latent and potent options to prevent and delay the progress of sarcopenia through ameliorating mitochondrial dysfunction. Melatonin  is a pineal hormone that is ubiquitously present in most organs and tissues, including skeletal muscle . Melatondiseases , 78, 153diseases , 155. Thrcopenia . HoweverOverall, the combination of physical exercise and protein supplementation is the most effective countermeasure for sarcopenia. However, nutritional and pharmacological interventions are more applicable for the majority of sarcopenia patients. Several potential therapeutic agents have been demonstrated to mitigate mitochondrial malfunction for the prevention and treatment of sarcopenia. Furthermore, gene therapy probably become a novel strategy for alleviating sarcopenia. METTL21c acts as a bone-muscle pleiotropic gene for sarcopenia. Although METTL21c’s biofunction is achieved by NF-κB signaling pathway, its potential role in quality control mechanisms of mitochondria deserves more investigation . AdditioSarcopenia is a prevalent and degenerative skeletal muscle disease that leads to poor quality of life in patients, particularly elderly individuals. Numerous pathophysiological alterations contribute to a progressive decline in muscle strength, muscle mass and physical performance, which are the predominant hallmarks of sarcopenia. Dyshomeostasis of mitochondrial quality control is one of the primary factors for the initiation and progression of sarcopenia. MQC requires coordination of mitochondrial proteostasis, biogenesis, dynamics and autophagy. Alterations in mitochondrial quality control exacerbate muscle atrophy and reduce muscle strength during aging concomitant with restricted locomotive function. The important regulators and signaling pathways in MQC may be involved in the etiology of sarcopenia and are comprehensively summarized in this review. Physical exercise, the most recommended therapeutic intervention for sarcopenia, improves skeletal muscle quantity and quality partially by facilitating the restoration of malfunctional mitochondria. Additionally, nutritional and pharmaceutical treatments ameliorate sarcopenia to a certain degree, although compelling evidence and mechanisms remain to be identified. Overall, dysfunctional MQC plays a causative role in sarcopenia and revealing latent mechanisms may shed light on efficient preventive and intervention strategies for patients with sarcopenia.


The isolation of podocytes in the urine and subsequent culture of urinary podocytes in vitro suggest that detachment of viable podocytes could be another important mechanism for podocyte loss. In this review, we summarize recent advances that address this controversial topic on the specific circumstances of podocyte loss.Glomerular podocytes are characterized by terminally differentiated epithelial cells with limited proliferating ability; thus, podocyte loss could not be fully compensated by podocyte regeneration. A large body of clinical studies collectively demonstrated that podocyte loss correlated with glomerular diseases progression. Both podocyte death and podocyte detachment lead to podocyte loss; however, which one is the main cause remains controversial. Up to date, multiple mechanisms are involved in podocyte death, including programmed apoptotic cell death (apoptosis and anoikis), programmed nonapoptotic cell death , immune-related cell death (pyroptosis), and other types of cell death (necroptosis and mitotic catastrophe-related cell death). Apoptosis is considered a common mechanism of podocyte loss; however, most of the data were generated  Glomerular podocytes account for approximately 30% of glomerular cells in number, which constitute glomerular filtration barrier (GFB) together with endothelial cell and glomerular basement membrane (GBM) . Podocytin vitro and the in vivo evidence of podocyte apoptosis is limited. The isolation of podocytes in the urine and subsequent culture of urinary podocytes in vitro suggest that detachment of viable podocytes could be another important mechanism for podocyte loss. In this review, we will summarize recent advances that address the mechanisms of podocytes loss.Podocyte loss can be caused by either podocyte death or podocyte detachment; however, which one is the main cause remain controversial. The major pathways involved in podocyte death include programmed apoptotic cell death (apoptosis and anoikis), programmed nonapoptotic cell death , immune-related cell death (pyroptosis), and other types of cell death (necroptosis and mitotic catastrophe-related cell death) . ApoptosPodocytes possess a complicated cellular structure, which consists of a cell body, a major process extending outwardly from their cell body, and a secondary process (foot process) surrounding the glomerular capillary loops. Major processes are restrained by microtubules and intermediate filaments, while foot processes are tethered by an actin-based cytoskeleton. Podocyte foot processes are connected by a functioning structure named slit diaphragm (SD), consisting of many proteins such as nephrin, Neph1, and podocin, which actively participate in podocyte signaling . AdditioThe cytoskeleton of podocytes and its interaction with GBM accounts for not only the attachment of the cells to the extracellular matrix but also the regulation of the capillary width, which is critical for determining the glomerular filtration rate . Many prThe cell cycle is a tightly regulated event that consists of four phases: G1 phase (ready for DNA synthesis), S phase (DNA replication), G2 phase (the gap between DNA synthesis and mitosis), and M phase (mitosis and cytokinesis). Cell enters a state of quiescence at the G0 phase. The cell’s nucleus divides in the mitosis process, while the cell’s cytoplasm divides in the cytokinesis process. A balance of cyclins and cyclin-dependent kinase (CDK)  and CDK inhibitors  governs the cell cycle . Differein situ on the glomerular basement membrane could result in cell detachment. Conversely, podocyte detachment leads to cell death. However, detachment of viable podocytes also occurs. In addition, we do not know whether podocytes detach because of their death or die because they lose their adhesion to GBM. Currently, in human kidney disease, podocytes injury was assessed mainly by the ultrastructural appearance of the secondary foot processes under transmission electron microscopy (EM), which are interdigitating and teethlike in normal condition but usually appear flat due to foot process effacement during podocyte injury. The assessment of podocyte death or detachment is not routinely performed in human renal biopsy specimens  transgenic mice  is a type of nonapoptotic cell death, which occurs in the absence of chromatin condensation but through a lysosome-dependent degradation pathway, accompanied by large-scale autophagic vacuolization of the cytoplasm . The detin vitro and in vivo  pathway is the best-characterized repressor of autophagy . The mecAltogether, these data suggest that autophagy dysregulation related cell death could be a major pathway for podocyte death, particularly in diabetic kidney disease.Necroptosis is a regulated cell death that shows the same characteristics as necrosis in morphology (lysis of cell membrane) and is different from the apoptosis signal pathway in its mechanism. A variety of molecules such as RIPK1 (receptor-interacting protein kinase 1), RIPK3 (receptor-interacting protein kinase 3) and its substrate MLKL (mixed lineage kinase domain-like pseudokinase) participate in the regulation of cell necroptosis . Westernvia RIPK1/RIPK3 pathway, evidenced by western blot assay of necrosis pathway markers such as RIPK3 and MLKL and scanning EM  nning EM . Sosna anning EM . Recentlnning EM . Thus, tPyroptosis is an inflammatory form of programmed cell death regulating immunity response . There avia the adaptor protein of CARD-PYRIN  or the neighboring cells, which could be regulated pathways . Gilmorepathways . Howeverpathways . Howeverpathways . HoweverDespite the above observations that favor the early and essential role of detachment in podocyte loss, there are two remaining issues regarding the urinary podocytes: 1) the identification of podocytes mainly relied on the podocalyxin-positive cells. Nevertheless, podocalyxin-positive cells are not necessarily podocytes since many other cells express podocalyxin, such as hematopoietic stem cells, vascular endothelial cells, platelets, and macrophages . 2) ThouThe “podocyte domino effect” is recently identified as a novel mechanism of progressive podocyte loss . Podocytin vivo and therefore may mask the fact that they are dead before detachment. Our understanding of the mechanisms of podocyte loss needs to be improved with more studies. Ultimately, we could develop effective measures to prevent podocyte loss as a therapy for glomerular disease.Podocyte number was correlated with the amount of proteinuria and extent of glomerulosclerosis. Due to its limited regeneration capacity, it is important to understand the underlying mechanism of podocyte loss, and therefore, we could develop effective measures to prevent their loss. Several common cell death pathways likely apply to podocyte loss in kidney diseases; however, podocytes may have unique pathways such as anoikis, podoptosis, and mitotic catastrophe. Podocytes may undergo cell death and detachment sequentially; however, the evidence of increased viable urinary podocytes in patients with glomerular disease supports that they may detach and stay alive. The dead podocytes are eliminated quickly by phagocytosis, so it is difficult to detect them


Epigenetic shifts are a hallmark of aging that impact transcriptional networks at regulatory level. These shifts may modify the effects of genetic regulatory variants during aging and contribute to disease pathomechanism. However, these shifts occur on the backdrop of epigenetic changes experienced throughout an individual’s development into adulthood; thus, the phenotypic, and ultimately fitness, effects of regulatory variants subject to developmental- versus aging-related epigenetic shifts may differ considerably. Natural selection therefore may act differently on variants depending on their changing epigenetic context, which we propose as a novel lens through which to consider regulatory sequence evolution and phenotypic effects. Here, we define genomic regions subjected to altered chromatin accessibility as tissues transition from their fetal to adult forms, and subsequently from early to late adulthood. Based on these epigenomic datasets, we examine patterns of evolutionary constraint and potential functional impacts of sequence variation . We find that while the signals observed with developmental epigenetic changes are consistent with stronger fitness consequences , they tend to have weaker effects on genetic risk associations for aging-related diseases. Conversely, we see stronger effects of variants with increased local accessibility in adult tissues, strongest in young adult when compared to old. We propose a model for how epigenetic status of a region may influence the effects of evolutionary relevant sequence variation, and suggest that such a perspective on gene regulatory networks may elucidate our understanding of aging biology. It has been suggested that the process of aging, and the concomitant manifestation of aging-related disease, is subject to both genetic and non-genetic factors impacting the regulatory networks (and subsequent behaviors) of aging cells , 2. NongAs aging can also be considered a continuation of development , the epiEpigenetic marks established during development can persist into adulthood , but theWhile epigenomes vary between cell types , 26 and Importantly, the epigenetic state of genetic variants  influences their regulatory effects, and subsequent association with heritable disease risk . Thus, gIn the present study, we seek to characterize common epigenetic trends between fetal and adult tissues, and subsequently examine the potential interaction of these developmental changes with later changes associated with epigenetic aging in adult tissues. We utilize our findings to propose a model for how evolutionary forces may have acted at these loci in humans, and how these forces in turn influence the distribution of mutations conferring heritable disease risk across a number of age-associated pathologies.To investigate epigenetic changes occurring over the course of post-natal development and aging, we focused on chromatin accessibility, as it reflects the regulatory potential of a genetic locus and can be considered a property of the epigenome which integrates a number of possible epigenetic phenomena  . We thusWe first identified chromatin regions exhibiting recurrent accessibility changes between fetal and adult samples across tissue types see , 2. We dTo gain insights into the roles these region sets have in transcriptional regulation, we next characterized the genomic distribution of our adult-biased and fetal-biased region sets using adult tissue epigenetic states  Supplem. We founcis-regulatory sequences, we next sought to identify the potential role of our regions in regulatory changes occurring during development and aging. We did this by considering promoter-level accessibility . We observe similar enrichment terms as well as significant overlaps with gene sets defined on the regulatory level . We found that genetic diversity in fetal-biased regions was markedly reduced  compared to genomic backgrounds, as well as to intronic and promoter-TSS elements . ConversOverall, natural selection appears to have acted upon regions subject to accessibility shifts in development and aging, modifying some loci  while protecting others  .Importantly, selective forces, both positive and negative, manifest phenotypically through the effects of random genetic mutations, which act to modify gene regulatory networks to varying degrees. We next examine this relationship.In our above analyses, the observed signals of consistent inter- and intra-species conservation in regions most associated with early development  follows with the expectation that variants negatively impacting early-life would be subject to stronger purifying selection , 53, 54.It has been suggested that the highly polygenic nature of complex traits and diseases reflects cumulative regulatory modification to a ‘core’ set of genes which functions most proximately in relevant biology . Across As variants in accessible non-coding regions likely have regulatory impacts generally , we confConsidering first developmental change, we found that variants in regions gaining nearby accessibility in adults  have greater association with disease than those in regions losing nearby accessibility  . UnexpecTaken together, these results would suggest that those variants most accessible in younger adults stand to have the greatest impact  on late-life disease risk – a finding that may have important implications for understanding the development of disease over adulthood  of regulatory elements. Subsequently, we found that the risk association of variants across a number of age-associated diseases also varied based on accessibility change in the vicinity of the variant. Much work has been done on understanding the relationship between sequence conservation and disease risk –66. For revalent , 67. Givrevalent  . Namely, those regions exhibiting the highest sequence constraint , left doSIRT6 and SIRT7 as having multiple variants falling nearby young-biased regions which contacted their respective gene promoters , should implicate sets of genes involved in aging biological processes. Our gene-level GWAS analyses using young-biased regions identified genes involved with histone deacetylation as being more consistently associated with aging-disease GWAS signal, a pattern which was diminished when considering gene-level associations in the absence of this epigenetic information , and wheromoters . Both thic aging , while Ske state . It may ke state .In this study we sought to describe how changing epigenetic context, defined here as changes to chromatin accessibility over both development and subsequent aging, influences the behavior of evolutionary forces and genetic disease risk at the sequence level. To address this question, we defined genomic regions whose chromatin accessibility consistently shift over the course of development and aging. Our approach to identify epigenetic shifts relies on the observation that chromatin accessibility broadly reflects the regulatory capacity at a given locus , though We performed several analyses suggesting that these regions reflect developmental and aging signatures from previous literature, including genomic features , epigenetic states  and histone mark data. Gene sets associated with developmentally-altered regions were enriched for immune system function and cellular proliferation terms, echoing an earlier study of fetal to adult epigenetic changes . FurtherGiven that epigenetic state impacts the potential regulatory effects of deleterious variants , we lookAccording to the fetal programming model, we would expect that regulatory regions most active during early development, both dictating developmental processes as well as responding to environmental perturbations , would hAccording to a model wherein epigenetic aging influences the phenotypic effects of regulatory mutations, we would expect that mutations with increased local accessibility in adult tissues, particularly aged adult tissues, would have stronger impacts on aging disease biology in these tissues (reflected in increased association with heritable disease risk). Here, we found that variants gaining nearby accessibility  have stronger associations across a number of aging-related diseases including several kinds of neoplasms, arthritis, and atherosclerosis. This finding suggests that the regulatory effects of deleterious variants may become ‘uncovered’ as tissues mature and follows with proposed links between development and ageing processes , 20, 24.We cannot rule out the effects of cell-type specific epigenetic (accessibility) shifts influencing the phenotypic impacts of regulatory sequence modifications on aging-associated disease risk. Similarly, it has been suggested that a facet of aging is ‘epigenetic drift’ – the accumulation of epigenomic aberrations that contribute to mis-regulation of gene regulatory networks, a component of which is tissue-specific , 80. HowRegulation of general aging-related mechanisms, as well as increases in heritable disease risk, represent phenotypes upon which evolutionary forces may act to modify aging and mortality rates. We found that young-biased regions were enriched for signals of positive selection, a number of which implicated relevant aging-associated genes, and exhibited increased phylogenetic and within-human sequence constraint. Given that these behaviors are intermediate between those observed with regions more accessible in fetal and older-adult tissues, we suggest the following model .Regulatory sequences most active during development are subjected to strong negative selection, both to maintain human-derived functional sequences and discourage subsequent modifications, as dysregulation of development would have the largest fitness consequences. Similarly, sequences most active during early adulthood are subjected to negative selection to maintain proper tissue maintenance and discourage disease. However, the strength of this selection is reduced, as we expect fitness benefits/costs to diminish with age as individuals reproduce less frequently –55. ThusDNase-I hypersensitivity datasets were obtained from ENCODE  for eighAdult DNase samples were further stratified in order to define age-altered chromatin regions, splitting samples used in the above analysis into those individuals younger than 50 (‘young-adult’) and those older than 50 (‘old-adult’), this age representing a roughly equal split of sample numbers. Not all tissues used in the initial fetal/adult comparison were represented in these age-stratified sets – thus we restricted the tissue comparisons to brain, heart, lung, muscle and stomach tissues. A similar computational method as that used in defining fetal- and adult-biased regions was applied here see . We compAll hg19 Refseq gene TSS were obtained from the UCSC Genome Browser  and paddPromoter capture datasets: Promoter-capture data was obtained from Jung et al., 2019 ; this daGene-set enrichment analyses: Gene sets generated in our analyses were tested for enrichment in different GO Biological Process terms using the ‘enrichGO’ function from the clusterProfiler  package ENCODE RNA-seq datasets: Processed per-gene quantification files, as generated by the ENCODE pipeline were obtained from the ENCODE web portal  see Sup.GTEx RNA-seq processing: Processed RNA-seq quantification files were obtained from the GTEx web portal  for the Human sequence variation datasets: Variation data from the 1000 Genomes Project phase 3 (1KGP)  n = 250.https://nealelab.github.io/UKBB_ldsc/downloads.html). These data were subsequently utilized across several bioinformatic analyses (see GWAS summary statistics data: To define a set of aging-associated diseases for use in our analyses, we first used broadly-defined categories as described in Chang et al., 2019 . This styses see .Additional computational methods, including implementations of statistical tests described in the Results, are described in detail in the Supplementary Information and MethodsSupplementary FiguresSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4


Affordable, sensitive, and scalable technologies are needed for monitoring antiretroviral treatment (ART) success with the goal of eradicating HIV-1 infection. This review discusses use of Sanger sequencing and next generation sequencing (NGS) methods for HIV-1 drug resistance (HIVDR) genotyping, focusing on their use in resource limited settings (RLS). Sanger sequencing remains the gold-standard method for detecting HIVDR mutations of clinical relevance but is mainly limited by high sequencing costs and low-throughput. NGS is becoming a more common sequencing method, with the ability to detect low-abundance drug-resistant variants and reduce per sample costs through sample pooling and massive parallel sequencing. However, use of NGS in RLS is mainly limited by infrastructure costs. Given these shortcomings, our review discusses sequencing technologies for HIVDR genotyping, focusing on common in-house and commercial assays, challenges with Sanger sequencing in keeping up with changes in HIV-1 treatment programs, as well as challenges with NGS that limit its implementation in RLS and in clinical diagnostics. We further discuss knowledge gaps and offer recommendations on how to overcome existing barriers for implementing HIVDR genotyping in RLS, to make informed clinical decisions that improve quality of life for people living with HIV. In an effort to end the HIV epidemic by 2030, the UNAIDS 95-95-95 goals aim to ensure that 95% of all people living with HIV know their status, 95% of all people diagnosed receive sustained antiretroviral treatment (ART), and 95% of all people receiving ART have sustainable viral suppression ,2. HIV-1Sanger sequencing is a population-based method that uses a di-deoxy chain termination chemistry to generate a single consensus sequence representative of the most common bases at each nucleotide position . This, hNGS also has the ability for massive parallel sequencing of individual input templates, generating incredible amounts of data per sequencing run ,14. Givepol gene, Illumina’s short read sequencing and lower instrument cost make it a front runner for HIVDR genotyping [Sanger sequencing platforms have largely been dominated by Applied Biosystems, with instruments ranging from 3100- to 3700-series, as well as the SeqStudio  instrument . The ABInotyping . Table 1Several in-house Sanger sequencing assays have been developed over the years for HIVDR genotyping. However, there is currently one commercially available Sanger sequencing assay approved by the US Food and Drug Administration (FDA) for HIVDR genotyping, namely ViroSeq HIV-1 Genotyping kit ,24,25,26A collaborative partnership between the Southern African Treatment and Resistance Network (SATuRN) and not-for-profit manufacturers (Life Technologies/Centers for Disease Control and Prevention) previously developed a discounted HIVDR genotyping assay that reliably detects mutations in the HIV-1 protease (PR) and reverse transcriptase (RT) genes from plasma and dried blood spot samples by Sanger sequencing ,29,30,31There is currently only one commercially available NGS assay approved by the FDA in November 2019 for clinical HIVDR genotyping, known as the Sentosa SQ HIV-1 Genotyping Assay ,50. ThisFor over two decades Sanger sequencing has been shown to be a highly reproducible and interpretable method for diagnosing HIVDR in clinical settings ,32,33. TIn contrast, there is a wider range of NGS platforms supplied by various manufacturers, using different sequencing chemistries  and achiAs new drugs to treat HIV-1 patients increase and extensive drug class resistance grows, HIVDR genotyping methods need to evolve to determine resistance to newer drug classes such as capsid inhibitors, entry inhibitors, nucleoside analogue reverse transcriptase translocation inhibitors and Rev inhibitors. For instance, as ART programs in RLS adopt use of integrase strand transfer inhibitors such as dolutegravir and possibly the long-acting injectable cabotegravir, sequencing of longer reads that cover all relevant HIV-1 viral genes  is required. Combined PCR amplification of these genes is difficult as they are spread apart. Genotyping PR and RT genes in a separate assay to the IN gene is an option; however, this doubles the HIVDR genotyping workload. Therefore, there is a need to design Sanger sequencing assays that detect all relevant mutations in a single amplicon, in order to simplify HIVDR genotyping for clinical diagnostics and research use in RLS.Although there are several advantages of Sanger sequencing over NGS for clinical diagnostic use, further technical innovations are still required to make Sanger sequencing affordable and available in RLS. The high cost per sample remains a major drawback for implementation of HIVDR genotyping in RLS. NGS can reduce the per sample costs through sample pooling which, however, results in longer turnaround times. Centralizing NGS for HIVDR genotyping could help address the challenge of longer turnaround times and higher per sample costs. Subsidizing of instrument and consumable costs by manufacturers for RLS, together with governmental support will also go a long way in reducing costs and establishing NGS in RLS.Requirements for expert bioinformatics analyses and high sequencing errors also remain major barriers for use of NGS in HIVDR genotyping. There is a need for international standards that guide NGS for HIVDR genotyping, as well as standardized data analysis pipelines to ensure reproducible, accurate and high-quality data outputs. This could be achieved through internal and external quality assurance programs , and valHyDRA, PASeq and MiCall are freely available web-based data analysis pipelines that require only minimal bioinformatics expertise and infrastructure . They alIn addition, understanding the clinical relevance of LA-DRVs detected by NGS remains elusive for use of NGS data in HIV-1 treatment clinical decisions . The SenHIVDR remains the greatest barrier to sustainable viral suppression on ART, whilst cost remains the greatest barrier to HIVDR genotyping in RLS. Available in-house and commercial sequencing assays should aim to offer accessible and relevant cost-effective HIVDR genotyping that can be used to make clinical decisions. In this review, we emphasize the need for Sanger sequencing assays to adapt to dynamic HIV-1 treatment programs, to simplify and make HIVDR genotyping affordable. We also highlight that NGS has great potential to achieve low-cost HIVDR genotyping useful for individual and diagnostic public-health use in RLS. Moreover, validation of wet-lab processes and data analysis pipelines is required for optimal detection and consistent interpretation of HIV resistance data for clinical utility. Ultimately, firm commitments and partnerships between the molecular diagnostics industry, local governments in RLS and global health agencies, are required to overcome HIVDR genotyping barriers that have often slowed down efforts by the UNAIDS to end the HIV epidemic .


Mastomys natalensis) in driving human infections, but control and prevention efforts remain hampered by a limited baseline understanding of the disease’s true incidence, geographical distribution and underlying drivers. Here, we show that Lassa fever occurrence and incidence is influenced by climate, poverty, agriculture and urbanisation factors. However, heterogeneous reporting processes and diagnostic laboratory access also appear to be important drivers of the patchy distribution of observed disease incidence. Using spatiotemporal predictive models we show that including climatic variability added retrospective predictive value over a baseline model (11% decrease in out-of-sample predictive error). However, predictions for 2020 show that a climate-driven model performs similarly overall to the baseline model. Overall, with ongoing improvements in surveillance there may be potential for forecasting Lassa fever incidence to inform health planning.Lassa fever is a longstanding public health concern in West Africa. Recent molecular studies have confirmed the fundamental role of the rodent host ( Lassa Fever is a rodent-borne viral haemorrhagic fever that is a public health problem in West Africa. Here, the authors develop a spatiotemporal model of the socioecological drivers of disease using surveillance data from Nigeria, and find evidence of climate sensitivity. Lassa virus  is a WHO-listed priority pathogen and a major focus of international vaccine development funding3 and, although often framed as a global health threat, LF is foremost a neglected endemic zoonosis. Lassa virus disease has a varied presentation, with many cases thought to be mild or asymptomatic4. More severe cases usually start with an unspecific fever and malaise, and occasionally progress to haemorrhagic symptoms, with fatalities in around 20% of severe cases. The disease has typically been characterised as having two main endemic foci in West Africa, one centred around Sierra Leone and Liberia, and the other in Nigeria, but in recent years most countries in the region have reported regular or sporadic cases4. Concurrently there has been a decline in surveillance-based case reports from the western hotspot around Sierra Leone, which may be related to the significant negative impacts of the recent Ebola epidemic on health systems and personnel. Conversely, Nigeria, which saw more limited impacts from Ebola, has continued to record a trend of increasing numbers of cases in this time.Between 2018 and 2020, Nigeria recorded its highest annual incidences of Lassa fever (LF) to date , prompting national and international healthcare mobilisation and raising concerns about an ongoing, systematic emergence of LF nationally5—are thought to arise directly from spillover from the LASV reservoir host, the widespread synanthropic rodent Mastomys natalensis, although with hospital-acquired infections potentially occurring in small clusters of human-to-human transmission8. M. natalensis is the most abundant agricultural rodent pest in sub-Saharan Africa and is generally found in high numbers in many human-dominated land types, with lower abundance in natural and forested ecosystems. Populations of this species have a strong seasonal dynamic that varies across habitat types, likely related to changes in food availability over time9. Unintentional interactions with people occur in a variety of settings from cropland and households, as well as intentional contact through hunting-related practices. Risk factors for spillover, while not well understood, are thought to include factors that increase the direct and indirect contact between rodents and people, including ineffective food storage, housing quality, and certain agricultural practices such as crop processing11. Evidence of correspondence between human case surges and seasonal rainfall patterns suggests that LF is a climate-sensitive disease12, whose incidence may be increasing with regional climatic change13.The significant majority of observed LF cases—including those from recent years in Nigeria4, and often-cited annual case estimates  are consequently extrapolations based on limited serological evidence from a handful of early studies15. This, alongside LF’s nonspecific presentation, means that many mild or subclinical infections (possibly 80% or more of infections) are thought to go undetected17. The patchy understanding of LF’s true annual incidence and drivers hinders diagnosis, treatment and disease control18 and provides a limited contextual understanding of whether the recent surges in reported cases have resulted from improvements in surveillance or a true emergence trend. To address these gaps, in this study we analyse the first long-term spatiotemporal epidemiological dataset of acute human LF case data, systematically collected over 8 years of surveillance in Nigeria. We use this dataset to characterise the epidemiology and spatial trends of LF in Nigeria between 2012 and 2019, and evaluate the drivers that explain the geographical distribution of LF occurrence and incidence. We then develop spatiotemporal predictive models to evaluate whether climatic variability can be used to predict interannual differences in the size and timing of outbreak peaks, to evaluate the scope for future forecasting of this high-burden disease.The present-day incidence and burden, however, remain poorly defined, because LASV surveillance has historically been opportunistic or focused on known endemic districts with pre-existing diagnostic capacity20 and Sierra Leone21, with some secondary peaks evident in early March and, increasingly, a small number of cases detected throughout the year , consists of weekly epidemiological reports (WERs) of acute human LF cases collected by all 774 local government authorities (LGAs) across Nigeria between January 2012 and December 2019 . The result of these improvements is potentially reflected in the smoother case accumulation curves in 2018–19 than observed previously  established in 2016, the opening of three additional LF diagnostic laboratories in 2017–19 . We adopt a two-level, hurdle model-based approach, and separately model the annual probability of LF occurrence  and is robustly associated with increasing poverty Fig. , which sM. natalensis, and indeed is supported by the year-on-year expansion of the endemic area in Nigeria as surveillance continues to be rolled-out1. Additionally, since LASV prevalence in rodents can vary widely over small geographical scales (e.g. between neighbouring villages)24, it is also possible that LF risk is highly discontinuous and localised. For example, fluctuations in rodent populations, pathogen dispersal, infection and immune dynamics, could lead to significant and challenging-to-predict variations in spillover risk over space and time. To identify underreported areas and target interventions, therefore, future surveys outside known endemic foci are urgently needed to understand unmeasured social or environmental factors influencing risk .Spatially projecting the fixed effects of the occurrence model . We first developed a baseline spatiotemporal model including spatially structured (state-level) and region-specific temporally-structured (year and week) random effects, and a fixed effect for travel time to Lassa diagnostic laboratory, to account for expansions of surveillance, intra-annual seasonality and baseline differences between states (Methods). To investigate additional effects of environmental conditions on interannual LF outbreak dynamics and evaluate the scope for forecasting, we conducted out-of-sample (OOS) based model selection for linear and nonlinear effects of climate covariates: air temperature, vegetation greenness , mean daily precipitation and Standardised Precipitation Index . These were averaged across a 60-day period starting at 0, 1, 2, 3 and 4-month lags prior to reporting week, to account for delayed effects of climate and any delays between infection and reporting . We considered candidate models for all lagged combinations of all four covariates, and identified the model that minimised OOS predictive error  on sequential 6-month holdout windows across the study period  focusing on the six states that comprise the main endemic foci of LF in the south  and north 2–2019 foThe best combination of climatic predictors included nonlinear effects of SPI (120–180 days lag), precipitation (60–120 days lag) and EVI (0–60 days lag), and substantially reduced OOS error relative to a random and reporting effects-only baseline model  or human agricultural and food storage practices4, all of which are important targets for future research. For example, declines in vegetation during the weeks preceding transmission could lead to synchronous food-seeking behaviour in rodents  and human behaviour changes relating to harvest and crop processing. The retrospective OOS predictive accuracy of the seasonal models , fixing all effects except climatic predictors at 2019 levels . Both models substantially underpredict the true number of cases observed in 2020 , potentially because neither effectively captures ongoing improvements in surveillance sensitivity or other potential unobserved events . The climate-driven model predicts a higher number of cases overall, likely because the model slightly better anticipates the longer duration of the Lassa season in 2020 than the baseline model .The climate-driven model results suggest that seasonal LF risk in endemic areas is linked to the distribution of rainfall in the preceding months  and with declines in vegetation in the preceding 0–60 days Fig. . TogetheM. natalensis, resulting in a strong association between LF cases and rainfall. Peak LF occurrence appears to be in areas experiencing around 1500 mm of rain annually, though incidence increases with rainfall, though with very wide uncertainty under very high rainfall conditions. While the role of host ecological suitability could be important, it may also be human behaviours that vary in response to climatic conditions, such as crop planting or farming techniques, that could be the principal or additional underlying cause of case variation. The trend of increasing incidence with higher rainfall, but the decreasing probability of disease occurrence, could be reflecting the correlation between rainfall and host habitat suitability in Nigeria. Areas with very high rainfall in the south are principally rainforest, which is unsuitable habitat for M. natalensis4. One possible implication for disease risk is that landscape conversion towards derived savannah or agriculture in these areas could drive increases in risk, as environmental conditions become more conducive for M. natalensis populations.Our results show that LF is a climate and land-use sensitive disease of poverty. Much of the environmental mediation of case numbers is likely driven by ecological dependencies of the principal host While the broad limits of the endemic area of LF occurrence and seasonal nature of observed LF cases may be driven by the ecological tendencies of the host or climate-responsive agricultural practices, the spatially discontinuous nature of incidence could be driven by several factors, for example, land-use patterns creating a mosaic of suitable and unsuitable host habitat, or heterogeneous human socioeconomic factors. Although our models identify an overall role of poverty, this is a higher level, compound metric that is likely to influence several more specific risk factors, such as the prevalence of rodent hunting, availability of food storage options, or the probability of having poor-quality housing that allows high levels of rodent ingress. The environmental covariates we examine in our models are, however, still limited in their ability to account for the fine-scale, LGA-level heterogeneity in case incidence; in future, incorporating more detailed socioeconomic and anthropological data may provide further insights. Additionally, it could be that reporting effort, which our models account for using random and distance-based effects, is driving the variation in observed cases at these finer spatial scales, and that more detailed data on reporting procedures would provide clearer insights into these spatial patterns.1 could be a further indication that less severe cases are going undetected. Efforts to better delimit the endemic area and total burden of LF would strongly benefit from investigation of areas with medium-to-high predicted suitability but currently lower observed case incidence, to understand if these are truly low incidence locations.Indicative of this, the highest reporting state, Edo, is not predicted to be a very high incidence state using socio-environmental factors alone, with high cases mostly explained by the effect of distance to the laboratory. From the comparison of the yearly maps Fig. , there iDuring the time period of data collection, West Africa experienced two particularly disruptive epidemics: the Ebola epidemic in 2013–15, and the ongoing global Covid-19 pandemic. Observed cases during the period of the Ebola epidemic are overestimated by our predictive models Fig.  suggesti32). However, the uncertain prospective performance shows that robust evaluation of climate-based forecasting approaches will be limited by the length and quality of the case time series used to develop the models. Improving these models year-on-year with new surveillance data, and including more precise information on agricultural practices, spatiotemporal variation in landscape characteristics and modelled rodent suitability, should assist in further identifying the drivers of large case surges and improving prospective prediction accuracy. Overall our findings have implications for ongoing disease management and targeting of LASV surveillance in rodents and humans toward environmentally suitable areas where LF is apparently absent, prioritising hotspot areas for future vaccination rollouts, and highlights the critical role of improvements in systematic human case surveillance across West Africa21 in helping to explain rising numbers of cases. With LF surveillance continuing to improve in Nigeria, these data and analyses provide a foundation for the future development of user-focused LF risk mapping and forecasting systems, to aid public health responses in this region.The improvement we show in retrospective predictive performance provided by a climate-driven model suggests that such an approach could in the future provide the basis for developing a forecast system in LF-endemic areas , Abakaliki (Ebonyi state) and Owo (Ondo state) . This regime includes post hoc follow-ups to ensure more accurate case counts, so our analyses use WER-derived case data from 2012 to 2016, and SitRep-derived case data from 2017 to 2019 .We visualised temporal and seasonal trends in suspected and confirmed LF cases within and between years, for both surveillance datasets. Weekly case counts were aggregated to country-level and visualised as both annual case accumulation curves, and aggregated weekly case totals Fig. . We also36), and LGA geographical areas in Nigeria are highly skewed and vary over >3 orders of magnitude . We therefore also aggregated all LGAs across Nigeria into 130 composite districts with a more even distribution of geographical areas, using distance-based hierarchical clustering on LGA centroids (implemented using hclust in R), with the constraint that each new cluster must contain only LGAs from within the same state . Weekly and annual suspected and confirmed LF case totals were then calculated for each aggregated district. We used these spatially aggregated districts to test for the effects of scale on spatial drivers of LF occurrence and incidence.Analyses of aggregated district data are sensitive to differences in scale and shape of aggregation ), which provides fast, stable and accurate posterior approximation for complex, spatially and temporally-structured regression models40, and has been shown to outperform alternative methods for modelling environmental phenomena with evidence of spatially biased reporting41.We analysed the full case time series Fig.  to charaM. natalensis distribution and population ecology , frequency and mode of human–rodent contact , both of the above  or likelihood of LF reporting . For each LGA we extracted the mean value for each covariate across the LGA polygon. The full suite of covariates tested across all analyses, data sources and associated hypotheses are described in Supplementary Table We collated geospatial data on socio-ecological and climatic factors that are hypothesised to influence either 42 from the Climate Hazards Infrared Precipitation with Stations (CHIRPS) project; this dataset is based on combining sparse weather station data with satellite observations and interpolation techniques, and is designed to support hydrologic forecasts in areas with poor weather station coverage 42. A recent study ground-truthing against weather station data showed that CHIRPS provides greater overall accuracy than other gridded precipitation products in Nigeria43. Air temperature daily minimum and maximum rasters were obtained from NOAA and were also averaged to calculate daily mean temperature. EVI, a measure of vegetation quality, was obtained from processing 16-day composite layers from NASA  . We obtained daily precipitation rasters for Africa44, average daily mean (Tmean), minimum (Tmin) and maximum (Tmax) temperature and EVI variables at sequential time lags prior to reporting week for seasonal modelling . SPI is a standardised measure of drought or wetness conditions relative to the historical average conditions for a given period of the year. SPI was calculated within a rolling 3-month window across the full 40-year historical CHIRPS rainfall time series (1981–2020) using the R package SPEI v.1.744.We derived several spatial bioclimatic variables to capture conditions across the full monitoring period (Jan 2012 to Dec 2019): mean precipitation of the driest annual month, mean precipitation of the wettest annual month, precipitation seasonality (coefficient of variation), annual mean air temperature, air temperature seasonality, annual mean EVI and EVI seasonality. We also calculated monthly total precipitation, 3-month SPI45, to proxy for the potential for homes to be infested with rodents. We accessed data on agricultural and urban land cover (population-weighted proportion of LGA area) for 2015 from processing ESA-CCI rasters.We accessed annual human population rasters at 100 m resolution from WorldPop. We accessed the proportion of the population living in poverty in 2010 (<$1.25 threshold) from WorldPop, to proxy for ability to access risk prophylaxis schemes (e.g. food storage boxes) and for potential susceptibility to disease as a consequence of lower nutrition and co-infection. We accessed modelled proportion of the population living in improved housing in 201546 to calculate LGA-level mean travel time to the nearest LF diagnostic laboratory (as a proxy for likelihood of sample testing for LASV) and nearest hospital47 . Such distance-based metrics are coarse approximations of complex processes and are subject to limitations. For example, differences in access to transport infrastructure and political unrest will have different effects on reporting in different areas of Nigeria, regardless of proximity to medical facilities, and clinical suspicion for LF will also be influenced by staff training and sensitisation. Furthermore, diagnostic centres are often established in areas where the disease is already recognised to occur  as a measure of LF incidence, since these years followed the establishment of updated systematic surveillance protocols and the associated geographical expansion of suspected case reports  where i during year t, and We model the annual occurrence of LF . Continuous covariates not log-transformed were scaled  prior to fitting linear fixed effects.For both models we considered linear coefficients , and excluding any that did not improve fit by a threshold of at least six DIC units. The best-fitting models with socio-ecological covariates explained substantially more of the variation in the data relative to baseline models  or low  documented incidence. Fixed and nonlinear effects direction and magnitude were robust in all holdout models, indicating that results were not overly driven by data from any one locality  across the full duration of surveillance (2012 to 2019). Low and/or variable surveillance effort outside known endemic areas could confound inference of temporal environmental drivers, so here we focus our analyses on states with case reporting records that span the entire monitoring period. These occur in two foci in the south  and north regions of Nigeria , which in total account for 87% of the total confirmed cases since 2012  as a Poisson process:i during week t, modelled as a log-link function of a linear combination of spatially and temporally-structured random effects and climate covariates:We fitted models to state-level LF time series from these six states , and extracting OOS predicted case counts for the holdout window. A model predictive error was calculated as RMSE of the difference between observed and OOS predicted case counts across the whole time series (2012–2019). We first conducted this procedure for a baseline model containing only random  and reporting effects (travel time to laboratory), which was used as a benchmark to compare against climate-driven model performance  and patient presentation at a medical facility. We included both precipitation and SPI as these reflect different, biologically relevant hydrometeorological phenomena: precipitation is a raw measure of rainfall, whereas SPI measures drought or wetness relative to historical trends at the same location and period of the year 44. We did not include the temporally-invariant covariates included in the spatial models, since the smaller number of states provides low comparative power to detect any spatial effects on incidence.To identify the combination of climate variables that minimised predictive error, we then conducted the same procedure for candidate models containing all combinations of four climate predictors at five different time lags: mean daily precipitation, SPI, EVI and mean daily air temperature, calculated across a 60-day window at time lags beginning from 0 days to 120 days prior to reporting week . We considered lagged climate variables to account for, firstly, delayed effects of seasonal environmental cycles on We then examined the calibration of the best climate-driven model through posterior predictive simulation, again using 6-month sequential holdout windows. Each sub-model was fitted, 2500 parameter samples were drawn from the approximated joint posterior distribution, and these were used to (1) calculate OOS posterior mean and intervals and (2) simulate the OOS Poisson predictive distribution (i.e. the range of plausible expected case counts given the model). We calculated the proportion of observed case counts falling within 67 and 95% OOS predictive intervals, overall and over time  from the model fitted to the full time series. We compared these predictions to 2020 preliminary state-wide confirmed case counts compiled for the NCDC Situation Reports, holding yearly random effects (Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary